Cellular And Molecular Neurobiology

NEUROSCIENCE LECTURE SUPPLEMENT Nachum Dafny, Ph.D., Professor Department of Neurobiology and Anatomy University of Texas Medical School at Houston

A. Cellular and Molecular Neurobiology

TABLE OF CONTENTS

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Overview of the Nervous System ...........................................................................1 Organization of Cell Types....................................................................................17 Resting Potentials and Action Potentials ...............................................................32 Propagation of the Action Potentials ....................................................................38 Synaptic Transmission at Skeletal Neuromuscular Junction ................................43 Synaptic Transmission in the Central Nervous System and Synaptic Plasticity...........................................................................................57

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OVERVIEW OF THE NERVOUS SYSTEM Nachum Dafny, Ph.D.

The human nervous system is divided into the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS, in turn, is divided into the brain and the spinal cord, which lie in the cranial cavity of the skull and the vertebral canal, respectively. The CNS and the PNS, acting in concert, integrate sensory information and control motor and cognitive functions.

The Central Nervous System (CNS) The adult human brain weighs between 1200 to 1500g and contains about one trillion cells. It occupies a volume of about 1400cc - approximately 2% of the total body weight, and receives 20% of the blood, oxygen, and calories supplied to the body. The adult spinal cord is approximately 40 to 50cm long and occupies about 150cc. The brain and the spinal cord arise in early development from the neural tube, which expands in the front of the embryo to form the main three primary brain divisions: the prosencephalon (forebrain), mesencephalon (midbrain), and rhombencephalon (hindbrain) (Figure 1A). These three vesicles further differentiate into five subdivisions: telencephalon, diencephalon, mesencephalon, metencephalon, and the myelencephalon (Figure 1B). The mesencephalon, metencephalon, and the myelencephalon comprise the brain stem.

Figure 1. Schematic lateral view drawing of human embryo at the beginning of the 3rd (A) and 5th (B) week of gestation.

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The telencephalon includes the cerebral cortex (cortex is the outer layer of the brain) which represents the highest level of neuronal organization and function (Figures 2A and 2B). The cerebral cortex consists of various types of cortices (such as the olfactory bulbs, Figure 1.2B) as well as closely related subcortical structures such as the caudate nucleus, putamen, globus, amygdala and the hippocampal formation (Figure 2C).

Figure 2. Lateral (A) and ventral (B) schematic drawing of the cerebral cortex. In C, drawing of subcortical structures.

The diencephalon consists of a complex collection of nuclei lying symmetrically on either side of the midline. The diencephalon includes the thalamus, hypothalamus, epithalamus and subthalamus (Figure 3).

Figure 3. Shows the main diencephalon nuclei.

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The mesencephalon (or midbrain) consists of several structures around the cerebral aqueduct such as the periaqueductal gray (or central gray), the mesencephalic reticular formation, the substantia nigra, the red nucleus (Figure 4), the superior and inferior colliculi, the cerebral peduncles, some cranial nerve nuclei, and the projection of sensory and motor pathways.

Figure 4. Schematic drawing of subcortical diencephalic and mesencephalic structures.

The metencephalon includes the pons and the cerebellum. The myelencephalon (spinal cordlike) includes the open and closed medulla, sensory and motor nuclei, projection of sensory and motor pathways, and some cranial nerve nuclei.

Figure 5. Schematic lateral view of the metencephalon and a spinal cord section with ventral and dorsal root fibers, and dorsal root ganglions.

The caudal end of the myelencephalon develops into the spinal cord. The spinal cord is an elongated cylindrical structure lying within the vertebral canal, which includes the central canal and the surrounding gray matter. The gray matter is composed of neurons and their supporting cells and is enclosed by the white matter that is composed of a dense layer of ascending and 3

descending nerve fibers. The spinal cord is an essential link between the peripheral nervous system and the brain; it conveys sensory information originating from different external and internal sites via 31 pairs of spinal nerves (Figure 5). These nerves make synaptic connections in the spinal cord or in the medulla oblongata and ascend to subcortical nuclei. The central nervous system, which includes the spinal cord and the brain, is the most protected organ in the human body. It is protected from the external environment by three barriers: skull, meninges, and CSF. The meninges are composed by three fibrous connective tissues (Figure.6). The most external is a dense collagenous connective tissue envelope known as the dura mater (Latin for “hard mother”). The second, or the intermediated membrane, is a delicate non-vascular membrane of fine collagenous layer of reticular fibers forming a web-like membrane, known as the arachnoid (Greek for “spider”). It is separated from the inner pia layer by subarachnoid space, which is filled with cerebrospinal fluid. The inner most delicate connective tissue membrane of collagenous is the pia mater, a thin translucent elastic membrane adherent to the surface of the brain and the spinal cord. Blood vessels located on the surface of the brain and the spinal cord are found on top of the pia matter. The meninges are subject to viral and bacterial infection known as meningitis, a life-threatening condition that requires immediate medical treatment.

Figure 6. Schematic drawing of the brain and spinal cord meninges.

The space between the skull and the dura is known as the epidural space. The space between the dura and the arachnoid is known as the subdural space. The space between the arachnoid 4

and the pia is known as the subarachnoid space. In this space, there is a clear liquid known as the cerebrospinal fluid (CSF). The CSF serves to support the CNS, and to cushion as well as protect it from physical shocks and trauma. The CSF is produced by the choroid plexus which is composed of a specialized secretory ependymal layer located in the ventricular system. The ventricular system is a derivative of the primitive embryonic neural canal. This system is an interconnected series of spaces within the brain containing the CSF (Figure 7).

Figure 7. Schematic drawing of ventricular system in four different angles.

In general, the CNS can be divided into three main functional components: the sensory system, the motor system, and homeostasis and higher brain functions. The sensory system consists of the somatosensory, viscerosensory, auditory, vestibular, olfactory, gustatory, and visual systems. The motor system consists of motor units, and the somatic (skeletal muscle) system, the spinal reflexes, the visceral (autonomic) system, the cerebellum, several subcortical and cortical sites, as well as the brain stem ocular motor control system. The homeostasis and higher functional system includes the hypothalamus, cortical areas involved in motivation, insight, personality, language, imagination, creativity, thinking, judgment, mental processing, and subcortical areas involved in learning, thought, consciousness, memory, attention, emotional state, sleep and arousal cycles.

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The Cerebral Hemispheres The telencephalon is the largest and most obvious parts of the human brain are the cerebral hemispheres. The cerebrum has an outer layer - the cortex, which is composed of neurons and their supporting cells, and in fresh brain, has a gray color thus called the gray matter. Under the gray matter there is the white matter, which is composed of myelinated ascending and descending nerve fibers, and in fresh brain have a white color. Embedded deep within the white matter are aggregation of neurons exhibiting gray color and known as subcortical nuclei. The cerebral hemispheres are partially separate from each other along the midline by the interhemispheric fissure (deep groove) the falx cerebri (Figure 8A); posteriorly, there is a transverse fissure that separates the cerebral hemisphere from the cerebellum, and contains the tentorium cerebellum. The hemispheres are connected by a large C-shaped fiber bundle, the corpus callosum, which carries information between the two hemispheres.

Figure 8. Schematic drawing of six cortical lobes: Dorsal view (A), Lateral view (B), Mid-sagittal section showing the limbic lobe (in green) (C), and Horizontal section showing the insular cortex (D).

For descriptive purposes each cerebral hemisphere can be divided into six lobes. Four of these lobes are named according to the overlying bones of the skull as follows: frontal, parietal, occipital and temporal (Figures 8A and 8B), the fifth one is located internally to the lateral sulcus – the insular lobes (Figure 8B and 8D), and the sixth lobe is the limbic lobe (Figure 8C) which contains the limbic system nuclei. Neither the insular lobe nor the limbic lobe is a true lobe. Although the boundaries of the various lobes are somewhat arbitrary, the cortical areas in each lobe are histologically distinctive. The surface of the cerebral cortex is highly convoluted with folds (gyri), separate from each other by elongate grooves (sulci). These convolutions allow for the expansion of the cortical surface area without increasing the size of the brain. On the lateral surface of the cerebral hemisphere there are two major deep grooves-sulci (or fissure), the lateral fissure (of Sylvian) and the central sulci (of Rolando), these sulci provide landmarks for topographical orientation (Figure 9A). The central sulcus separates the frontal lobe from the parietal lobe and runs from the superior margin of the hemisphere near its midpoint obliquely downward and forward until it nearly meets the lateral fissure (Figures 8A and 8B). The lateral fissure, separating the frontal and parietal lobes from the temporal lobe, begins inferiorly in the basal surface of the brain and extends laterally posteriorly and upward, separating the frontal and parietal lobes from the temporal lobe (Figure 9A). The frontal lobe is the portion which is rostral to the central sulcus 6

and above the lateral fissure, and it occupies the anterior one third of the hemispheres (Figures 8 and 9). The boundaries of the parietal lobe are not precise, except for its rostral border – the central sulcus. The occipital lobe is the portion which is caudal to the parietal lobe (Figures 8 and 9). Along the lateral surface of the hemisphere, an imaginary line connecting the tip of the parietal-occipital sulcus and the preoccipital notch (Figure 9A); separate the parietal lobe from the occipital lobe. On the medial surface of the hemisphere (Figure 9B), parieto-occipital sulcus forms the rostral boundary of the parietal lobe. The temporal lobe lies ventral to the lateral sulcus, and on its lateral surface, it displays three diagonal oriented convolutions-the superior, middle, and inferior temporal gyri (Figure 9A). The insula lies in the depths of the lateral sulcus. It has a triangular cortical area with gyri and sulci (Figures 8B & 2D, and Figure 9A). The limbic lobe consists of several cortical and subcortical areas (Figure 9B).

Figures 9A and 9B. Lateral schematic drawing of the different cortices, sulci and gyri (A) and mid-sagittal drawing emphasizes the limbic lobe (in green) (B).

The cerebral cortex is a functionally organized organ. A functional organized system is a set of neurons linked together to convey a specific type(s) of information to accomplish a particular task(s). It is possible to identify on the cerebral cortex primary sensory areas, secondary sensory areas, primary motor area, premotor area, supplementary motor area and association areas, which are devoted to the integration of motor and sensory information, intellectual activity, thinking and comprehension, execution of language, memory storage and recall. The frontal lobe is the largest of all the brain lobes and is comprised of four gyri, precentral gyrus that parallels to the central sulcus, and three horizontal gyri: the superior, middle, and inferior frontal gyri. The inferior frontal gyrus is comprised of three parts: the orbital, the triangular and opercular. The term opercular refers to the “lips of the lateral fissure. Finally, 7

the straight gyrus (gyrus rectus) and the orbital gyri form the base of the frontal lobe (Figure 9B). Four general functional areas are in the frontal lobe. They are the primary motor cortex, where all parts of the body are represented, the premotor and supplementary motor areas. A region concerned with the motor mechanisms of speech formulation comprised of the opercular and triangular parts of the inferior frontal gyrus are known as Broca’s speech area, and the remainder of the prefrontal cortex is involved in mental activity, personality insight, foresight, and reward. The orbital portion of the prefrontal cortex is important in the appropriate switching between mental sets and the regulations of emotion. The parietal lobe is comprised of three gyri: postcentral gyrus, superior and inferior parietal gyri (Figure 9A). The postcentral gyrus is immediately behind the central sulcus which forms its anterior boundary. The postcentral gyrus comprises the primary somatosensory cortex which is concerned with somatosensory reception, integration and processing sensory information from the surface of the body and from the viscera, and is important for the formulation of perception. Caudal to the postcentral gyrus is the inferior parietal gyrus. The intraparietal sulcus separates the posterior parietal gyrus from the inferior parietal gyrus. The inferior parietal gyrus represents the cortical association area which integrates and processes sensory information from multiple modalities such as auditory and visual information. The inferior parietal gyrus, which is known as Wernicke's area, is also important for language and reading skills, whereas the superior parietal gyrus is concerned with body image and spatial orientations. The temporal lobe is formed by three obliquely oriented gyri: the superior, middle, and inferior temporal gyri (Figure 9A). Inferomedial to the inferior temporal gyrus are the occipitotemporal and the parahippocampal gyri, which are separated by the collateral sulcus. The upper surface of the superior temporal gyrus, which extends into the lateral fissure, is called the transverse temporal gyrus (of Heschl) and is the primary auditory cortex. The caudal part of the superior temporal gyrus, which extends up to the parietal cortex, forms part of Wernicke’s area. Wernicke’s area is concerned, in part, with processing the auditory information and is important in the comprehension of language. The inferior part of the temporal lobe (i.e., the occipitotemporal gyri) is involved in visual and cognitive processing. More medially is the parahippocampal gyrus, which is involved in learning and memory. Portions of the frontal, parietal, and temporal lobes, which are adjacent to the lateral sulcus and overlie the insular cortex, are known as the operculum. The inferomedial surface of the temporal lobe is made up of the uncus and the parahippocampal gyrus medially. The inferior surface of the temporal lobe rests on the tentorium cerebelli. The occipital lobe is the most caudal part of the brain, lies on the tentorium cerebelli (Figure 9A) and is comprised of several irregular lateral gyri. On its medial surface, there is a prominent fissure – the calcarine fissure and parieto-occipital sulcus. The calcarine fissure (sulcus) and the parieto-occipital sulcus also define a cortical region known as the cuneus. The cuneus sulcus divides the occipital lobe into the cuneus dorsally and ventrally into the lingual gyrus. The occipital lobe contains the primary and higher-order visual cortex. The insula lobe is located deep inside the lateral fissure and can be seen only when the temporal and the frontal lobes are separated. The insula is characterized by several long gyri and sulci, the gyri breves and gyri longi. There is some evidence that the insular cortical areas are involved in nociception and regulation of autonomic function (Figures 8B and 8D). 8

The limbic lobe is not a true lobe and is comprised of several cortical regions such as the cingulate and parahippocampal gyri, some subcortical areas like the hippocampus, amygdala, septum, and other areas with their respective ascending and descending connections (Figures 8C and 9B). The limbic lobe is involved in memory and learning, drive related behavior, and emotional function. There are subcortical areas in the telencephalon like the basal ganglia and the amygdaloid nucleus complex. The corpus callosum is a collection of nerve fibers which connect the two hemispheres. The corpus callosum is divided into rostrum (head), body, the most rostrally part is the genu (knee) with connecting the rostrum and the body, and the splenium at the caudal extremity (Figure 10). Behavioral studies have shown that the corpus callosum play an important role in transferring information from one hemisphere to the other.

