Biomedical subjects

R R Mize

Publications and source records attributed to R R Mize.

At least 19 recordsLinked to original sources

PubMed1992

The calcium binding proteins parvalbumin and calbindin-D 28K form complementary patterns in the cat superior colliculus.

Parvalbumin (PV) and calbindin-D 28K (CaBP) are calcium binding proteins involved in calcium regulation in the brain. In some regions they coexist in the same neuron, while in other regions they are found in different cell types. We have studied the distribution and morphology of PV labeled neurons in the cat superior colliculus (SC) with antibody immunocytochemistry and compared this labeling to that of CaBP. PV neurons were concentrated in a dense tier within the deep superficial gray and upper optic layers. Scattered PV neurons also were found within the deep layers of SC. By contrast, CaBP neurons were concentrated in three tiers: one within the zonal and upper superficial gray layers, a second within the deep optic and upper intermediate gray layers (IGL), and a third within the deep gray layer. The distribution of PV neurons is thus complementary to that of CaBP neurons, with the CaBP cell tiers bordering the dense tier of PV neurons. PV neurons varied in size and morphology. The average diameter of labeled cells was 20 microns, almost twice the size of CaBP neurons. The cells were predominantly round, vertical fusiform, or stellate, and included the very large neurons found scattered in the IGL. Horseradish peroxidase injections into the lateral geniculate nucleus, the lateral posterior nucleus, the opposite superior colliculus, the dorsal lateral pontine gray nucleus, and two descending pathways--the crossed predorsal bundle and the tecto-ponto-bulbar tracts--each labeled SC neurons that were also labeled by PV. A large percentage (84%) of projection neurons contained PV. This result also differs from CaBP neurons in SC, most of which are interneurons. Two antigen double-label experiments did not produce any cells that contained both PV and CaBP. The two calcium binding proteins thus reveal a unique sublaminar organization in SC that consists of alternating small cell interneuron groups and large cell projection neuron groups.

Animals↗

PubMed1992

The organization of GABAergic neurons in the mammalian superior colliculus.

GABA is an important inhibitory neurotransmitter in the mammalian superior colliculus. As in the lateral geniculate nucleus, GABA immunoreactive neurons in SC are almost all small and are distributed throughout the structure in all mammalian species studied to date. Unlike the LGN, GABA-labeled neurons in SC have a variety of morphologies. These cells have been best characterized in cat, where horizontal and two granule cell morphologies have been identified. Horizontal cells give rise to one class of presynaptic dendrite while granule C cells give rise to another class of spine-like presynaptic dendrite. Granule A cells may be the origin of some GABAergic axon terminals. GABA containing synaptic profiles form serial synapses, providing a possible substrate for disinhibition. The distribution of GABAA and GABAB receptor subtypes appears similar to that of GABA neurons, with the densest distribution found within the superficial gray layer. However, antibody immunocytochemistry of the beta 2 and beta 3 subunits of the GABAA receptor reveals that it is located at both synaptic and non-synaptic sites, and may be associated with membrane adjacent to terminals with either flattened or round vesicles. A few GABA containing neurons in SC colocalize the pentapeptide leucine enkephalin or the calcium binding protein calbindin. However, none appear to co-localize parvalbumin, a situation different from GABA containing interneurons in the LGN and visual cortex. The diversity of GABA neurons in SC rivals that found in visual cortex, although unlike visual cortex, the pattern of co-occurrence does not distinguish GABA cell types in SC. The superior colliculus also differs from both LGN and visual cortex in that GABA and calbindin immunoreactivity is not altered by either long-term occlusion and/or short-term enucleation in adult Rhesus monkeys. No consistent differences have been found in the optical density of GABA labeling in either cells or neuropil. To conclude, GABA neurons in the superior colliculus share some properties like those in LGN and others like those in visual cortex. In other properties, they differ from GABA neurons in both the LGN and visual cortex. The GABA systems in the superior colliculus are similar in all mammalian species studied, suggesting that they are phylogenetically conserved systems which are not amenable to plastic alterations, a situation different to that in the geniculostriate system.

