James E. Vaughn

Organization and morphological characteristics of cholonergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase

Choline acetyltransferase (ChAT), the acetylcholine (ACh) synthesizing enzyme, has been localized immunocytochemically with a monoclonal antibody in light and electron microscopic preparations of rat central nervous system (CNS). The antibody was an IgG1 subclass immunoglobulin that removed ChAT activity from solution. The specificity of the antibody and immunocytochemical methods has been confirmed by the demonstration of ChAT-positive neurons in a number of well-characterized cholinergic systems. For example, ChAT-positive reaction product was present in the cell bodies of spinal and cranial nerve motoneurons, as well as in their axons and terminations as motor end-plates in skeletal muscle. In addition, the somata of preganglionic sympathetic and parasympathetic neurons were ChAT-positive. The specificity of staining was further supported by a lack of reaction product in several groups of neurons thought to use neuroactive substances other than acetylcholine. No specific staining was observed in control specimens. The findings indicated that ChAT had an extensive intraneuronal distribution in many cholinergic neurons, being present in cell bodies, dendrites, axons and axon terminals. ChAT-positive somata were found in the medial septum and diagonal band, the medial habenula, and the basal nucleus of, the forebrain, 3 regions that are sources of cholinergic afferents to the hippocampus, interpeduncular nucleus and cerebral cortex, respectively. In addition, positively stained cell bodies were present within the cerebral cortex. ChAT-positive punctate structures were observed in the ventral horn of the spinal cord, where electron microscopic studies demonstrated that some of these structures were synaptic terminals. Other regions containing numerous ChAT-positive puncta included the hippocampus, the interpeduncular nucleus and the cerebral cortex. The light microscopic appearance of these putative cholinergic terminals varied among different brain regions. Large punctate structures related to well-defined post-synaptic elements were characteristic of some regions, such as the ventral horn of the spinal cord, while smaller punctate structures and varicose fibers with a diffuse pattern of organization distinguished other regions, such as the cerebral cortex.

Immunocytochemical localization of glutamic acid decarboxylase in neuronal somata following colchicine inhibition of axonal transport.

Abstract The enzyme that synthesizes the neurotransmitter γ-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD), has been immunocytochemically localized in the somata and dendrites of certain neurons in rat cerebellum and Ammoms horn following colchicine injections into these two brain regions. In the cerebellum. GAD-positive reaction product was observed in the somata and proximal dendrites of Purkinje, Golgi II, basket and stellate neurons. Occasional staining of the proximal portions of axons was also observed in these colchicine-injected preparations. None of the somata or dendrites of these same cell types exhibited reaction product in preparations that were not pretreated with colchicine, although the axon terminals of these neurons were GAD-positive. In Ammons horn, the somata of a few cells that are classified as probable basket and other short-axon neurons contained detectable concentrations of GAD in preparations that were not pretreated with colchicine. However, following colchicine injections into the Ammons horn, there was approximately a five-fold increase in the number of GAD-positive somata of basket and other short-axon neurons. There was also a substantial increase in the extent of dendritic staining exhibited by these neurons. Control injections of saline and lumicolchicine produced the same results as those observed in preparations which were not pretreated with colchicine. Thus, the results from the control injections indicate that the increases in somal and dendritic staining are due to a colchicine-mediated inhibition of the somatofugal transport of GAD rather than to a non-specific effect of the drug and/or the injection procedure. The results of the present study permit the direct identification of the neuronal somata in the cerebellum and Ammons horn whose synaptic terminals probably use GABA as their neurotransmitter. On the basis of the present findings, a reasonable explanation for the failure of earlier immunocytological studies to detect somal GAD in certain GABAergic neurons is that the axonal transport of GAD appears to occur at a sufficiently rapid rate to limit the somal concentration of GAD to low, undetectable levels.