Figure 10. The corpus callosum and its different parts.

The Diencephalon The second major derivative of the prosencephalon is the diencephalon. The diencephalon is the most rostral structure of the brain stem; it is embedded in the inferior aspect of the cerebrum. The posterior commissure is the junctional landmark between the diencephalon and the mesencephalon. Caudally, the diencephalon is continuous with the tegmentum of the midbrain. During development the diencephalon differentiates into four regions: thalamus, hypothalamus, subthalamus and epithalamus (Figure 11). The epithalamus comprises the stria medullaris habenular trigone, pineal gland and the posterior commissure (Figure 11).

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Figure 11. Midsagittal drawing showing the main structures of the diencephalon.

The Brain Stem The brain stem consists of mesencephalon (midbrain), metencephalon, and myelencephalon. The metencephalon and myelencephalon together compose the rhombencephalon (hindbrain), which divides into pons, and medulla oblongata (Figure 12). Mesencephalon (midbrain) is continuous with the diencephalons rostrally and with the pons caudally. The midbrain is the smallest part of the brain stem, being about 2 cm in length. It consists of a tectum posteriorly, a tegmentum inferiorly, and a base anteriorly. The tectum forms the roof of the cerebral aqueduct, which connects the third ventricle with the fourth ventricle and the tegmentum its floor. The base of the midbrain consists of the cerebral peduncle, which contain nerve fibers descending from the cerebral cortex. The nuclei of the 3rd (oculomotor), the 4th (trochlear) and part of the 5th (trigeminal) are located in the midbrain tegmentum. The red nucleus and the substantia nigra, two prominent nuclei, are also found in the midbrain tegmentum. The midbrain tectum is formed by two pairs of rounded structures: the superior and inferior colliculi. The superior and inferior colliculi (Figure 12) are involved in visual and auditory functions respectively.

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Figure 12. Midsagittal drawing of the brain stem.

Pons. The pons is continuous with the midbrain and is composed of two parts, the pontine tegmentum (located internally) and the basilar pons. At the level of the pons, the cerebral aqueduct has expanded to form the fourth ventricle (Figure 12). The cerebellum is situated posterior to the pons and forms part of the roof (tectum) of the forth ventricle. The pons contains nuclei that receive axons from various cortical areas. Projections from the axons of these pontine neurons form large transverse fiber bundles which traverse the pons and ascend to the contralateral cerebellum via the middle cerebellar peduncles. Also, within the pons base and tegmentum are longitudinally ascending and descending fibers. The nuclei of the 5th (trigeminal), 6th (abducens), 7th (facial) and the 8th (vestibulocochlear) nerves are located in the pons tegmentum. Medulla Oblongata (myelencephalon is also known as the medulla). The medulla lies between the pons rostrally and the spinal cord caudally. It is continuous with the spinal cord just above to foramen magnum and the first spinal nerve. The posterior surface of the medulla forms the caudal half of the fourth ventricle floor and the cerebellum, its roof (Figure 12). The base of the medulla is formed by the pyramidal-descending fibers from the cerebral cortex. The medulla tegmentum contains ascending and descending fibers and nuclei from the 9th (glossopharyngeal), 10th (vagus), 11th (accessory) and the 12th (hypoglossal) nerves. The corticospinal fibers (pyramid) are alongside the anterior median fissure, and decussate (cross the midline) to the contralateral side on their way to the spinal cord. Other prominent structures in the medulla are the inferior olive, and the inferior cerebellar peduncle. The medulla contains nuclei which regulate respiration, swallowing, sweating, gastric secretion, cardiac, and vasomotor activity. The arterial blood supply to the brain is derived from two arterial systems: the carotid system and the vertebrobasilar system. A series of an anastomotic channels lying at the base of the brain, known as the circle of Willis, permits communication between these two systems (Figure 13).

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Figure 13. Schematic drawing showing the main arterial blood supplies to the brain.

The arterial blood supply to the spinal cord is derived from two branches of vertebral artery, the anterior and two posterior spinal arteries which run the length of the spinal cord and form an irregular plexus around it (Figure 14).

Figure 14. Schematic drawing of the spinal cord arterial blood supply.

The Peripheral Nervous System (PNS) The PNS includes 31 pairs of spinal nerves, 12 pairs of cranial nerves, the autonomic nervous system and the ganglia (groups of nerve cells outside the CNS) associated with them. Also included in the PNS are the sensory receptor organs. The receptor organs are scattered in all parts of the body, sense and perceive changes from external and internal organs, then transform this information to electrical signals, which are carried via an extensive nervous network to the CNS (Figure 15). The cranial and spinal nerves contain nerve fibers that conduct information to-afferent-(Latin for carry toward) and from-efferent (Latin for carry away) the CNS. Afferent fibers convey sensory information from sensory receptors in the skin, mucous membranes, and internal organs and from the eye, ear, nose and mouth to the CNS; the efferent fibers convey signals from cortical and subcortical centers to the spinal cord and from there to the muscle or autonomic ganglia that innervate the visceral organs. The afferent (sensory) fibers enter the spinal cord via the dorsal (posterior) root, and the efferent (motor) fibers exit the spinal cord via 12

the ventral (anterior) root. The spinal nerve is formed by the joining of the dorsal and the ventral roots. The cranial nerves leave the skull and the spinal cord nerves leave the vertebrae through openings in the bone called foramina (Latin for opening).

Figure 15. Schematic drawing of the peripheral nervous system.

The PNS is divided into two systems: the visceral system and the somatic system. The visceral system is also known as the autonomic system. The autonomic nervous system (ANS) is often considered a separate entity; although composed partially in the PNS and partially in the CNS, it interfaces between the PNS and the CNS. The primary function of the ANS is to regulate and control unconsciousness functions including visceral, smooth muscle, cardiac muscle, vessels, and glandular function (Figure 16). The ANS can be divided into three subdivisions: 1. The sympathetic (or the thoracolumbar) subdivision associated with neurons located in the spinal gray between the thoracic and the upper lumbar levels; 2. The parasympathetic (or craniosacral) subdivision is associated with the 3rd, 7th, 9th and the 10th cranial nerves as well as with the 2nd, 3rd, and 4th sacral nerves; 3. The enteric subdivision is a complex neuronal network within the walls of the gastrointestinal system and contains more neurons than the spinal cord. The visceral (autonomic) system regulates the internal organs outside the realm of conscious control. The PNS component of the somatic system includes the sensory receptors and the neurons innervating them and their nerve fibers entering the spinal cord. The visceral and the somatic nervous system are primarily concerned with their own functions, but also work in harmony with other aspects of the nervous system.

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Figure 16. Schematic drawing showing the autonomic nervous system. C, T, L and S indicate the cervical, thoracic, lumbar and sacral levels of the spinal cord, respectively. III, VII, IX, X indicate the 3rd, 7th, 9th and 10th crania nerves respectively.

Orientation to the Central Nervous System In this section you will be introduced to representative sections through the human CNS. They will acquaint you with prominent structures that will help you to recognize the level and orientation of the section and provide landmarks for locating nuclei and tracts involved in sensory and motor functions. Directional terms are used in describing the locations of structures in the CNS.

Figure 17. Orientation of the central nervous system of the spinal cord and different brain sections.

Keep in mind that certain terms were developed to describe the nervous system of quadrupeds and may have a slightly different meaning when applied to bipeds. For example, the ventral surface of the quadruped spinal cord is comparable to the anterior surface of the biped (Figure 14

18). In the following descriptions, the terms are applied to a standing human. The terms rostral and anterior refer to a direction towards the face/nose. The terms caudal and posterior refer to a direction towards the buttocks/tail. The terms inferior and superior generally refer to spatial relationships in a vertical direction (Figure 18). A coronal section is parallel to the vertical plane and a midcoronal section would divide the head into anterior and posterior halves (Figure 19). The sagittal section is also parallel to the vertical plane, but a midsagittal section would divide the head into right and left halves. The horizontal (axial) section is parallel to the horizontal plane and a mid horizontal section would divide the head into superior and inferior halves. Transverse or cross sections of the spinal cord of humans are taken in a plane perpendicular to the vertical, i.e., in the horizontal plane of the head. Most electromagnetic imaging techniques produce images of the brain in the coronal, horizontal (axial) and sagittal planes. The representative sections are transverse sections through the spinal cord and brain stem and coronal sections through the telencephalon and diencephalon (Figure 17).

Figure 18. A schematic illustration showing the brain direction.

Figure 19. A schematic illustration showing the three planes of brain section.

Transverse Section through the Spinal Cord. This section was taken at the level of the thoracic spinal cord (Figure 17A). The spinal cord neuron (gray matter) form a central core taking a butterfly configuration that is surrounded by nerve fibers (white matter). In the left and right halves of the spinal cord, the gray matter is organized into a dorsal horn and ventral horn with the intermediate gray located between them. In the thoracic spinal cord, which is illustrated in this figure, a lateral horn extends laterally from the intermediate gray (Figure 17A). The spinal cord white matter is subdivided into the posterior white column, the anterior white column and the lateral white column. The anterior white commissure joins the two halves of the spinal cord and is located ventral to the intermediate gray. The dorsal root fibers enter the spinal cord at the 15

dorsolateral sulcus and the fibers of the ventral root fibers exit the spinal cord in numerous fine bundles through the ventral funiculus (see Figures 1-5). Transverse Section through the Medulla. (Figure 17B) This is a section taken at the level of the upper medulla. Landmark structures include the fourth ventricle, hypoglossal nucleus, inferior cerebellar peduncle, inferior olivary complex and the pyramids. As in the spinal cord section, the fiber tracts, the inferior cerebellar peduncle and pyramids, appear dark in this section while the nuclei in the inferior olivary complex appear light. Transverse Section through the Pons. (Figure.17C) This is a section taken at the level of the mid pons. Landmark structures include the fourth ventricle, the pons tegmentum, which includes the abducens nuclei; the pons base, which includes the corticofugal fibers and pontine nuclei; and the middle cerebellar peduncles. Coronal Section through the Rostral Telencephalon. (Figure 17D) This is a section taken at the level of the decussation of the anterior commissure. Landmark structures include the head of the caudate nucleus, the anterior limb of the internal capsule, the globus pallidus and putamen (all-important in controlling motor functions). The anterior commissure, a fiber bundle connecting the right and left frontal lobes, can be seen decussating (crossing the midline). The corpus callosum forms a thick band of decussating nerve fibers located above the lateral ventricles. Below the telencephalon afferent nerve fibers from each eye decussate in the optic chiasm and join uncrossed fibers to form the optic tract. Coronal Section through the Midbrain-Diencephalon Junction. (Figure 17E) This is a section taken at the level junction of the midbrain with the diencephalon. Notice that the plane of section differs from those of previously viewed sections. At this level, a landmark structure of the diencephalon is the thalamus, which surrounds the third ventricle. The posterior limb of the internal capsule separates the thalamus from the surrounding telencephalic structures, i.e., the globus pallidus and putamen. Lateral to the putamen is the insula while more dorsomedially the corpus callosum overlies the cavities of the lateral ventricles. Below the third ventricle are the red nucleus, substantia nigra and crus cerebri of the midbrain, which are the continuation of the internal capsule. Section through the Midbrain. (Figure 17F) This section aims to show the main midbrain nuclei which include the tectum (superior colliculi) the periaqueductal gray, the red nuclei, substantia nigra and the cerebral peduncles.

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ORGANIZATION OF CELL TYPES Jack C. Waymire, Ph.D.

The human nervous system is estimated to consist of roughly 90 billion nerve cells. There are hundreds of different types of neurons based on morphology alone. Further neurons that look similar may have strikingly different properties- for example the neurotransmitter(s) they respond to or utilize. This lecture reviews the cellular components of nervous tissue. You should be able to describe neurons and glia, their morphological components, as seen with the light and electron microscope, and some of the fundamental functional roles these cell types play in the nervous system. I

NEURONS

A. Structure of a model neuron. The morphology of an individual neuron is intimately related to its particular role in processing and transmitting information. Neurons interact with sensory receptors (which themselves may be neurons or epithelial derivatives), with other neurons, and with effectors such as skeletal muscle fibers and the smooth muscle components of the viscera. Ignoring the wide variety of neurons that exist in the nervous system we can describe a generalized neuron which has features shared by most neurons, as seen in the light microscope. This neuron is diagrammed in Fig. 1. It has the basic subcellular organelles needed to carry out metabolic functions.

Figure 1. Diagrammatic representation of a neuron: This neuron makes functional contacts with other neurons via axon collateral and with muscle fibers via the motor endplates. Neurons may contact blood vessels, neurons, glands and muscles.