Animals↗

PubMed1992

Visual deprivation fails to reduce calbindin 28kD or GABA immunoreactivity in the rhesus monkey superior colliculus.

Antibody labeling of the calcium-binding protein calbindin 28kD (CaBP) and gamma-aminobutyric acid (GABA) is altered by short-term monocular deprivation in the lateral geniculate nucleus and visual cortex of adult primates. It is not known whether these alterations occur in other subcortical visual structures. We therefore have examined antibody labeling to CaBP and GABA in the superior colliculus (SC) of visually deprived Rhesus monkeys. One group was monocularly enucleated as adults. The other monkeys experienced different types of monocular and binocular deprivation from birth, including occlusion of one eye, and/or surgically induced aphakia, optically corrected with extended-wear contact lenses, or an intraocular lens implant. Some of these monkeys also had one eye enucleated prior to perfusion. In the SC of normal monkeys, CaBP-immunoreactive neurons formed three laminar tiers within SC, one within the zonal layer (ZL) and upper superficial gray layer (SGL), another bridging the optic and intermediate gray layers, and a third within the deep gray layer. CaBP neurons within the upper tier had small pyriform or stellate morphologies while those in the deeper tiers were slightly larger neurons, most with a stellate morphology. GABA-immunoreactive neurons were densely distributed within the SGL and more sparsely distributed within the deeper layers. These cells were mostly small neurons with horizontal, pyriform, or stellate morphologies. Neither monocular enucleation nor occlusion nor aphakia combined with continuous occlusion of the fellow eye produced any visible reduction in antibody labeling in cells or neuropil within the SC. Full-field measures of labeling intensity (optical density) within the ZL and upper SGL revealed no consistent differences between the SC contralateral or ipsilateral to the affected eye in either CaBP- or GABA-labeled sections. Measures of the optical density, number, and size of labeled neurons also showed no consistent effects of enucleation and/or occlusion. We therefore conclude that the retino-geniculostriate and retino-collicular systems differ in their response to deprivation which is likely due to the significant overlap of retinal axons from the two eyes that occurs in the SC of the Rhesus monkey.

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PubMed1992

Monocular enucleation reduces immunoreactivity to the calcium-binding protein calbindin 28 kD in the rhesus monkey lateral geniculate nucleus.

The calcium-binding proteins calbindin (CaBP) and parvalbumin (PV) are important in regulating intracellular calcium in brain cells. PV immunoreactivity is reduced by enucleation in the lateral geniculate nucleus (LGN) and by enucleation and visual deprivation in the striate cortex of adult monkeys. The effects of enucleation and visual deprivation on CaBP immunoreactivity in the LGN are not known. We therefore have studied cells and neuropil in the LGN that are labeled by antibodies to CaBP in normal and visually deprived Rhesus monkeys to determine if there is an effect on this calcium-binding protein. One group of monkeys had one eye removed 2 weeks to 4.3 years before sacrifice. A second group had one eye occluded with opaque lenses from infancy without enucleation. A final group had one eye occluded long-term followed by short-term enucleation 2 weeks before sacrifice. In normal monkeys, CaBP-immunoreactive neurons were found throughout the LGN. They were sparsely distributed within the six main laminae, and more densely distributed within layer S and the interlaminar zones (ILZ). The labeled ILZ neurons had a distinct morphology, with fusiform somata and elaborate dendritic trees that were confined primarily to the ILZ. Most CaBP-labeled neurons in the main layers had dendrites that radiated in all directions from the soma. ILZ and main layer cells labeled by CaBP thus probably represent two different cell types. Monocular enucleation with or without occlusion produced a significant reduction in antibody labeling in the deafferented laminae. Field measures revealed an average 11.5% reduction in optical density in each deafferented lamina compared to its adjacent, nondeprived layer. The differences in field optical density between deprived and nondeprived layers were statistically significant. CaBP neurons were still visible, but the optical density of antibody labeling in these cells also was reduced. Occlusion without enucleation had no effect. Thus, deafferentation, but not light deprivation, reduces concentrations of CaBP in monkey LGN. This effect is different than that seen in striate cortex of adult monkeys, where visual deprivation as well as enucleation alters CaBP immunoreactivity.