GABA neurons are the major cell type of the nucleus reticularis thalami

Glutamic acid decarboxylase (GAD), the synthesizing enzyme for the neurotransmitter gamma-aminobutyric acid (GABA), has been localized in a large number of neuronal somata within the nucleus reticularis thalami (NR) of rat brain by light microscopic immunocytochemical methods. GAD-positive staining of neuronal somata and proximal dendrites is observed in the NR of normal (untreated) rats, and this staining is substantially enhanced following colchicine injection into the lateral cerebral ventricle. GAD-positive neuronal cell bodies are prominent throughout the dorsoventral and rostrocaudal extents of the NR and, thus, form a band around the entire lateral aspect of the thalamus. In the lateral part of the NR, oval-shaped neurons with elongated GAD-positive dendritic processes are oriented parallel to the narrow axis of the NR and lie perpendicular to the penetrating fascicles of unstained thalamocortical and corticothalamic fibers. Semithin (2 micrometers) sections confirm that GAD-positive reaction product is contain within the cytoplasm of cell bodies and proximal dendrites. In addition, GAD-positive punctate structures, representing axon terminals, are present in the neuropil and, occasionally, are observed in close proximity to positively-stained neuronal somata. This finding suggests that GABA-mediated inhibition of GABA neurons may occur in the NR. The large number of GAD-positive cell bodies within the NR contrasts with a paucity of positively-stained somata in the more internally located thalamic nuclei. Within these nuclei, GAD-positive punctate structures that represent GABAergic synaptic sites are a characteristic feature. Since previous anatomical studies have demonstrated that a large proportion or reticularis neurons project into the thalamus, it is suggested that many of these GAD-positive punctate structures are the axon terminals of reticularis neurons. Through these projections, reticularis neurons may contribute to GABA-mediated inhibition within many of the thalamic nuclei.

GABAergic terminals are presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord

Multiple, dorsal rhizotomies were performed unilaterally at lumbar levels L1–L4 in adult rats. Following 24–48 h degeneration periods and fixation by intracardiac perfusions, spinal cord were removed and transversely cut into 150 μm thick sections. These sections were incubated in immunocytochemical reagents for the peroxidase-labeling of glutamic acid decarboxylase (GAD), the enzyme that synthesizes the neurotransmitter γ-aminobutyric acid (GABA). The sections were then prepared for electron microscopic examination, while other sections were processed for light microscopic, GAD immunocytochemistry and for Fink-Heimer staining of degenerating axons and axon terminals. Thirty-six hours following dorsal rhizotomies, the sections that were prepared for the light microscopic study of terminal degeneration showed large numbers of degenerating profiles in the ipsilateral substantia gelatinosa while degenerating profiles were virtually absent contralaterally. In electron microscopic preparations, degenerating primary afferent terminals were commonly observed at the centers of rosettes where they formed synaptic contacts with other axon terminals and with surrounding dendrites. Several types of synaptic relationships were observed in the rosettes which involved both GAD-positive and degenerating primary afferent terminals. Such synaptic relationships included those in which: (a) a single GAD-positive terminal was presynaptic to the central, primary afferent terminal, (b) two different GAD-positive terminals formed synapses with opposite sides of the same central, primary afferent terminal and were also closely apposed to the surrounding dendrites of the rosette, and (c) a GAD-positive terminal was presynaptic to a primary afferent terminal and both types of terminals were presynaptic to the same dendrite of the rosette. The synaptic relationships described in this study are discussed with respect to their possible functional roles in such GABA-mediated phenomena as: (a) primary afferent depolarization, (b) the dorsal root reflex and (c) primary afferent hyperpolarization. Our observations support the concept that GABAergic axon terminals are involved in the synaptic circuits which produce presynaptic inhibition and presynaptic facilitation of the primary afferent input to the dorsal spinal cord. Collectively the observed synaptic relationships could provide a morphological substrate that is compatible with an inhibitory surround system in the substantia gelatinosa.

The fine structural localization of glutamate decarboxylase in synaptic terminals of rodent cerebellum

Abstract Glutamic acid decar☐ylase (GAD), the enzyme that synthesizes the putative neurotransmitter γ-aminobutyric acid (GABA), has been localized by peroxidase labeling antibody techniques at the light and electron microscopic levels in rodent cerebellum. Specific anti-GAD peroxidase product was highly localized in certain synaptic terminals in close association with the membranes of synaptic vesicles and mitochondria but not within these organelles. GAD-positive terminals were seen on the somata and proximal dendrites of neurons in the deep cerebellar nuclei. Other positive terminals were presumed Golgi type II endings of synaptic glomeruli in the granular layer. Positive terminals were also seen in the molecular layer, including presumed basket cell endings which contained product on smooth membrane cisternae in the preterminal axon as well as around synaptic vesicles and mitochondria. These observations correlate well with a large body of evidence that certain synaptic connections in the cerebellum are inhibitory and that many, if not all, of the presynaptic components of these connections may use GABA as their neurotransmitter.