1. Cell Body. The region of the neuron containing the nucleus is known as the cell body, soma, or perikaryon. The cell body is the metabolic "center" of the neuron. Structures situated toward this center are designated proximal, while structures away from the cell body are said to be distal. A membrane surrounds the entire neuron, including the cell body and the cytoplasmic processes (dendrites and axon).

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Figure 2. Diagrammatic representation of the neuron cell body or perikaryon emphasizing the endoplasmic reticulum, Golgi apparatus and cytoskeleton.

The metabolic center of a neuron is the cell body, or soma illustrated in Figure 2. The interior of the soma, consists of cytoplasm, a gel within a microtrabecular lattice formed by the microtubules and associated proteins that make up the cytoskeleton. These microstructures of the cytoplasm are described in greater detail in the Glossary. Energy producing metabolism and the synthesis of the macromolecules used by the cell to maintain its structure and execute its function are the principal activities of the neuronal soma. As described in Dr. Byrne’s lectures, it also acts as a receptive area for synaptic inputs from other cells. Embedded within the neuronal cytoplasm are the organelles common to other cells, the nucleus, nucleolus, endoplasmic reticulum, Golgi apparatus, mitochondria, ribosomes, mitochondria lysosomes and peroxisomes. Many of these cell inclusions are responsible for the expression of genetic information controlling the synthesis of cellular proteins and enzymes involved in energy production, growth, and replacement of materials lost by attrition. Dendrite. The membrane of the neuron can function as a receptive surface over its entire extent; however, specific inputs (termed afferents) from other cells are received primarily on the surface of the cell body and on the surface of the specialized processes known as dendrites (as shown in Figure 3). The dendritic processes may branch extensively and is often covered with projections known as dendritic spines. Spines provide a tremendous increase in the surface area available for synaptic contacts. The dendritic processes of neurons are essentially expansions of cytoplasm containing most of the organelles found in the cell body. Dendrites contain numerous orderly arrays of microtubules and fewer neurofilaments (see below). The microtubules associated proteins (MAPs) in the dendrite are higher in molecular weight than those found in the 2.

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axon. An example is MAP2. In addition microtubules in dendrites have their positive ends toward the cell soma. Mitochondria are often arranged longitudinally. Rough endoplasmic reticulum and ribosomes are present in large, but are not small dendrites. The shape and extent of the "dendritic tree" of an individual neuron is indicative of the quantity and variety of information received and processed by that neuron.

Figure 3. Diagrammatic representation of the neuron dendrite, emphasizing the areas of contact by other afferent inputs to the neuron.

Information is received by the dendrite through an array of receptors on dendrite surfaces that react to transmitters released from the axon terminals of other neurons. Dendrites may consist of a single twig-like extension from the soma or a multi-branched network capable of receiving inputs from thousands of other cells. For instance, an average spinal motor cell with a moderate-sized dendritic tree, both in the number of branches and their length, may receive 10,000 contacts, 2,000 on the soma and 8,000 on the dendrites. 3. Axon. The other type of process in our idealized neuron is the axon shown in Figure 4. Each neuron has only one axon and it is usually straighter and smoother than the dendritic profiles. Axons also contain bundles of microtubules and neurofilaments and scattered mitochondria. The MAPs in axon have a lower molecular weight than those in the axon. A predominant MAP in axons is tau. Beyond the initial segments, the axoplasm lacks rough endoplasmic reticulum and free ribosomes. The branches of axons are known as axon collaterales. The axon itself is often surrounded by a membranous myelin sheath formed by non- neuronal cells. The myelin sheath acts to insulate the plasmalemma of the axon in a way that necessitates the saltatory spread of the depolarization of the plasmalemma and increases the speed of conduction of the nerve impulse.

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Figure 4. Diagrammatic representation of the neuron axon emphasizing the areas of microtubules, neurofilaments coursing within the cytoplasm.

4. Initial Segment and Axon Hillock. The cone-shaped region of the cell body from which the axon originates in an idealized neuron is the axon hillock. This areae is free of ribosomes and most other cell organelles, with the exception of cytoskeletal elements and organelles that are being transported down the axon. The neurofilaments become clustered together as fascicles. The region between the axon hillock and the beginning of the myelin sheath is known as the initial segment. This is the anatomical location for the initiation of the action potential. The area under the axolemma in this region has material that stains darkly when viewed by EM. This is shown in Figure 5. At the distal-most end of the axon or its collaterales are small branches whose tips are button-shaped cytoplasmic enlargements, called terminal boutons or nerve endings.

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Figure 5. Diagrammatic representation of the neuron initial segment emphasizing the areas where the action potential is initiated.

5. Nerve Ending. That part of the plasma membrane of the nerve ending which is specialized to form functional contacts with other cells is termed the synapse. When neurons interact with muscle fibers, the region of functional contact is called the neuromuscular junction or motor endplate. According to the classical definition of synapses, when a nerve ending synapses on a dendrite or soma of a second neuron they are termed an axodendritic or axosomatic synapse, respectively. However, almost all possible combinations of pre- and postsynaptic elements have been found in the central nervous system. These different types of synapse are designated by combining the name of the structure of the presynaptic element with that of the postsynaptic structure. For example, when the transfer of information occurs from an axon to axon or from one terminal to another, the synapse involve is called an axo-axonic synapse. Cellular elements at the typical nerve ending Regions of functional contacts between neurons (synapses) have distinct morphological characteristics. Though a great deal of variation exists in the size and shape of boutons of individual neurons, synapses can be identified by the presence of the following: (1) A presynaptic complement of membrane-bound synaptic vesicles exists. These are spherical vesicles in excitatory nerve ending, shown in Figure 6. In inhibitory neurons the synaptic vesicle are often flattened. (2) The nerve ending often has aggregations of dense material in the cytoplasm immediately adjacent to the membrane on one or both sides of the junction (these are known as pre- or postsynaptic densities-although this is not an absolute 21

criterion. This dense material on the presynaptic side is thought to be the site of vesicle attachment preceding neurotransmitter release. (3) There is a distinct synaptic cleft or intercellular space of approximately 20-40 nm; and (4) Many mitochondria are present, especially in the nerve terminal.

Figure 6. Left This is a diagrammatic representation of an excitatory synapse, showing the spherical vesicles and the presynaptic density. Right This is a diagrammatic representation of an inhibitory synapse, showing the flat vesicles and the presynaptic density.

B. Variations in neuronal structure.. Numerous variations of the "model" neuron described above exist. An important modification, which occurs especially in receptor neurons, involves the designation of a neuronal process as a dendrite or as an axon. Classically, the axon has been identified as the myelinated or unmyelinated process that transmits signals away from the cell body. The classical view of the dendrite is that of an unmyelinated tube of cytoplasm which carries information toward the cell body. However, this distinction does not hold for ALL neurons. Some cells have a myelinated process that transmits signals toward the cell body. Morphologically the "dendrite" and the "axon" may, therefore, be indistinguishable. Neither the position of the cell body nor the presence or absence of myelin is always a useful criterion for understanding the orientation of the neuron. The region of impulse initiation is more reliable guide to understanding the functional focal point of the cell. This region is analogous to the initial segment of the model neuron, discussed above. Routinely the fiber or process, which contains the initial segment or trigger zone, is referred to as an axon. Note that the trigger zone does not have to be immediately adjacent to the cell body. One of the most important variations is the differences in the ways neurons make synapses with other neurons. This will be covered by Dr. Byrne when he discusses the fact that synapses can be on any element of the postsynaptic cell. As we will discuss in the next lecture, the nature of the synapse varies also. Some nerve endings such as those of monoamine neurons make very diffuse synaptic contacts and have no classic synaptic cleft.

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Figure 7. Comparison of variations in the structure of neurons.

C. Naming neuronal types. A number of conventions have evolved to classify and name neurons. One of the oldest, devised by Golgi in the late 1800’s,.is based on the complexity of the dendritic tree of the neuron. Through this approach cells are classified as unipolar, bipolar and multipolar neurons as shown in Figure 8. Unipolar cells have only one cell process. These are primarily found in invertebrates but a form of this type of cells is the vertebrate sensory neurons. Because these cell start out developmentally as bipolar neurons and then become unipolar as they mature, they are called pseudounipolar cells. Bipolar cells are present in the retina and the olfactory bulb. Multipolar cells make up the remainder of neuronal types and are, consequently, the most numerous type. These have been further sub-categorized into Golgi type I cells that are small neurons, usually interneurons, and Golgi type II cells that are large multipolar neurons. Cells are also named for their shape (e.g. pyramidal cells) or for the person who first described them (e.g. Purkinje cells). And more recently cells have been named for their function or the neurotransmitter they contain (e.g. CNS norepinephrine cells groups are designated A1-A12). This is possible because of the development of histochemical and immunocytochemical methods to specifically identify the neurotransmitter type used by neurons.

Figure 8. Two variations in cell morphology: Left is the pyramidal cell named for its characteristic pyramid shape. This cell is prominent in the cerebral cortex. Right is the cell soma and dendrites of the Purkinje cell found in the cerebellum and named for the scientist, Purkinje.

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D.

Glossary

Endoplasmic reticulum is a labyrinthine, membrane bounded compartment in the cytoplasm where lipids are synthesized and membrane bound proteins are made. In some regions of the neuron ER is devoid of ribosomes and is termed smooth ER. The smooth ER is involved in Ca2+ buffering and in the biosynthesis and recycling of synaptic vesicles as will be discussed in the next lecture. Endosome is a membrane-bounded organelle that carries materials ingested by endocytosis and passes them to lysosomes and peroxisomes for degradation. Golgi apparatus is a collection of stacked, smooth-surfaced membrane bound organelles where proteins and lipids made in the endoplasmic reticulum are modified and sorted. Lysosomes contain enzymes that digest compounds that originate inside or outside the cells. They are involved in converting proteins to amino acids and glycogen to glucose, the basic nutrient of neurons. Their enzymes act at acidic pH. As will be described later they also serve as vesicles for reverse transport from axon terminals to the soma. Many lysosomes become degraded to lipofuscin granules, which accumulate as the organism ages and are regarded as neuronal refuse. Budding off the Golgi apparatus form lysosomes in a variety of membrane-bound shapes and sizes, ranging in size between 250 and 700 nm in diameter. Microfilaments are 7 nm in diameter filaments arranged as a paired helix. of two strands of globular actin. Microfilaments are especially prominent in synaptic terminals, in dendritic spines and in association with the axolemma. Microtubules are 20 to 25 nm diameter tubular structures that run in loose bundles around the nucleus and funnel into the base of the axonal and dendritic processes where they form parallel arrays distributed longitudinally. They are made up of dimers of α and β tubulin subunits and contain associated proteins known as microtubule associated proteins (MAPS). The MAPS regulate the polymerization of tubulin subunits to form the microtubules. The dimers of α and β tubulin subunits polymerize to form protofilaments arranged in an α helix such that 13 dimer subunits make up each full turn of the α helix. In addition microtubules are not continuous, each microtubule is composed of numerous 100 μm units. Microtubules are involved in axoplasmic transport. Function in axoplasmic transport that will be discussed later. Mitochondria are distributed ubiquotously throughout the cytoplasm of the entire nerve cell and are especially plentiful at presynaptic specializations. Neurofilaments are a type of intermediate filament found in nerve cells. Neurofilaments are involved with the maintenance of the neuron shape and mechanical strength. Although neuronal neurofilaments are classified as intermediate filaments, their

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composition in neurons is different than found in other cells. They are composed of three subunits that are arranged to form a 10-nm diameter tubule. It is the neurofilament that stains with heavy metal to permit the visualization of neuronal shape. Neurofilaments run in loose bundles around the cell nucleus and other organelles and funnel into the base of the axonal and dendritic processes where they form parallel arrays distributed longitudinally. Neurofilaments are more abundant than microtubules in axons whereas microtubules are more abundant than neurofilaments in dendrites. It is the neurofilaments that undergo modification in the Alzheimer's disease to form neurofibrillary tangles, as will be discussed later in the Neuroscience course. Nucleolus is in the center of the nuclei of all neurons. It is a prominent, deeply stained spherical inclusion about one-third the size of the nucleus. The nucleolus synthesizes ribosomal RNA, which has a major role in protein synthesis. Nucleus of the neuron is large and round and is usually centrally located. In some cells, masses of deeply staining chromatin are visible in the nucleus. The nuclear membrane of neurons is like that of other cells - a double membrane punctuated by pores (nuclear pores) which are involved in nuclear-cytoplasmic interactions. The nucleus in neurons is spherical and ranges in diameter from 3 and 18 micrometers depending on the size of the neuron. Neurons with long axons have a larger cell body and nucleus. As in other cells, the principal component of the nucleus is deoxyribonucleic acid (DNA), the substance of the chromosomes and genes. Peroxisomes are small membrane bounded organelles that use molecular oxygen to oxidize organic molecules. They contain some enzymes produce, and others that degrade hydrogen peroxide. Plasma membrane of the neuron appears in the electron microscope as a typical bilayered cellular membrane, approximately 10 nm thick. Postsynaptic density is darkly staining material of postsynaptic cell adjacent to the synapse. Receptors, ion channels and other signaling molecules are likely bound to this material. Presynaptic density is the region of darkly staining material adjacent the synapse where synaptic vesicle are hypothesized to dock prior to fusion with the presynaptic membrane. Ribosomes are particles composed of ribosomal RNA and ribosomal protein that associates with mRNA and catalyzes the synthesis of proteins. When ribosomes are attached to the outer membranes of the ER the organelle is termed rough ER. The rough ER, in laminae with interspersed ribosomes is visible with the light microscope as Nissl substance. In light microscopic preparations, the appearance of Nissl substance varies in different types of neurons. It may appear as densely stained ovoids or as finely dispersed particles or aggregations of granules.