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PubMed1991

The calcium binding protein calbindin-D 28K reveals subpopulations of projection and interneurons in the cat superior colliculus.

The calcium binding protein calbindin-D 28K (CaBP) has been localized in the cat superior colliculus (SC). Four important features of SC organization have been revealed by using CaBP immunocytochemistry. 1) CaBP neurons formed three laminar tiers in SC, one within the upper one half of the superficial gray layer (SGL), the second bridging the deep optic (OL) and intermediate gray layers (IGL), and the third within the deep gray layer (DGL). 2) CaBP labeled several classes of interneuron in SC. In the upper CaBP tier, the labeled neurons were all small, but they varied in morphology and included horizontal, pyriform, and stellate neurons. A unique class of interneuron was labeled by anti-CaBP in the OL-IGL tier. This cell was stellate-like with highly varicose dendrites and broad dendritic trees. Other labeled neurons in the intermediate and deep tiers included nonvaricose stellate neurons and rare large neurons in the DGL. 3) A few anti-CaBP neurons were projection neurons. Virtually no CaBP neurons were retrogradely labeled after injections of HRP into the predorsal bundle and dorsolateral midbrain tegmentum or into the lateral posterior nucleus. However, 2.4% of anti-CaBP neurons were retrogradely labeled after HRP injections into the dorsal and ventral lateral geniculate nuclei. These represented 14.7% of all neurons projecting to the LGN complex. 4) A small percentage of CaBP neurons co-localized GABA. A two-chromagen double-labeling technique showed that about 4.0% of labeled neurons were labeled by both antibodies. In summary, antibodies to CaBP densely labeled subpopulations of neurons in the cat SC, most of which were interneurons, some of which projected to the LGN, and a few of which co-localized GABA.

Animals↗

PubMed1991

Organization of neurons labeled by antibodies to gamma-aminobutyric acid (GABA) in the superior colliculus of the Rhesus monkey.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is found in the superior colliculus (SC) of many mammalian species. In cat, several distinct classes of putative GABAergic neuron have been identified using antibodies directed against the neurotransmitter. It is not known whether these classes are found in other species. To study this, we examined the distribution, morphology, ultrastructure, and synaptic organization of GABA immunoreactive neurons in the SC of the Rhesus monkey (Macaca mulatta). Antibody-labeled neurons were distributed throughout the monkey SC, but were most densely concentrated within the zonal and superficial gray layers (32.5% of the total). These neurons were all small cells ranging from 6.6-16.3 microns in average diameter, and had granule, pyriform, and horizontal morphologies. Four types of labeled profile were identified in single ultrathin sections with the electron microscope. Presynaptic dendrites (PSDs) contained pleomorphic vesicles, received synaptic input from unlabeled axon terminals, and sometimes formed symmetric synaptic contacts with postsynaptic profiles. Two subtypes were found. One type contained loose accumulations of synaptic vesicles throughout the profile and had a distinctive varicose shape. The other type contained small discrete clusters of synaptic vesicles near the site of synaptic apposition. The former were much more common. Profiles with typical axon terminal morphology were also found. These profiles usually contained numerous flattened vesicles and formed symmetric synapses with postsynaptic profiles, both dendrites and cell bodies. Some conventional dendrites and myelinated axons were also labeled. Serial ultrathin section reconstructions revealed that PSDs formed complex synaptic relationships with other elements. Retinal terminals, identified by their characteristic pale mitochondria, established synaptic contacts with both types of PSD. These PSDs also established contact with each other, providing a possible anatomical substrate for disinhibition. We conclude that the monkey SC has multiple GABAergic cell types, similar to those found in cat, and may represent an organization common to both mammals and some other vertebrate species. The circuitry established by these cell types may provide a mechanism for disinhibition as well as inhibition in the mammalian SC.