Immunocytochemical localization of glutamate decarboxylase in rat substantia nigra

L-Glutamate decarboxylase (GAD, EC 4.1.1.15), the enzyme which catalyzes the alpha-decarboxylation of L-glutamate to form gamma-aminobutyric acid (GABA), was localized both light and electron microscopically in rat substantia nigra by an immunoperoxidase method. Large amounts of GAD-positive reaction produce were seen throughout the substantia nigra in light microscopic preparations, and it appeared to be localized in punctate structures that were apposed to dendrites and somata. Electron microscopic studies revealed that most of the axon terminals in the substantia nigra were filled with GAD-positive reaction product and formed both axodendritic and axosomatic synapses. Many dendrites were extensively surrounded by GAD-positive terminals which most commonly formed symmetric synaptic junctions, although some formed asymmetric synpatic junctions. The results of this investigation are consistent with biochemical, pharmacological and physiological data which have indicated that neurons of the neostriatum and globus pallidus exert a GABA-mediated, postsynaptic inhibition upon the neurons of the substantia nigra. These findings provide another example in the vertebrate central nervous system where Golgi I projection neurons are inhibitory and use GABA as their neurotransmitter.

Glutamate decarboxylase localization in neurons of the olfactory bulb.

Glutamate decarboxylase (GAD), the enzyme that synthesizes the neurotransmitter gamma-aminobutyric acid (GABA), has been localized in the rat olfactory bulb by immunocytochemical methods with both light and electron microscopy. The light microscopic results demonstrated GAD-positive puncta concentrated in the external plexiform layer and in the glomeruli of the glomerular layer. In addition, GAD-positive reaction product stained the dentrites and somata of granule and periglomerular cells. The electron microscopic observations confirmed the presence of GAD-positive reaction product within granule and periglomerular somata and dendrites. In electron micrographs of the external plexiform layer, the gemmules which arise from the distal dentrites of granule cells were also observed to be filled with reaction product, and these structures corresponded in size and location to the puncta observed in light microscopic preparations. The gemmules were observed to form reciprocal dendrodentritic synaptic junctions with mitral cell dentrites which lacked reaction product. In the glomeruli, GAD-positive reaction product was observed in the dentritic shafts and gemmules of periglomerular cells which also formed reciprocal dendrodentritic synaptic contacts with mitral/tufted cell dentrites. The localization of GAD in known inhibitory neurons of the olfactory bulb supports the case that these local circuit neurons use GABA as their neurotransmitter. The present study demonstrates that GAD molecules located within certain neuronal somata and dentrites can be visualized with antisera prepared against GAD that was purified from synaptosomal fractions of mouse brains. This finding suggests that the lack of GAD staining within somata and dentrites of GABA-ergic neurons noted in previous studies of the cerebellum and spinal cord was probably due to low GAD concentrations, rather than to antigenic differences among GAD molecules located in different portions of the neuron. A striking differences among GAD molecules located in different portions of the neuron. A striking difference between the granule and periglomerular neurons of the olfactory bulb and the neurons of the cerebellum and spinal cord is that the former have presynaptic dentrites while the latter do not. Since GAD-positive reaction product can be detected in the somata and dentrites of GABA-ergic neurons which have presynaptic dentrites, it is suggested that these neurons may differ from other GABA-ergic neurons with respect to either transport or metabolism of GAD.

The cytoarchitecture of gabaergic neurons in rat spinal cord

Glutamic acid decarboxylase (GAD), the enzyme that synthesizes the transmitter gamma-aminobutyric acid (GABA), has previously been localized within synaptic terminals in rat spinal cord by immunocytochemistry. In the present study, GAD was localized within the somata and dendrites of GABA neurons following colchicine injections into rat lumbar spinal cord. All regions of the spinal gray matter contained GAD-positive somata except the motoneuron pools (lamina IX). GAD-positive somata also were observed in the ependymal layer and in the dorsolateral funiculus. Small GAD-positive somata, averaging 9 X 13 micrometer in size, were located in laminae I-III, and the size of GAD-positive somata increased for cells located in progressively more ventral laminae, reaching a maximum in lamina VII where somal size averaged 12 X 19 micrometer. Lamina I contained two classes of GAD-positive cell bodies; lenticular shaped, intermediate size neurons that were reminiscent of stalked cells, and a smaller cell type that was elongated in the sagittal plane. GAD-positive somata in laminae II and III had the size and position of islet cells. In laminae IV-VI, GAD-positive somal profiles averaged 12 X 17 micrometer in size. Lamina IV neurons were concentrated along laminar edges, while those in laminae V and VI were distributed more homogeneously. In lamina VIII, GAD-positive cell bodies appeared in groups of 3 or 4 and were smaller than those in lamina VII. Lamina X contained GAD-positive somal profiles averaging 12 X 16 micrometer in size. In the ependymal layer, there were two types of cerebrospinal fluid (CSF)-contacting neurons that contained GAD; one spherical and the other elongated. Both types sent extensions into the central canal where these processes expanded into 4-5 micrometer-wide end bulbs. CSF-contacting cells with sizes and shapes similar to the GAD-positive ones were seen to receive synapses in electron micrographs. The widespread distribution of GABA neurons in spinal cord was suggestive of diverse functions for these cells, encompassing conventional synaptic roles and, perhaps, an involvement in hormonally modulated communication via GABAergic, CSF-contacting neurons.