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Synaptic vesicles are small membrane bound spherical organelles in the cytoplasm of neurons that contain neurotransmitter and various proteins necessary for neurotransmitter secretion. Vesicle containing inhibitory neurotransmitter are often flat or elliptical whereas vesicles that contain excitatory neurotransmitter are usually more spherical. Synapse is the cell-cell communication junction that allows signals to pass from a nerve cell to another cell. The synaptic cleft is the gap between the pre- and postsynaptic cells' membranes. In a chemical synapse the signal is carried by a diffusable neurotransmitter. The cleft between the presynaptic cell and the postsynaptic cells is 20 to 40 nm wide and may appear clear or striated. Recent studies have indicated that the cleft is not an empty space per se, but is filled with carbohydrate-containing material. II.

Glial cells and function

The most numerous cellular constituents of the central nervous system are the nonneuronal, neuroglial ("nerve glue") cells that occupy the space between neurons. The estimate is that there are roughly 360 billion glial cells. These cells comprise 80-90% of the cells in the CNS. We will cover the general classifications of the neuroglial cells and describe some of the general properties that distinguish neuroglia from neurons. Neuroglia differ from neurons in several general ways:

(1) do not form synapses, (2) have essentially only one type of process, (3) retain the ability to divide, and (4) are less electrically excitable than neurons. Neuroglia are most reliably classified and distinguished from neurons, at the light microscopic level, with alkaline (basic) dyes showing nuclear morphology. In addition, several metal stains are used show the shape of the cell and cytoplasmic architecture. Characteristics of nuclei, including size, shape, staining intensity and distribution of chromatin, are used to distinguish cell types in pathological material. Cell body characteristics, including size, shape, location, and branching pattern and density of processes, are also used. Neuroglia are divided into two major categories based on size, the macroglia and the microglia. The macroglia are of ectodermal origin and consist of astrocytes, oligodendrocytes and ependymal cells. Microglia cells are probably of mesodermal origin. A comparison of the various neuroglial types is shown in Figure 9.

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Figure 9. Types of neuroglia.

A. Macroglia. Protoplasmic Astrocytes Protoplasmic astrocytes are found primarily in gray matter. With silver or glial specific stains, their cell bodies and processes are very irregular. The processes may be large or very fine, sometimes forming sheets that run between axons and dendrites, and may even surround synapses. These fine sheet-like processes give the protoplasmic astrocyte cell body a "fuzzy" or murky appearance under the light microscope. Within the cytoplasm bundles of fine fibrils may be seen. The nucleus of a protoplasmic astrocyte is ellipsoid or bean-shaped with characteristic flecks of chromatin. Specific types of intercellular junctions have been noted between the processes of protoplasmic astrocytes. These probably mediate ion exchange between cells.

Fibrous Astrocytes Fibrous astrocytes are found primarily in white matter, have a smoother cell body contour than do protoplasmic astrocytes as seen with glial-specific

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stains, and have processes that tend to emerge from the cell body radially. These processes are narrower and branch to form end feet on blood vessels, ependyma, and pia. Consequently the processes of fibrous astrocytes do not form sheets and do not tend to conform the shape of the surrounding neurons or vascular elements. The major distinguishing feature of fibrous astrocytes, as the name suggests, is an abundance of glial fibrils arranged in parallel arrays in the cytoplasm and extending into the processes. In Nissl stains, the fibrous astrocytes have a nucleus essentially the same as that of the protoplasmic type with a flecked appearance. Intercellular adherences have also been observed between fibrous astrocytes. Both types of astrocytes function to support the neurons in their immediate vicinity. They provide a physical barrier between cells, maintain the ionic and pH equilibrium of the extracellular space around neurons and continually modify the chemical environment of the neighboring cells. They form a complete lining around the external surface of the CNS (glial limitans) and around blood vessels (perivascular feet). During development they form scaffolding along which nerve cells migrate to achieve their mature structure. During injury the astrocytes proliferate and phagocytize dead cells. This often leads to the formation of glial scare in response to injury. In addition to these general functions astrocytes also act in more specialized ways to facilitate neuron function. They metabolize neurotransmitter by removing them from the synaptic cleft. For example the amino acid glutamate is taken up by astrocytes and inactivated by conversion to glutamine. As will be discussed by Dr. Waxham the glutamine is transported to the neuron to be re-synthesized into glutamate. More recent evidence indicates that the astrocytes can dramatically change size as part of their physiological regulation of the neuronal environment. These functions will be discussed in later lectures.

Figure 10. Astrocyte with end feed projecting to the surface of neurons, blood vessels, ependyma and the meninges. No single astrocyte would project to all of these structures.

Oligodendroglia Oligodendrocytes are also located in both gray and white matter. They are the predominant cell type in white matter. In gray matter, oligodendroglia are usually located near neurons and, hence, are known as perineuronal satellite cells. Cell

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bodies of oligodendroglia are often located near capillaries, but they lack the definite perivascular end feet characteristic of astrocytes. In white matter, oligodendroglia are often located as rows of cells between groups of neuronal processes. These are termed interfascicular oligodendroglia and are involved in the formation and maintenance of the myelin surrounding the neuronal processes nearby. The processes of oligodendrocytes are fewer and more delicate than astrocytes, and the cell body shape is polygonal to spherical. Utilizing alkaline dyes to stain the nucleus, the oligodendrocyte nucleus is smaller than that of the astrocyte, eccentrically located in the cell body, and contains clumps of chromatin. The cytoplasm of oligodendrocytes tends to be darker than that of astrocytes with silver stains, and does not contain glial fibrils (although they do contain microtubules).

Figure 11. Diagram showing an oligodendrocyte (left) and the fact that one oligodendrocyte myelinates several internodal regions (right).

The role of oligodendroglia in the central nervous system, particularly of the interfascicular oligodendrocytes, is the formation and maintenance of myelin. Myelin is the sleeve of membranous material, that wraps the neuronal axon to facilitate the conduction of the action potential through saltatory conduction. Myelin is composed of concentric layers of membranes compacted against one another with an internal (i.e. against the nerve fiber) and an external collar of cytoplasm. It is estimated that a single oligodendrocyte contributes to the myelination of several adjacent nerve processes. Moreover, more than one oligodendrocyte contributes to the myelination of a single internode of an axon. The lamellae of myelin membranes result from the spiral wrapping of the axon by cytoplasmic processes of interfascicular oligodendroglia. As will be covered in more detail by Dr. Byrne, along the length of the nerve process the myelin sheath is interrupted at intervals by nodes of Ranvier. Each segment of myelin between the nodes is formed by more than one glial cell. Also the oligodendrocyte forming a particular myelin internode (i.e. the myelin between two nodes) is seldom seen directly adjacent to the myelin-wrapped process. This is because thin cytoplasmic bridges connect the region of the oligodendrocyte cell body to the external wrap of myelin. It is important to note that the region of the axon exposed at the node of Ranvier is not bare. It may be the site of branching of the axon, the site of synaptic contacts, or it may be

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covered with various glial processes. The axon in this region usually contains concentrations of organelles, especially mitochondria. In the PNS Schwann cells are responsible for the myelin formation. These cells myelinate axons differently than the interfascicular oligodendroglia. They migrate around the axon, laying a membrane covering around the axon by squeezing out the cytoplasm of the Schwann cell. Also every internode of a PNS axon represents a single Schwann cell. In addition unmyelinated axons in the PNS are also inclosed by in channels formed by Schwann cells.

Figure 12. Diagrammatic representation of how single Schwann cells myelinate each internodal region.

Ependyma Ependymal cells are derived from the early germinal epithelium lining the lumen of the neural tube and thus are also ectodermal derivatives (along with neurons, astrocytes, and oligodendrocytes). Ependymal cells line the ventricles of the brain and the central canal of the spinal cord. They are arranged in a single-layered columnar epithelium, and have many of the histological characteristics of simple epithelium, varying from squamous to cuboidal, depending upon their location. The ependyma forming the ventricular lining do not connect to a basal lamina, but rest directly upon underlying nervous tissue. The surface facing the ventricle contains many microvilli and cilia. These cilia move cerebrospinal fluid (CSF) in the ventricles. The lateral borders of the ependymal cells are relatively straight and form junctions with adjacent cells.

Figure 13. Diagrammatic representation of the arrangement of ependymal cells to form the ciliated lining of the ventricles.

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Ependymal cells are modified in various regions of the ventricles into layers of cuboidal epithelium, which do lie on a basement membrane (formed by an outgrowth of the pia) over a rich bed of vasculature and connective tissue. This is the choroid plexus we studied in the Laboratory that is responsible for the secretion, uptake and transport of substances to and from the CSF. B. Microglia Microglia, in contrast to the other types of glial cells, originate from embryonic mesoderm. They are present throughout the central nervous system, but tend to be inconspicuous in mature normal tissue and are difficult to identify with the light or electron microscope. They are more abundant in gray matter, and may compromise up to 5-10% of the neuroglia in the cerebral cortex. The general appearance of microglia is similar to oligodendrocytes although they are smaller, and have undulating processes with spine-like projections. Microglial nuclei are elongated or triangular and stain deeply with alkaline dyes.

Following damage to nervous tissue, microglia proliferate and migrate to the site of injury where they clear cellular debris by phagocytosis. The reacting microglia have a swollen form with shortened processes and are difficult to discriminate from phagocytes from the periphery or migrating perivascular cells. It is estimated that at least one third of the phagocytes appearing in the area of a lesion are of CNS origin.

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Resting Potentials and Action Potentials

Despite the enormous complexity of the brain, it is possible to obtain an understanding of its function by paying attention to two major details: • •

First, the ways in which individual neurons, the components of the nervous system, are wired together to generate behavior. Second, the biophysical, biochemical, and electrophysiological properties of the individual neurons.

A good place to begin is with the components of the nervous system and how the electrical properties of the neurons endow nerve cells with the ability to process and transmit information.

Introduction to the Action Potential

Figure 1.1

Important insights into the nature of electrical signals used by nerve cells were obtained more than 50 years ago. Electrodes were placed on the surface of an optic nerve of an invertebrate eye. (By placing electrodes on the surface of a nerve, it is possible to obtain an indication of the changes in membrane potential that are occurring between the outside and inside of the nerve cell.) Then 1-sec duration flashes of light of varied intensities were presented to the eye; first dim light, then brighter lights. Very dim lights produced no changes in the activity, but brighter lights produced small repetitive spike-like events. These spike-like events are called action potentials, nerve impulses, or sometimes simply spikes. Action potentials are the basic events the nerve cells use to transmit information from one place to another.

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Features of Action Potentials The recordings in the figure above illustrate three very important features of nerve action potentials. First, the nerve action potential has a short duration (about 1 msec). Second, nerve action potentials are elicited in an all-or-nothing fashion. Third, nerve cells code the intensity of information by the frequency of action potentials. When the intensity of the stimulus is increased, the size of the action potential does not become larger. Rather, the frequency or the number of action potentials increases. In general, the greater the intensity of a stimulus, (whether it be a light stimulus to a photoreceptor, a mechanical stimulus to the skin, or a stretch to a muscle receptor) the greater the number of action potentials elicited. Similarly, for the motor system, the greater the number of action potentials in a motor neuron, the greater the intensity of the contraction of a muscle that is innervated by that motor neuron. Action potentials are of great importance to the functioning of the brain since they propagate information in the nervous system to the central nervous system and propagate commands initiated in the central nervous system to the periphery. Consequently, it is necessary to understand thoroughly their properties. To answer the questions of how action potentials are initiated and propagated, we need to record the potential between the inside and outside of nerve cells using intracellular recording techniques.

Intracellular Recordings from Neurons The potential difference across a nerve cell membrane can be measured with a microelectrode whose tip is so small (about a micron) that it can penetrate the cell without producing any damage. When the electrode is in the bath (the extracellular medium) there is no potential recorded because the bath is isopotential. If the microelectrode is carefully inserted into the cell, there is a sharp change in potential. The reading of the voltmeter instantaneously changes from 0 mV, to reading a potential difference of -60 mV inside the cell with respect to the outside. The potential that is recorded when a living cell is impaled with a microelectrode is called the resting potential, and varies from cell to cell. Here it is shown to be -60 mV, but can range between -80 mV and -40 mV, depending on the particular type of nerve cell. In the absence of any stimulation, the resting potential is generally constant.

Figure 1.2

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Figure 1.3

It is also possible to record and study the action potential. Here the cell has already been impaled with one microelectrode (the recording electrode) which is connected to a voltmeter. The electrode records a resting potential of -60 mV. The cell has also been impaled with a second electrode called the stimulating electrode. This electrode is connected to a battery and a device that can monitor the amount of current that flows through the electrode. Changes in membrane potential are produced by closing the switch and by systematically changing both the size and polarity of the battery. If the negative pole of the battery is connected to the inside of the cell, there will be an instantaneous change in the amount of current that flows through the stimulating electrode, and the membrane potential becomes transiently more negative. This result should not be surprising. The negative pole of the battery makes the inside of the cell more negative than it was before. A change in potential that increases the polarized state of a membrane is called a hyperpolarization. The cell is more polarized than it was normally. Use yet a larger battery and the potential becomes even larger. The resultant hyperpolarizations are graded functions of the size of the stimuli used to produce them.

