Robert P. Barber

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.

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.

Immunocytochemical localization of glutamic acid decarboxylase in the dorsal lateral vestibular nucleus: evidence for an intrinsic and extrinsic GABAergic innervation.

Immunocytochemical methods were used to localize the gamma-aminobutyric acid (GABA) synthesizing enzyme glutamic acid decarboxylase (GAD) in normal and partially deafferented dorsal lateral vestibular nucleus (dLVN) of the rat. Normal dLVN exhibited more intense staining than the ventral part of the nucleus (vLVN), and this was mainly due to more numerous GAD+ axon terminals surrounding dLVN somata and within neuropil. Small GAD+ somata were also present. Electron microscopy suggested the existence of two types of GAD+ synaptic terminals. The major type conformed to descriptions of Purkinje cell terminals, whereas the minor type did not. Ablation of the anterior cerebellar vermis resulted in a 73% reduction in the number of GAD+ terminals, the percentage decrease being greater around neuronal somata (83%) than within neuropil (70%). The presence of GAD+ somata within the nucleus and the persistence of some GAD+ terminals after extensive lesions of the Purkinje cell afferents suggest that both extrinsic and intrinsic GABAergic neurons may contribute to information processing within the dLVN.

The genetic organization of neuron number in the granule cell layer of the area dentata in house mice

This report concerns variations in neuron number within the granule cell layer of the area dentata that occur among inbred strains of house mice. There is genetically associated variability in the total number of neurons present, with a very substantial range of estimated values. Systematic strain variations in the orientation of the granule cell layer are also present. When statistical corrections for variations in orientation are made, associations between the neuron numbers of subdivisions of the granule cell layer are consistent with the presence of common genetic determination of neuron number throughout the entire lamina.

Species-specific second antibodies reduce spurious staining in immunocytochemistry

Spurious staining related to the second (linking) antibodies was observed in immunocytochemical specimens processed with an unlabeled antibody method. Some of this staining was suspected to result from species cross-reactivity of the second antibodies with endogenous immunoglobulin Gs in the tissue. Therefore, species-specific second antibodies were obtained, and the staining patterns of tissue processed with such antibodies were compared with those of tissue processed with standard (nonspecies-specific) second antibodies. In these studies, a monoclonal antibody to choline acetyltransferase (ChAT) was utilized as the primary antibody, and a similarly prepared monoclonal antibody that did not react with ChAT served as a control antibody. Spurious staining that included staining of discrete tissue and cellular components as well as amorphous background staining was present in both control and experimental tissue processed with standard second antibodies. Such staining was virtually eliminated in tissue processed with species-specific second antibodies. In specimens from the central nervous system, for example, species-specific second antibodies greatly reduced dark staining within the area postrema, in the pia-arachnoid membranes, and around blood vessels as well as the staining of small dot-like structures within some large neurons. In addition, the general level of background staining was reduced in both adult and developing tissues, thus permitting clearer visualization of many positively stained structures. In peripheral tissues such as skeletal muscle, spurious staining of connective tissue elements was eliminated, allowing the observation of previously occluded ChAT-positive structures such as nerve fibers and motor end-plates. Thus, species-specific second antibodies appear to be very useful for immunocytochemistry, particularly when the primary antibody and the tissue to be studied are from closely related species.