Morphological diversity of immunocytochemically identified GABA neurons in the monkey sensory-motor cortex

SummaryGABAergic neurons have been identified in monkey sensory-motor cerebral cortex by light microscopic, immunocytochemical localization of the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD). All GAD-positive neurons are non-pyramidal cells. Their somata are present in all layers and are evenly distributed across layers II-VI of the motor cortex (area 4), but are found in greater concentrations in layers II, IV and VI of all areas of first somatic sensory cortex (SI; areas 3a, 3b and 1–2). GAD-positive puncta (putative axon terminals) are present throughout the sensory-motor cortex, and they are found immediately adjacent to the somata, dendrites and presumptive axon initial segments of GAD-negative pyramidal cells. In addition, they are observed in close approximation to the somata of both large and small GAD-positive neurons. In area 4, the density of puncta is highest in the superficial cortical layers (layers I-III) and gradually declines throughout the deeper layers. In SI, the highest densities of puncta are present in layer IV, while moderately high densities are found in layers I-III and VI. In areas 3a and 3b, the puncta in layers IV and VI are particularly numerous and form foci that exhibit greater density than adjacent regions.GAD-positive neurons withlarge somata, 15–33 μ in diameter, are present in layers IIIB-VI of all areas. Such cells have many primary dendrites that radiate in all directions. In addition they have axons that ascend either from the superficial aspect of the somata or from primary dendrites, and that exhibit horizontal collateral branches. These neurons closely resemble the large basket cells (Marin-Padilla, 1969; Jones, 1975), and they may give rise to many of the GAD-positive endings surrounding the somata and proximal dendrites of pyramidal cells in layers III-VI. In addition,small GAD-positive somata are present in all layers, but they are most numerous in layers II and IIIA of all areas and in layer IV of SI. The somata and proximal dendrites of these cells vary from a multipolar shape with small, beaded dendrites, found primarily in layer IV, to bitufted and multipolar shapes with larger, smooth dendrites. The diversity of somal sizes and locations, the variety of dendritic patterns, and the different distributions of GAD-positive puncta, all combine to suggest that several different morphological classes of intrinsic comprise the GABA neurons of monkey cerebral cortex.

Fine structural relationships between neurites and radial glial processes in developing mouse spinal cord

SummaryNeurites have been observed in various types of fine structural associations with developing radial glial processes in the lateral marginal zones of embryonic mouse spinal cords. The least complex of the neurite-glial relationships involves a simple apposition of dendrites and dendritic growth cones with radial glial processes. We interpret this relationship to mean that dendrites, growing from the somata of the lateral motor nucleus, may preferentially move along the interface between radial glial processes and the adjacent axons of the marginal zone. A somewhat more specialized neurite-glial association is the occurrence ofpuncta adhaerentia between neurites and radial glial processes. Finally, the most elaborate morphological relationship observed is that of synapse-like contacts between axons and radial glial processes. These ‘axoglial synapses’ begin to appear at E11 and E12, are most numerous in E13 and E14 spinal cords, but they can not be found after embryonic day 15. Based on these observations, we make the following speculation: the formation of synaptic junctions occurs in a hierarchy of steps with a process of trial and error primarily involved in the initial stages of synapto-genesis, while more specific requirements are involved in the later steps of synaptic differentiation. Thus, the formation of ‘axoglial synapses’ may reflect errors in development while their disappearance may result when more specific mechanisms of synaptic differentiation become operational.