When the positive pole of the battery is connected to the electrode, the potential of the cell becomes more positive when the switch is closed. Such potentials are called depolarizations. The polarized state of the membrane is decreased. A larger battery produces even larger depolarizations. Again, the magnitude of the response is proportional to the size of the stimulus. However, an unusual event occurs when the size of the depolarization reaches a level of membrane potential called the threshold. A totally new type of signal is initiated; the action potential. Note that if the size of the battery is increased even more, the amplitude of the action potential is the same as the previous one. If the suprathreshold stimulus was long enough, however, a train of action potentials would be elicited. The action potentials would continue to fire as long as the stimulus continues, with the frequency of firing being proportional to the magnitude of the stimulus. Action potentials are not only initiated in an all-or-nothing fashion, but they are also propagated in an all-or-nothing fashion. An action potential initiated in the cell body of a motor neuron in the spinal cord will propagate in an undecremented fashion all the way to the synaptic terminals of that motor neuron.

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Components of the Action Potentials The action potential consists of several components. The threshold is the value of the membrane potential which, if surpassed, leads to the all-or-nothing initiation of an action potential. The initial or rising phase of the action potential is called the upstroke. The region of the action potential between the 0 mV level and the peak amplitude is the overshoot. The return of the membrane potential to the resting potential is called the repolarization phase. There is also a phase of the action potential during which time the membrane potential is actually more negative than the resting potential. This phase of the action potential is called the undershoot or the hyperpolarizing afterpotential.

Ionic Mechanisms of Resting Potentials Before examining the ionic mechanisms of action potentials, it is first necessary to understand the ionic mechanisms of the resting potential. The two phenomena are intimately related. The story of the resting potential goes back to the early 1900's when Julius Bernstein suggested that the resting potential (Vm) was equal to the potassium equilibrium potential (EK). Where

The key to understanding the resting potential is the fact that ions are distributed unequally on the inside and outside of cells, and that cell membranes are selectively permeable to different ions. K+ is particularly important for the resting potential. The membrane is highly permeable to K+. In addition, the inside of the cell has a high concentration of K+ ([K+]i) and the outside of the cell has a low concentration of K+ ([K+]o). Thus, K+ will naturally move by diffusion from its region of high concentration to its region of low concentration. Consequently, the positive K+ ions leaving the inner surface of the membrane leave behind some negatively charged ions. That negative charge attracts the positive charge of the K+ ion that is leaving and tends to "pull it back". Thus, there will be an electrical force directed inward that will tend to counterbalance the diffusional force directed outward. Eventually, an equilibrium will be established; the concentration force moving K+ out will balance the electrical force holding it in. The potential at which that balance is achieved is called the Nernst Equilibrium Potential.

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An experiment to test Bernstein's hypothesis that the membrane potential is equal to the Nernst Equilibrium Potential (i.e., Vm = EK) is illustrated to the left. The K+ concentration outside the cell was systematically varied while the membrane potential was measured. Also shown is the line that is predicted by the Nernst Equation. The experimentally measured points are very close to this line. Moreover, because of the logarithmic relationship in the Nernst equation, a change in concentration of K+ by a factor of 10 results in a 60 mV change in potential. Figure 1.4

Note, however, that there are some deviations in the figure at left from what is predicted by the Nernst equation. Thus, one cannot conclude that Vm = EK. Such deviations indicate that another ion is also involved in generating the resting potential. That ion is Na+. The high concentration of Na+ outside the cell and relatively low concentration inside the cell results in a chemical (diffusional) driving force for Na+ influx. There is also an electrical driving force because the inside of the cell is negative and this negativity attracts the positive sodium ions. Consequently, if the cell has a small permeability to sodium, Na+ will move across the membrane and the membrane potential would be more depolarized than would be expected from the K+ equilibrium potential.

Goldman-Hodgkin and Katz (GHK) Equation When a membrane is permeable to two different ions, the Nernst equation can no longer be used to precisely determine the membrane potential. It is possible, however, to apply the GHK equation. This equation describes the potential across a membrane that is permeable to both Na+ and K+.

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Note that is the ratio of Na+ permeability (PNa) to K+ permeability (PK). Note also that if the permeability of the membrane to Na+ is 0, then alpha in the GHK is 0, and the GoldmanHodgkin-Katz equation reduces to the Nernst equilibrium potential for K+. If the permeability of the membrane to Na+ is very high and the potassium permeability is very low, the [Na+] terms become very large, dominating the equation compared to the [K+] terms, and the GHK equation reduces to the Nernst equilibrium potential for Na+.

If the GHK equation is applied to the same data in Figure 1.4, there is a much better fit. The value of alpha needed to obtain this good fit was 0.01. This means that the potassium K+ permeability is 100 times the Na+ permeability. In summary, the resting potential is due not only to the fact that there is a high permeability to K+. There is also a slight permeability to Na+, which tends to make the membrane potential slightly more positive than it would have been if the membrane were permeable to K+ alone. Figure 1.5

Membrane Potential Laboratory Click here to go to the interactive Membrane Potential Laboratory to experiment with the effects of altering external or internal potassium ion concentration and membrane permeability to sodium and potassium ions. Predictions are made using the Nernst and the Goldman, Hodgkin, Katz equations.

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Propagation of the Action Potential

Changes in the Spatial Distribution of Charge Once an action potential is initiated at one point in the nerve cell, how does it propagate to the synaptic terminal region in an all-or-nothing fashion? This figure shows a schematic diagram of an axon and the charge distributions that would be expected to occur along the membrane of that axon. Positive charges exist on the outside of the axon and negative charges on the inside. Now consider the consequences of delivering some stimulus to a point in the middle of the axon. If the depolarization is sufficiently large, voltagedependent sodium channels will be opened, and an action potential will be initiated. Consider for the moment "freezing" the action potential at its Figure 3.1 peak value. Its peak value now will be about +40 mV inside with respect to the outside. Unlike charges attract, so the positive charge will move to the adjacent region of the membrane. As the charge moves to the adjacent region of the membrane, the adjacent region of the membrane will depolarize. If it depolarizes sufficiently, as it will, voltage-dependent sodium channels in the adjacent region of the membrane will be opened and a "new" action potential will be initiated. This charge distribution will then spread to the next region and initiate other "new" action potentials. One way of viewing this process is with a thermal analogue. You can think of an axon as a piece of wire coated with gunpowder (the gunpowder is analogous to the sodium channels). If a sufficient stimulus (heat) is delivered to the wire, the gunpowder will ignite, generate heat, and the heat will spread along the wire to adjacent regions and cause the gunpowder in the adjacent regions to ignite.

Determinants of Propagation Velocity There is a great variability in the velocity of the propagation of action potentials. In fact, the propagation velocity of the action potentials in nerves can vary from 100 meters per second (580 miles per hour) to less than a tenth of a meter per second (0.6 miles per hour). Why do some axons propagate information very rapidly and others slowly? In order to understand how this process works, it is necessary to consider two so-called passive properties of membranes, the time constant and the space or length constant. Why are these called passive properties? They have nothing to do with any of the voltagedependent conductances discussed earlier. They have nothing to do with any pumps or exchangers. They are intrinsic properties of all biological membranes.

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Time Constant. First, consider a thermal analogue. Place a block of metal at 10oC on a hotplate at 100oC. How would the temperature change? It will increase from its initial value of 10oC to a final value of 100oC. But the temperature will not change instantly. In fact, it would change as an exponential function of time. An analogous situation occurs in nerve cells, when they receive an instantaneous stimulus. The figure at right represents an idealized nerve cell. The recording electrode initially measures a potential of -60 mV (the resting potential). At some point in time (time 0), the switch is closed. The switch closure occurs instantaneously and as a result of the instantaneous closure, instantaneous current flows through the circuit. (This is equivalent to slamming the block of metal on the hotplate.) Note that despite the fact that this stimulus changes instantly, the change in potential does not occur instantaneously. It takes time for the potential to change from its initial value of -60 mV to its final value of -50 mV. There is a total of 10 mV depolarization, but the change occurs as an exponential function of time.

Figure 3.2

There is a convenient index of how rapidly exponential functions change with time. The index is denoted by the symbol and called the time constant. It is defined as the amount of time it takes for the change in potential to reach 63% of its final value. In this example, the potential changes from -60 to -50 and the 63% value is -53.7 mV. Thus, the time constant is 10 msec. The smaller the time constant, the more rapid will be the change in response to a stimulus. Therefore, if this neuron had a time constant of 5 msec, then in 5 msec the membrane potential would reach -53.7 mV. The time constant is analogous to the 0 to 60 rating of a high performance car; the lower the 0 to 60 rating, the faster the car. The lower the time constant, the faster or more rapidly a membrane will respond to a stimulus. The effects of the time constant on propagation velocity will become clear below. The time constant is a function of two properties of membranes, the membrane resistance (Rm) and the membrane capacitance (Cm). Rm is the inverse of the permeability; the higher the permeability, the lower the resistance, and vice versa. Membranes, like the physical devices known as capacitors, can store charge. When a stimulus is delivered, it takes time to charge up the membrane to its new value.

Space Constant. Consider another thermal analogue. Take a long, metal rod that is again initially at 10oC and consider the consequences of touching one end of the rod to a 39

hotplate which is at 100oC (Assume that it is placed there for a certain amount of time to allow the temperature changes to stabilize). How would the temperature be distributed along the length of the rod? There would be a gradient that could be described by an exponential function because of the physical processes involved.

Figure 3.3

An analogous situation occurs in nerve cells. The figure at left represents an idealized nerve cell in which recordings are made from different regions along the axon at 1 mm increments. The cell body is impaled with a stimulating electrode connected to a battery, the value of which changes the potential of the cell body to -50 mV (the equivalent of putting a 10oC rod on a 100oC hot plate). This axon, even though it initially had a spatially uniform resting potential of -60 mV, now has a potential of -50 mV in the soma because that is the region in which the stimulus is applied. However, the potential is not -50 mV all along the axon; it varies as a function of distance from the soma. One mm away the potential is -56 mV; at 2 mm away it is even closer to -60 mV; and far enough along the axon, the potential of the axon is -60 mV, the resting potential. Just as there is an index for how a change in potential changes with the time (the time constant), there is also an index denoted by the symbol λ (called the space constant or the length constant) which is an indication of how far a potential will spread along an axon in response to a subthreshold stimulus at another point. In Figure 3.3, the space constant or length constant is 1 mm. In 1 mm the potential will change by 63% of its final value. If λ was greater than 1 mm, the potential would spread a greater distance. If λ was 1/2 mm, the potential would spread less along the axon. Thus, whereas the time constant is an index of how rapidly a membrane would respond to a stimulus in time, the space constant is an index of how well a subthreshold potential will spread along an axon as a function of distance.

The length constant can be described in terms of the physical parameters of the axon, where d is the diameter of the axon, Rm is, as before, the membrane resistance, the inverse of the permeability, and Ri is the internal resistance (resistance of the axoplasm). Ri is an indicator of the ability of charges to move along the inner surface of the axon.

Propagation Velocity. How are the time constant and the space constant related to propagation velocity of action potentials? The smaller the time constant, the more rapidly

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a depolarization will affect the adjacent region. If a depolarization more rapidly affects an adjacent region, it will bring the adjacent region to threshold sooner. Therefore, the smaller the time constant, the more rapid will be the propagation velocity. If the space constant is large, a potential change at one point would spread a greater distance along the axon and bring distance regions to threshold sooner. Therefore, the greater the space constant, the more rapidly distant regions will be brought to threshold and the more rapid will be the propagation velocity. Thus, the propagation velocity is directly proportional to the space constant and inversely proportional to the time constant. There are separate equations that describe both the time constant and the space constant. The insight above allows us to make a new equation that combines the two.

The equation provides insights into how it is possible for different axons to have different propagation velocities. One way of endowing an axon with a high propagation velocity is to increase the diameter. However, there is one serious problem in changing the propagation velocity by simply changing the diameter. To double the velocity, it is necessary to quadruple the diameter. Clearly there must be a better way of increasing propagation velocity than by simply increasing the diameter. Another way to increase the propagation velocity is to decrease the membrane capacitance. This can be achieved by coating axons with a thick insulating sheath known as myelin. One potential problem with this approach is that the process of covering the axon would cover voltage-dependent Na+ channels. If Na+ channels are occluded, it would be impossible to generate an action potential. Instead of coating the entire axon with the myelin, only sections are coated and some regions called nodes are left bare.

Propagation in Myelinated Fibers Propagation in myelinated fibers works as illustrated in Figure 3.4. Start with an action potential at a node on the left. In the absence of myelin, the action potential would propagate actively through the simple mechanisms discussed above, but the myelin occludes all the voltage-dependent sodium channels. (In fact, myelinated axons do not even have sodium channels in the internodal region.) Rather, the potential change at one node spreads in the internodal region along the axon passively just as the temperature would spread along a long metal rod. The potential spreads, but gets smaller (decrements), just as a temperature change induced at one end of a rod would get smaller as it spreads along a rod.

Figure 3.4

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Now consider the point at which the passively spreading potential reaches the next node. A "new" action potential will be initiated. The stimulus for this action potential is the depolarization that emerges from the end of the myelin. Think of the gunpowder analogue again, but this time coat the rod with some insulation and put gunpowder only at the bare regions. Because of the insulation, a temperature change produced by the ignition of the gunpowder will spread effectively along the metal rod. The temperature will be sufficient to ignite the gunpowder at the next region and the process will repeat itself.

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Synaptic Transmission at the Skeletal Neuromuscular Junction and Mechanisms of Neurotransmitter Release

The synapse is a specialized structure that allows one neuron to communicate with another neuron or a muscle cell. There are billions of nerve cells in the brain and each nerve cell can make and receive up to 10,000 synaptic connections with other nerve cells. Also, the strength of the synapse is modifiable. Changes in the strength of synapses endow the nervous system with the ability to store information.

Anatomy of the Neuromuscular Junction The synapse for which most is known is the one formed between a spinal motor neuron and a skeletal muscle cell. Historically, it has been studied extensively because it is relatively easy to analyze. However, the basic properties of synaptic transmission at the skeletal neuromuscular junction are very similar to the process of synaptic transmission in the central nervous system. Consequently, an understanding of this synapse leads to an understanding of the others. Therefore, we will first discuss the process of synaptic transmission at the skeletal neuromuscular junction.

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The features of the synaptic junction at the neuromuscular junction are shown in the figure at left. Skeletal muscle fibers are innervated by motor neurons whose cell bodies are located in the ventral horn of the spinal cord. The terminal region of the axon gives rise to very fine processes that run along skeletal muscle cells. Along these processes are specialized structures known as synapses. The particular synapse made between a spinal motor neuron and skeletal muscle cell is called the motor endplate because of its specific structure.

Figure 4.1

The synapse at the neuromuscular junction has three characteristic features of chemical synapses in the nervous system. First, there is a distinct separation between the presynaptic and the postsynaptic membrane. The space between the two is known as the synaptic cleft. The space tells us there must be some intermediary signaling mechanism between the presynaptic neuron and the postsynaptic neuron in order to have information flow across the synaptic cleft. Second, there is a characteristic high density of small spherical vesicles. These synaptic vesicles contain neurotransmitter substances. Synapses are also associated with a high density of mitochondria. Third, in most cases, there is a characteristic thickening of the postsynaptic membrane, which is due at least in part to the fact that the postsynaptic membrane has a high density of specialized receptors that bind the chemical transmitter substances released from the presynaptic neuron. Additional details on the morphological features of synaptic junctions is provided in Chapter 8, Part 7 and Chapter 11, Part 4.

Physiology of Synaptic Transmission at the Neuromuscular Junction

4.2

This figure illustrates in a very schematic way how it is possible to study the physiology of synaptic transmission at the skeletal neuromuscular junction in great detail. A piece of muscle and its attached nerve are placed in a small experimental chamber filled with an appropriate Ringer solution. The resting potential of the muscle cell is recorded with a microelectrode. Electrodes are also placed on the surface of the nerve axon. Brief electric Figure shocks cause action potentials to be initiated, which propagate to the synaptic terminal.

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Figure 4.3

The figure above illustrates two types of potential changes that were recorded in such an isolated nerve-muscle preparation. The experiment also illustrates the properties of a powerful drug, curare, which has proven to be very useful in studying the process of synaptic transmission at the skeletal neuromuscular junction. Part A illustrates the sequence of potential changes recorded in the muscle cell as a result of stimulating the motor axon. The arrow indicates the point in time when the shock is delivered to the motor axon. Note that there is a quiescent period of time after the shock. The delay is due to the time it takes for the action potential in the motor axon to propagate from its site of initiation. After the delay, there are two types of potentials recorded in the muscle cell. First, there is a relatively slowly changing potential that will be the focus of the following discussion. If that slow initial potential is sufficiently large, as it normally is in skeletal muscle cells, a second potential, an action potential, is elicited in the muscle cell. Action potentials in skeletal muscle cells are due to ionic mechanisms similar to those discussed previously. Specifically, there is a voltage-dependent change in Na+ permeability followed by a delayed increase in K+ permeability. (For smooth muscle cells and cardiac muscle cells the ionic mechanisms are different, however.) The underlying event that triggers the action potential can be revealed by taking advantage of curare, an arrow poison used by some South American Indians. A low dose of curare (Part B) reduces the underlying event, but it is still not sufficiently reduced to fall below threshold. If a somewhat higher dose of curare is delivered (Part C), the slow underlying event becomes subthreshold. The underlying signal is known as the endplate potential (EPP) because it is a potential change recorded at the motor endplate. Generally, it is known as an excitatory postsynaptic potential (EPSP). Curare blocks the endplate potential because it is a competitive inhibitor of acetylcholine (ACh), the transmitter released at the presynaptic terminal. Curare does not block the voltage-dependent Na+ conductance or the voltage-dependent K+ conductance that underlies the muscle action potential. Curare affects the stimulus (the EPSP) which normally leads to the initiation of the muscle action potential. An animal that is poisoned

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with curare will asphyxiate because the process of neuromuscular transmission at respiratory muscles is blocked. Normally, the magnitude of the endplate potential is quite large. It more than reaches threshold; it goes beyond threshold. In fact, the amplitude of the endplate potential is about 50 mV, but only about 30 mV is needed to reach threshold. The extra 20 mV is called the safety factor. Note that even if the endplate potential were to become somewhat smaller, the EPP would reach threshold and there would still be a one-to-one relationship between an axon potential and the motor axon and an action potential in the muscle cell.

Propagation of the EPP What are the properties of the EPP and how does it compare with the properties of the action potential? Is the endplate potential due to a voltage-dependent change in Na+ and K+ permeabilities like the action potential? Is the endplate potential propagated in an all-ornothing fashion like the action potential? The figure on the right illustrates an experiment that examines the propagation of the endplate potential. The muscle fiber is impaled repeatedly with electrodes at 1 mm intervals. (Note that the endplate potential is small because this experiment is done in the presence of a low concentration of curare so the endplate potential can be recorded without the complications of triggering an action potential.) The endplate potential is not propagated in an all-or-nothing fashion. It does spread along the muscle, but it does so with decrement. Thus, the spread of the endplate potential from its site of initiation to other sites along the muscle cell occurs passively and with decrement, just as a subthreshold potential change in one portion of the axon spreads along the axon, or just as a change in temperature at one point on a metal rod spreads along the rod.

Figure 4.4

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Overview of the Sequence of Events Underlying the EPP

Figure 4.5

What are the other steps in the process of chemical synaptic transmission? Figure 4.5 provides an overview. A nerve action potential that is initiated in the cell body of a spinal motor neuron propagates out the ventral roots and eventually invades the synaptic terminals of the motor neurons. As a result of the action potential, the chemical transmitter acetylcholine (ACh) is released into the synaptic cleft. ACh diffuses across the synaptic cleft and binds to special receptors on the postsynaptic or the postjunctional membrane. The binding of ACh to its receptors produces a conformational change in a membrane channel that is specifically permeable to both Na+ and K+. As a result of an increase in Na+ and K+ permeability, there is a depolarization of the postsynaptic membrane. That depolarization is called the endplate potential or more generally the EPSP. If the EPSP is sufficiently large, as it normally is at the neuromuscular junction, it leads to initiation of an action potential in the muscle cell. The action potential initiates the process of excitation contraction coupling and the development of tension. The duration of the endplate potential is about 10 msec. Two factors control the duration of the EPSP at the neuromuscular junction. First, ACh is removed by diffusion. Second, a substance in the synaptic cleft, called acetylcholinesterase (AChE), hydrolyzes or breaks down ACh.

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Role of AChE

Figure 4.6

An important family of substances, one of which is neostigmine, inhibits the action of AChE. Neostigmine blocks the action of AChE, and thereby makes the endplate potential larger and longer in duration. This figure illustrates two endplate potentials. One was recorded in saline and curare and a second recorded after neostigmine was added to the solution. (Curare is added so that the properties of the EPP can be studied without triggering an action potential in the muscle cell.) After applying neostigmine the endplate potential is much larger and longer in duration.

Myasthenia Gravis Myasthenia gravis is associated with severe muscular weakness because of a decrease in the number of acetylcholine receptors in the muscle cell. If the endplate potential is smaller, the endplate potential will fail to reach threshold. If it fails to reach threshold, there will be no action potential in the muscle cell and no contraction of the muscle, which causes muscular weakness. Neostigmine and other inhibitors of AChE are used to treat patients with myasthenia gravis. These agents make the amount of acetylcholine that is released more effectively reach the remaining acetylcholine receptors.

Iontophoresis of ACh Iontophoresis is an interesting technique that can be used to further test the hypothesis that ACh is the neurotransmitter substance at the neuromuscular junction. If ACh is the transmitter that is released by this synapse, one would predict that it should be possible to substitute artificial application of the transmitter for the normal release of the transmitter. Since ACh is a positively charged molecule, it can be forced out of a microelectrode to simulate the release of ACh from a presynaptic terminal. Figure 4.7

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Indeed, minute amounts of ACh can be applied to the vicinity of the neuromuscular junction. This figure compares an EPP produced by stimulation of the motor axon and the response to ejections of ACh. The potential change looks nearly identical to the endplate potential produced by the normal release of ACh. This experiment provides experimental support for the concept that ACh is the natural transmitter at this synapse.

Figure 4.8

The response to the ejection of ACh has some other interesting properties that are all consistent with the cholinergic nature of the synapse at the skeletal neuromuscular junction. Neostigmine makes the response to the iontophoresis of ACh longer and larger. Curare reduces the response because it competes with the normal binding of ACh. If ACh is ejected into the muscle cell, nothing happens because the receptors for acetylcholine are not in the inside; they are on the outside of the muscle cell. Application of acetylcholine to regions of the muscle away from the end-plate produces no response because the receptors for the ACh are concentrated at the synaptic region.

To test your understanding so far, consider how an agent such as TTX would affect the generation of both an EPP and the response of a muscle fiber to the iontophoretic application of ACh? TTX has no effect on the response to ACh, but it does block the EPP. The reason the response to ACh is unaffected is clear, but many expect that if there is no effect here, there should be no effect on the EPP either. Tetrodotoxin does not affect the binding of acetylcholine to the receptors and therefore will not affect the response to direct application of ACh. However, tetrodotoxin will affect the ability of an action potential to be elicited in the motor axon. If an action potential cannot be elicited in the motor axon, it cannot cause the release of transmitter. Thus, tetrodotoxin would totally abolish the EPP. The block would not be due to a block of ACh receptors, but rather to a block of some step prior to the release of the transmitter.

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Ionic Mechanisms of the EPP Bernard Katz and his colleagues were pioneers in investigating mechanisms of synaptic transmission at the neuromuscular junction. They suggested that the channel opened by ACh was one that had equal permeability to both Na+ and K+. Because it was equally permeable to Na+ and K+, Katz suggested that, as a result of the opening of these channels, the membrane potential would move toward 0 mV. (A value of alpha in the GHK equation equal to one, which when substituted into the equation, yields a potential of about 0 mV.)

Figure 4.9

The experiment shown in the figure on the left tests that concept. The muscle cell has been penetrated with a recording electrode as well as another electrode that can be connected to a suitable source of potential in order to artificially change the membrane potential. Normally, the membrane potential is about -80 mV [Skeletal muscle cells have higher (more negative) resting potentials than most nerve cells.] Again, a small amount of curare is added so that the EPP is small. Katz noticed in these experiments that the size of the EPP changed dramatically depending upon the potential of the muscle cell. If the membrane potential is moved to 0 mV, no potential change is recorded whatsoever. If the membrane potential is made +30 mV, the EPP is inverted. So three different stimuli produce endplate potentials that are very different from each other.

The lack of a response when the potential is at 0 mV is particularly informative. Consider why no potential change is recorded. Presumably, the transmitter is being released and binding to the receptors. The simple explanation for a lack of potential change is that the potential at which the opening of ACh channels are trying to reach has already been achieved. If the membrane potential is made more positive than 0 mV, then the EPP is inverted. No matter what the potential, the change in permeability tends to move the membrane potential towards 0 mV! If the resting potential is more negative than 0 mV, there is an upward deflection. If it is more positive, there is a downward deflection. If it is already at 0 mV, there is no deflection.

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This potential is also called the reversal potential, because it is the potential at which the sign of the synaptic potential reverses. The experiment indicates that, as a result of ACh binding to receptors, specific channels become equally permeable to Na+ and K+. This permeability change tends to move the membrane potential from wherever it is initially towards a new potential of 0 mV. Why does the normal endplate potential never actually reach 0 mV? One reason is that the sequence of permeability changes that underlie the action potential "swamp out" the changes produced by the EPP. But even if an action potential was not triggered, the EPP still would not reach 0 mV. This is because the ACh channels are only a small fraction of the total number of channels in muscle fibers. The K+ channels that endow the muscle cells with its resting potential are present as well. Their job is to try to maintain the cell at the resting potential.

Figure 4.10 (.gif version of graphic)

The channel opened by ACh is a member of a general class of channels called ligandgated channels or ionotropic receptors. As illustrated in Figure 4.10, the transmitter binding site is part of the channel itself. As a result of transmitter binding to the receptor (generally two molecules are necessary), there is a conformational change in the protein allowing a pore region to open and ions to flow down their electrochemical gradients. Additional details of the channel are presented in Chapter 12, Part 5.

Role of Calcium in Transmitter Release Calcium is a key ion involved in the release of chemical transmitter substances. Bernard Katz and his colleagues examined its role using the skeletal nerve muscle synapse. Electrodes were placed near the presynaptic terminal to initiate an action potential in the terminal (Figure 5.1). The preparation was perfused with a solution free of calcium. In order to precisely control the delivery of calcium, another microelectrode was filled with calcium. Since Ca2+ is positively charged, it can be delivered to the vicinity of the synaptic terminal by briefly closing a switch connected to a battery in such a way that the positive pole forces minute amounts of calcium out of the electrode. In the absence of Ca2+ ejection, stimulation of the motor neuron produced no EPSP.

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CLICK HERE TO SEE SERIES OF RELATED GRAPHICS

Figure 5.1

Just before the presynaptic axon was stimulated a second time, the switch was briefly closed to eject a small amount of calcium in the vicinity of the presynaptic terminal. A normal EPSP was recorded. The experiment was repeated a third time, but now the calcium ejection occurred after the presynaptic axon was stimulated. There was no EPSP. This experiment demonstrates that calcium must be present before or during the action potential in the presynaptic terminal. Based on this experiment and others like it, Katz and colleagues proposed the calcium hypothesis for chemical synaptic transmission.

Calcium Hypotheses for Chemical Synaptic Transmission

Figure 5.2

The figure above illustrates some of the key features of the calcium hypothesis for chemical synaptic transmission at the neuromuscular junction, but this hypothesis is generally applicable to all chemical synapses in the nervous system. There are two parts

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to this hypothesis. First, the depolarization of the presynaptic terminal leads to an increase in Ca2+ permeability. Just as there are voltage-dependent Na+ and K+ channels, there are also voltage-dependent Ca2+ channels. The structure of the voltage-dependent channels are very similar to the structure of the voltage-dependent sodium channels. Indeed, just a few amino acids can make the difference between a channel being selectively permeable to calcium and one that is selectively permeable to sodium. The Ca2+ channel is normally closed, but if there is a depolarization of the membrane (caused by a presynaptic action potential), the channel opens and the opening of the channel allows calcium influx. The second part of the calcium hypothesis for chemical synaptic transmission involves the consequences of the Ca2+ influx. The opening of the Ca2+ channel allows for calcium to flow down its concentration gradient from the outside to the inside of the synaptic terminal. This influx leads to an increase in the concentration of the Ca2+ in the presynaptic terminal. Through mechanisms that are not fully understood, calcium in the presynaptic terminal leads to the release of the chemical transmitter substance.

Quantal Nature of Transmitter Release How does the increase in the intracellular concentration of Ca2+ cause transmitter release? The answer to this question came from an experiment which initially seems unrelated to the issue. Using high amplification of the electrical recording system, Katz noticed small deflections that occurred spontaneously and randomly at a rate of about once every 50 msec (Panel A of the figure to the right). These small deflections had interesting properties. • •

• • •

First, they occurred in the absence of any stimulus. Second, they were small with an average amplitude of about 0.5 mV. The distribution could be fit by a single gaussian function, indicating that the events arose from a common underlying process. Third, these events could only be recorded in the vicinity of the synaptic junction. Fourth, they were blocked by curare. Fifth, they were enhanced by neostigmine.

Figure 5.3

Based on these considerations, Katz called these 53

events miniature endplate potentials or MEPPs. They appeared very similar to endplate potentials, but they were only about 0.5 mV in amplitude compared to the 50 mV amplitude of the normal EPP. Katz suggested that MEPPs were due to the spontaneous and random release of ACh. This idea intuitively makes good sense. If there is an abundance of ACh in the presynaptic perhaps some will leak out and diffuse across the cleft, bind to ACh receptors, and produce a small potential change. ACh is likely to be spontaneously released occasionally because there is a basal level of calcium in the presynaptic terminal. Each vesicle actually contains enough transmitter to open about 1,000 individual AChsensitive channels. This information is necessary to answer "Test Your Knowledge" Question 5 on page 108 of the Lecture Supplement.

(The designation MEPP has a very specific meaning. It refers to those small endplate potentials that occur randomly in the absence of any stimulation. For example, small endplate potentials (EPPs) can be recorded in the presence of curare or low levels of extracellular Ca2+ , but they are not MEPPs.) Katz suggested, as a result of the experiment illustrated in Figure 5.3, that the normal EPP is due to the summation effects of many vesicles being released at the same time. One vesicle produces a potential of about 0.5 mV. The release of 100 of those vesicles at the same time could produce a potential which is 100 times as great (50 mV). The illustration below (Figure 5.4) shows one of these vesicles in the process of fusing with the membrane and releasing its contents into the synaptic cleft through a process called exocytosis. For illustrative purposes, each synaptic vesicle is shown to contain three molecules of transmitter. In reality, each vesicle contains about 10,000 molecules of transmitter. Vesicles ready to be released are found in a region near the presynaptic terminal membrane called the releasable pool. Newly synthesized vesicles are found in the storage pool. The process by which a vesicle migrates from the storage pool to the releasable pool is called mobilization. After fusing with the membrane and releasing its contents, the membrane is recycled to form new synaptic vesicles. This process is called recycling. Additional details of this process are found in Chapter 11.

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Figure 5.4 (.gif version of graphic)

Figure 5.5

An experiment by Katz that further supported the quantal hypothesis for chemical synaptic transmission is shown above. The extracellular concentration of calcium was lowered to reduce the size of the evoked endplate potential. Because less Ca2+ is in the

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extracellular medium, less Ca2+ will be available to enter through the voltage-dependent Ca2+ channels. At the arrow, the electrical shock was delivered to the motor axon. Eight successive stimuli were delivered to the presynaptic terminal. EPSPs with stars are the miniature endplate potentials (MEPPs). Note that they are uncorrelated with the stimulus. The evoked endplate potentials are small and highly variable. Sometimes the EPP was 1.6 mV in amplitude; sometimes there was no EPP at all. Sometimes the EPP was 0.4 mV. Katz noticed that these amplitudes showed a specific kind of distribution. The smallest evoked responses were 0.4 mV. He called these responses "units". Other times he recorded EPPs that were about 0.8 mV and called such responses "doubles" because they were twice the unit, and sometimes responses were 1.6 mV. Figure 5.5 is a plot of the number of times an EPP of various amplitudes was observed. Katz noticed that the amplitude of the smallest EPP that could be evoked was the same amplitude (0.5 mV) as the amplitude of the MEPP. Based on these results Katz proposed the quantal hypothesis for chemical synaptic transmission. An action potential in the presynaptic cell produces an influx of Ca2+ which promotes the exocytosis of synaptic vesicles from the presynaptic terminal. There is a statistical variability in the amount of vesicles that can be released. When the extracellular calcium concentration is low, sometimes there is not enough calcium to release any vesicles. At other times, there is enough calcium to cause the release of one vesicle and other times two vesicles, or three vesicles, and so forth. Each peak is therefore an integral multiple of the next, indicating that these vesicles are released in a quantized fashion. With normal levels of calcium, there is sufficient influx of Ca2+ to release about 100 vesicles, which produce an endplate potential (EPP) of about 50 mV.

Figure 5.6 Place cursor over graphic to see labels.

Figure 5.6, above, illustrates a summary of the steps involved in the process of synaptic transmission at the neuromuscular junction.

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Synaptic Transmission in the Central Nervous System and Synaptic Plasticity

Synaptic Transmission in a Simple Reflex Circuit One of the simplest behaviors mediated by the central nervous system is knee-jerk or stretch reflex. In response to a neurologist's hammer to the patella tendon, there is a reflex extension of the leg. This figure illustrates the neurocircuitry that controls that reflex response. The stretch to the patella tendon stretches the extensor muscle. More specifically, it stretches a group of specific receptors known as muscle spindle receptors or simply stretch receptors. The stretch elicits action potentials in the stretch receptors which then propagate over type 1A afferent fibers, the somata of which are located in the dorsal root ganglion. Processes of these sensory neurons then enter the spinal cord and make synaptic connections with two types of cells. First, a synaptic connection is formed with the extensor motor neuron located in the ventral horn of the spinal cord. As the result of synaptic activation of this motor neuron, action potentials are elicited in the motor neuron and Figure 6.1 propagate out the ventral roots, ultimately invading the terminal regions of the motor axon (i.e., the neuromuscular junction), causing release of acetylcholine, depolarization of the muscle cell, formation of an action potential in the muscle cell, and a subsequent contraction of the muscle.

The sensory neurons also make synaptic connections with another type of neuron in the spinal cord called an interneuron. Interneurons are so named because they are interposed between one type of neuron and another. The particular interneuron shown is an inhibitory interneuron. As a result of its activation through the process of synaptic transmission, action potentials are elicited in the interneuron. An action potential in the inhibitory neuron leads to the release of a chemical transmitter substance that inhibits the flexor motor neuron, thereby preventing an improper movement from occurring. This particular reflex is known as the monosynaptic stretch reflex because this reflex is mediated by a single excitatory synaptic relay in the central nervous system. 57

Ionic Mechanisms of EPSPs Synaptic Potentials The figure at right illustrates how it is possible to experimentally examine some of the components of synaptic transmission in the reflex pathway that mediates the stretch reflex. Normally, the sensory neuron is activated by a stretch to the stretch receptor, but this process can be bypassed by injecting a depolarizing current into the sensory neuron. That stimulus initiates an action potential in the sensory neuron which leads to a change in the potential of the motor neuron. This potential is known as an excitatory postsynaptic potential (EPSP); excitatory because it tends to depolarize the cell, thereby tending to increase the probability of firing an action potential in the motor neuron and postsynaptic because it is a potential recorded on the postsynaptic side of the synapse.

Figure 6.2

The ionic mechanisms for the EPSP in the spinal motor neuron are essentially identical to the ionic mechanisms for the EPSP at the neuromuscular junction. Specifically, the transmitter substance diffuses across the synaptic cleft and binds to specific ionotropic receptors on the postsynaptic membrane, leading to a simultaneous increase in the sodium and potassium permeability (See Figure 4.10). The mechanisms for release are also identical to those at the neuromuscular junction. An action potential in the presynaptic terminal leads to the opening of voltage dependent Ca2+ channels, and the Ca2+ influx causes transmitter substance to be released.

Differences between the EPSP at the Skeletal Neuromuscular Junction and EPSPs in the CNS There are two fundamental differences between the process of synaptic transmission at the sensorimotor synapse in the spinal cord and the process of synaptic transmission at the neuromuscular junction. First, transmitter substance released by the sensory neuron is not ACh but rather the amino acid glutamate. Indeed, there are many different transmitters in the central nervous system - up to 50 or more and the list grows every year. Fortunately, these 50 or more transmitter substances can be conveniently grouped into four basic categories: acetylcholine, monoamines, peptides, and the amino acids. Second, in contrast to the 50-mV amplitude of the synaptic potential at the neuromuscular

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junction, the amplitude of the synaptic potential in a spinal motor neuron, as a result of an action potential in a 1A afferent fiber, is only about 1 mV.

Temporal and Spatial Summation If the amplitude of the postsynaptic potential is only 1 mV, how can an action potential in the motor neuron be triggered and the reflex function? Note that a 1-mV EPSP is unlikely to be sufficient to drive the membrane potential of the motor neuron to threshold to fire a spike. If there is no spike, there will be no contraction of the muscle. The answer is that the stretch of the muscle fires multiple action potentials in many different stretch receptors. In fact, the greater the stretch, the greater is the probability of activating more stretch receptors. This process is referred to as recruitment. Therefore, multiple 1A afferents will converge onto the spinal motor neuron and participate in its activation. This is not the whole answer, however. Recall that the greater the intensity of the stimulus, the greater is the number of action potentials elicited in a sensory receptor. The greater the stretch, the greater the number of action potentials elicited in a single sensory neuron and the greater number of EPSPs produced in the motor neuron from that train of action potentials in the sensory cell. The processes by which the multiple EPSPs from presynaptic neurons summate over space and time are called temporal and spatial summation.

Figure 6.3

Temporal summation. A single action potential in sensory neuron 1 produces a 1mV EPSP in the motor neuron. Now consider the consequences of firing two action potentials in quick succession (See figure above). Two EPPs are elicited, the second of which summates on the falling edge of the first. As a result of two action potentials, a summated potential about 2 mV in amplitude occurs. If there were three presynaptic action potentials, and they occurred rapidly enough, the total potential would be about 3 mV, and so forth. Temporal summation is strictly a passive property of nerve cells. Special ionic conductive mechanisms are not needed to explain it. The potentials summate because of the passive properties of the nerve cell membrane, specifically the ability of membranes to store charge. The membrane temporarily stores the charge of the first PSP and then the charge from the second PSP is added to it to produce a potential

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twice as large at first. This process of temporal summation is very much dependent upon the duration of the synaptic potential. The temporal summation occurs when the presynaptic action potentials occur in quick succession. The time frame is dependent upon the passive properties of the membrane, specifically the time constant.

Spatial summation. Now consider a motor neuron that receives two inputs. An action potential produced in sensory neuron 1 produces a 1-mV EPSP and a single action potential in sensory neuron 2 also produces a 1-mV EPSP. If action potentials are produced simultaneously in sensory neuron 1 and in sensory neuron 2, the EPSPs summate to produce a summated EPSP which is twice that of the individual EPSPs. Spatial summation in nerve cells occurs because of the space constant, the ability of a charge produced in one region of the cell to spread to other regions of the cell.

IPSPs Whether a neuron fires in response to a synaptic input is dependent upon how many action potentials are being fired in any one afferent input, as well as how many individual afferent pathways are activated. The decision to fire also depends on the presence of inhibitory synaptic inputs. Artificially depolarizing the interneuron to initiate an action potential produces a transient hyperpolarization of the membrane potential of the motor neuron (See Figure 6.2). The time course of this hyperpolarization looks very similar to that of an EPSP, but it is reversed in sign. The synaptic potential in the motor neuron is called an inhibitory postsynaptic potential (IPSP) because it tends to move the membrane potential away from the threshold, thereby decreasing the probability of this neuron initiating an action potential.

Ionic Mechanisms for IPSPs The membrane potential of the flexor motor neuron is about -65 mV, so one might predict that the IPSP would be due to an increase in the permeability or the conductance of an ion whose equilibrium potential is more negative than -65 mV. One possibility is potassium. Potassium does mediate some inhibitory synaptic potentials in the central nervous system, but not at the particular synapse between a spinal interneuron and spinal motor neuron. At this particular synapse, the IPSP is due to a selective increase in chloride permeability. Note that the equilibrium potential for chloride is about -70 mV. The transmitter released by the spinal interneuron binds to a special class of ionotropic receptors which are normally closed, but open and become selectively permeable to chloride ions as a result of the binding of the transmitter. As a result of the increase in Clpermeability, the membrane potential moves from its resting value of -65 mV towards the Cl- equilibrium potential. (Note that in principle, decreasing the resting conductance of Na+ could also produce an IPSP.)

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Transmitter Substance of the Spinal Inhibitory Neuron What about the transmitter substance that is released by the inhibitory interneuron in the spinal cord? The transmitter substance is glycine, an amino acid which is used frequently in the central nervous system as a transmitter that produces inhibitory actions. It is not the most common, however. The most common transmitter with inhibitory actions is gamma amino butyric acid (GABA).

Metabotropic Synaptic Responses In addition to the responses mediated by ionotropic receptors, there is an entirely separate class of synaptic potentials that have durations with orders of magnitude greater than the durations of the classical EPSPs. These are so-called slow synaptic potentials and they are mediated by metabotropic receptors. Slow synaptic potentials are not observed at every postsynaptic neuron but they are certainly observed at many. The figure below illustrates a postsynaptic neuron which receives two inputs. An action potential in neuron 1 produces an excitatory postsynaptic potential or EPSP in the postsynaptic cell whose duration is about 20 msec. Neuron 2 can also produce a postsynaptic potential but its duration is more than three orders of magnitude longer than that of the conventional type of synaptic potential. The mechanism of these slow synaptic responses involves changes in metabolism of the cell.

Figure 6.4

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Figure 6.5

One mechanism for a slow synaptic potential is shown in the illustration at left (Figure 6.5) and in Figure 12.11. In contrast to the ionotropic receptor for which the receptors are actually part of the channel complex, the channels that produce the slow synaptic potentials are not directly coupled to the transmitter receptors. Rather, the receptors are separate from the channel. These receptors are known as metabotropic because they involve changes in the metabolism of the cell and, in general, changes in activation of specific second messenger systems. The figure at left illustrates an example of one type of response that involves the cyclic AMP cascade. Slow PSPs are in some cases mediated by cyclic AMP but they are also mediated by other protein kinases. For the response in Figure 6.5, the transmitter activates G proteins that lead to the increased synthesis of cyclic AMP. Cyclic AMP then leads to the activation of cyclic AMP-dependent kinase (PKA), which phosphorylates a channel protein or a component of the channel and then produces a conformational change in the channel and a change in its ionic permeability. In contrast to a direct conformational change produced by the binding of a transmitter to the receptor channel complex (seen in responses mediated by ionotropic receptors), the conformational change is produced by phosphorylation. The particular channel is one that is selectively permeable to K+ and is normally open. As a result of the channel phosphorylation by PKA, the channel closes and becomes less permeable to K+. Since the normal resting potential is due to a balance of Na+ and K+, decreasing the K+ conductance favors the effects of the Na+ conductance and a depolarization is produced.

It is interesting to point out that the activation of metabotropic receptors can produce effects which are much longer than several hundred seconds. For example, protein kinase A can diffuse in the nucleus where it can phosphorylate proteins (i.e., transcription factors) that regulate gene expression.

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Neurotoxins The discovery of certain toxins has greatly facilitated the analysis of voltage and chemically gated channels as well as the process of synaptic transmission. The following table illustrates some that have been particularly useful. SOME IMPORTANT NEUROTOXINS tetrodotoxin (TTX):

Fish toxin that blocks the pore of voltage-dependent Na+ channels.

µ-conotoxin (µCTX):

Fish-hunting cone snail toxin with properties similar to TTX.

saxitoxin (STX):

Toxin from marine dinoflagellates with properties similar to TTX. STX is also known as paralytic shellfish poison.

ω-conotoxin (ωCTX):

Fish-hunting cone snail toxin that blocks certain types of voltage-dependent Ca2+ channels.

funnel web spider toxin (ω-Aga):

Toxin from funnel web spider which blocks certain types of voltage-dependent Ca2+ channels.

apamin:

Bee venom toxin that blocks certain types of Ca2+-activated K+ channels.

charybdotoxin (ChTX):

Scorpion venom toxin that blocks pore of some Ca2+-activated K+ channels and voltage-dependent K+ channels.

curare (dtubocuraine):

Plant toxin that is a competitive inhibitor of nicotinic ACh receptors.

α-bungarotoxin:

Snake toxin that is competitive and highly irreversible inhibitor of nicotinic ACh receptors.

picrotoxin:

GABAA receptor blocker isolated from the seed of Anamirta cocculus.

strychnine:

Glycine receptor blocker isolated from the seed of the East Indian tree Strychnos nux-vomica.

tetanus toxin:

Clostridial neurotoxin with zinc-dependent protease activity; Cleaves synaptic vesicle proteins in the CNS and thereby blocks release of neurotransmitters.

botulinum toxin:

Clostridial neurotoxin with zinc-dependent protease activity; Cleaves synaptic vesicle proteins at the neuromuscular junction and thereby blocks release of ACh.

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Synaptic Plasticity Historically, it was generally thought that the role of the synapse was to simply transfer information between one neuron and another neuron or between a neuron and a muscle cell. In addition, it was thought that these connections, once established during development, were relatively fixed in their strength, much like a solder joint between two electronic components. One exciting development in neurobiology over the past twenty years is the realization that most synapses are extremely plastic; they are able to change their strength as a result of either their own activity or through activity in another pathway. Many think that this synaptic plasticity is central to understanding the mechanisms of learning and memory. There are two general forms of synaptic plasticity, intrinsic and extrinsic. Intrinsic mechanisms, also known as homosynaptic mechanisms, refer to changes in the strength of a synapse that are brought about by its own activity. (Homo from the Greek meaning the same.) Extrinsic plasticity, or heterosynaptic plasticity, is a change in the strength of a synapse brought about by activity in another pathway.

Figure 7.1

Homosynaptic Plasticity. There are two types of intrinsic or homosynaptic plasticity, synaptic depression and synaptic facilitation. Synaptic depression and facilitation are not always found at the same synapse. Some synapses exhibit one but not the other, whereas some synapses exhibit both. Figure 7.1B illustrates homosynaptic plasticity at the synapse between a 1A afferent fiber and a spinal motor neuron. An action potential in the sensory neuron produces an EPSP in the motor neuron. A second action potential in the sensory neuron, 200 msec after the first, produces an EPSP that is smaller than that produced by the first action potential. This phenomenon is called synaptic depression. The efficacy of synaptic transmission is not constant; it varies depending upon the frequency of stimulation. The mechanisms of synaptic depression vary but one common mechanism is depletion of the available transmitter. The second of two action potentials will release less transmitter because less transmitter is available to be released. (See Figure 7.2A) The figure at left illustrates the second form

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of homosynaptic plasticity, synaptic facilitation. This particular example is known as paired-pulse or twin-pulse facilitation. Two action potentials in the presynaptic cell produce two EPSPs in the postsynaptic cell. The first action potential produces a 1 mV EPSP, but the second action potential, which occurs about 20 msec after the first, produces an EPSP that is larger than the EPSP produced by the first. In this example, it is twice as large as the first one. The net EPSP is 3 mV, due to the process of temporal summation.

One mechanism contributing to twin pulse facilitation is residual calcium. An action potential leads to the opening of Ca2+ channels and the influx of Ca2+, which leads to the release of transmitter. Now consider the fate of the calcium after the first action potential (Figure 7.2B). Ca2+ levels will decline back to their initial level, but this recovery will not occur instantaneously. Thus, if a second action potential is initiated at a time during which the calcium has not yet recovered to its basal level, the calcium influx associated with the second spike will add to the "residual calcium" that is left over from the first. The net effect is that the total concentration of calcium will be greater after the second spike than it was after the first, and more transmitter will be released. Figure 7.2

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Figure 7.3

Another intrinsic type of synaptic plasticity is called post-tetanic potentiation (PTP). It is an extreme example of facilitation defined as a relatively persistent (minutes) enhancement of synaptic strength following a brief train of spikes (a tetanus). Heterosynaptic Forms of Synaptic Plasticity. Just as there are two types of homosynaptic plasticity, there are two types of heterosynaptic plasticity. Before discussing heterosynaptic plasticity, it is useful to review the types of synapses that are present in the central nervous system. Three broad categories of synapses are found in the central nervous system. (See also Chapter 8, Part 7 NOTE: Selecting this link moves you forward. After viewing, you will need to press the browser's "Back" button to return to this page.)

Axosomatic synapses are synapses that are made onto the soma or cell body of a neuron. Axodendritic synapses, probably the most prominent kind of synapses, are synapses that one neuron makes onto the dendrite of another neuron.

Figure 7.4

Axoaxonic synapses are synapses made by one neuron onto the synapse of another neuron. Axoaxonic synapses mediate presynaptic inhibition and presynaptic facilitation.

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Figure 7.5

The figure at left illustrates the two major types of heterosynaptic plasticity; presynaptic inhibition and presynaptic facilitation. Presynaptic inhibition is not an esoteric phenomenon. It is very prominent in the spinal cord and regulates the propagation of information to higher brain centers. An action potential in the presynaptic cell produces an EPSP in the postsynaptic cell. The modulatory cell (M1) makes an axoaxonic synapse with the presynaptic cell. After firing cell M1, the EPSP in the postsynaptic cell is smaller. This phenomenon is called presynaptic inhibition, because cell M1 regulates the ability of the presynaptic cell to release transmitter. The modulatory transmitter engages metabotropic type receptors that then activate a second messenger system that phosphorylates Ca2+ channels in such a way that the Ca2+ channels open less readily. There are fewer Ca2+ channels to be opened, therefore there is less Ca2+ influx. Less Ca2+ influx leads to less transmitter release and a smaller EPSP.

The phenomenon complementary to presynaptic inhibition is presynaptic facilitation. The scheme is the same, but the mechanisms are different. M2 is capable of increasing the strength of the synaptic pathway. Whereas the mechanism for presynaptic inhibition is a decrease in Ca2+ influx produced by affecting calcium channels directly, the mechanism for presynaptic facilitation is not due to the direct modulation of a Ca2+ channel, but rather to an indirect effect on the Ca2+ channel brought about by modulation of a K+ channel. As a result of the activation of a second messenger cascade by M2, there are fewer K+ channels available to be opened in the presynaptic terminal. The action potential is broader and there is a greater amount of time for the Ca2+ influx to occur. The Ca2+ influx occurs for a longer time, therefore more transmitter can be released. In both presynaptic inhibition and presynaptic facilitation, the Ca2+ current is modulated. But in one case the Ca2+ channel is modulated directly (presynaptic inhibition) and in the other case (presynaptic facilitation), the Ca2+ channel is modulated indirectly. Long-Term Potentiation (LTP). A very enduring form of synaptic plasticity is called long-term potentiation (LTP). It can have both homosynaptic and heterosynaptic components. An electric shock to afferent fibers produces an EPSP. If the pathway is repeatedly stimulated (e.g., every minute), the amplitude of EPSP is constant. A tetanus produces post-tetanic potentiation (PTP) that dies away after several minutes. What is left is a very enduring enhancement of the EPSP. There is excitement about LTP because it is the kind of mechanism necessary to store memory.

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Figure 7.7 Figure 7.6

Figure 7.8 (.gif version of graphic)

Figure 7.9 (.gif version of graphic)

The NMDA-type receptor is critical for some forms of LTP, in particular LTP at the CA3-CA1 synapse in the hippocampus. The postsynaptic spines of CA1 neurons have two types of glutamate receptors; NMDA-type glutamate receptors and the non-NMDAtype glutamate receptors (Figures 7.8 and 7.9). Both receptors are permeable to Na+ and K+, but the NMDA-type has two additional features. • •

First, in addition to being permeable to Na+, it also has a significant permeability to Ca2+. Second, this channel is normally blocked by Mg2+.

Even if glutamate binds to the channel and produces a conformational change, there is no efflux of K+ or influx of Na+ or Ca 2+ because it is "plugged up" by the Mg2+. Thus, a weak stimulus will not open this channel because it is blocked by Mg2+. A weak stimulus will produce an EPSP, but that EPSP will be mediated by the non-NMDA receptor. Now consider the consequences of a tetanus. Because of the tetanus, there will be spatial and temporal summation of the EPSPs produced by the multiple afferent synapses on the common postsynaptic cell. Consequently, the membrane potential of the postsynaptic neuron will become very depolarized. Since the inside of the cell becomes positive, the 68

positively charged Mg2+ is "thrust" out of the channel (Figure 7.10). Ca2+ then enters the spine through the NMDA receptor. That Ca2+ activates various protein kinases, which then trigger long-term changes.

Figure 7.10 (.gif version of graphic)

Summary

Figure 7.11

A given postsynaptic neuron receives synaptic input from a number of different sources. There are the traditional type of axosomatic and axodendritic synapses. These can be either excitatory or inhibitory. In addition, the synaptic responses can be mediated by both ionotropic or metabotropic receptors. The presynaptic cells can be modulated through presynaptic inhibition and presynaptic facilitation. Consider that any one postsynaptic cell makes and receives 10,000 connections with other cells and that this module can be recapitulated in each of the billions of cells in the nervous system. It is this enormous pattern of synaptic connections and the plasticity that occurs at each one of these synapses which makes the nervous system so extraordinary.

It is very difficult to overestimate the importance of synaptic transmission. It is critical to the basic functioning of the nervous system and appears to be critical in learning and memory. Also, changes in synaptic transmission seem to be central to understanding a number of neurological disorders such as myasthenia gravis and Parkinson's disease. Synaptic transmission is central to understanding mental diseases such as schizophrenia, anxiety, and depression. A major theme of neuroscience is to identify the specific transmitter systems involved in these brain diseases and design appropriate interventions. Finally, most of the psychoactive drugs function by affecting some aspects of synaptic transmission.

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