Animals↗

PubMed1991

A unique neuronal organization in the cat pretectum revealed by antibodies to the calcium-binding protein calbindin-D 28K.

The pretectum is an important center for visual reflexes. However, the location, boundaries, and connections of individual nuclei of the pretectum are incompletely understood. In cat, the traditionally defined nuclear boundaries have been placed in doubt by recent evidence showing that the retinal input to the pretectum forms four continuous projection zones that do not match the cytoarchitectural boundaries of individual pretectal nuclei defined by previous studies. We now show that antibodies to the calcium-binding protein calbindin-D 28K (CaBP) label clusters of neurons within the pretectum that match the zones of retinal termination. Four obvious cell clusters within the pretectum were labeled by CaBP antisera. Computer three-dimensional reconstruction of these cell clusters revealed that they form four distinct but continuous zones that run the rostrocaudal length of the pretectum in a medial-to-lateral direction. By combining anterograde HRP labeling of retinal terminals with CaBP immunocytochemistry, these CaBP-labeled cell clusters were found virtually to overlap the retinal projection zones. The CaBP-labeled neurons included both multipolar and fusiform morphologies, and most were medium- to large-sized cells. HRP retrograde transport studies showed that many CaBP-labeled neurons in the clusters projected to the LGN, while none projected to the inferior olive (IO). GABA-immunoreactive neurons were also found within the CaBP cell clusters, but these neurons were smaller than most CaBP-labeled neurons, and none were retrogradely labeled following HRP injections into the LGN or IO. Two-color antibody double-labeling experiments did not reveal any GABA neurons within the clusters that colocalized CaBP. In summary, calbindin is a precise marker of neuron clusters that overlap the retinal projection zones in the cat pretectum. Many of these CaBP neurons project to the LGN, and none contain GABA.

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PubMed1989

[3H]muscimol labels neurons in both the superficial and deep layers of cat superior colliculus.

We examined the pattern of [3H]muscimol labeling in cat superior colliculus to determine if it matches that of [3H] gamma-aminobutyric acid ([3H]-GABA) labeling or GABA antibody immunoreactivity. Injections in the superficial layers labeled cell bodies in only the superficial layers. Of 204 labeled cells, 68% were located within the upper 200 microns of the superior colliculus, 31% within the deep superficial gray layer, and only 1% below that layer, a pattern similar to that seen with [3H]GABA labeling. By contrast, an injection in the deep layers of the colliculus resulted in cell labeling in both the superficial and deep layers, including 68% in the superficial gray layer, 24% in the optic layer, and 8% in the intermediate and deep gray layers. This pattern approximates that seen with GABA immunocytochemistry. We conclude that the pattern of accumulation of [3H]muscimol depends critically upon the location of the injection and reasonably matches the pattern of GABA immunoreactivity if the injection involves the deep layers of the colliculus.

Animals↗

PubMed1989

Enkephalin-like immunoreactivity in the cat superior colliculus: distribution, ultrastructure, and colocalization with GABA.

The distribution of enkephalin (ENK) immunoreactivity has been examined in the cat superior colliculus (SC) by means of light and electron microscope immunocytochemistry. The antisera were directed against leucine enkephalin but also recognized methionine enkephalin. Colocalization of ENK with gamma aminobutyric acid (GABA) was studied with a two-chromagen double-labeling technique. Enkephalin antiserum labeling was highly specific. Dense neuropil labeling was found only in a thin band 75-100 microns wide within the upper superficial gray layer of SC. Negligible neuropil labeling was seen deeper, except for patches of label within the intermediate gray layer. Intensely labeled neurons also had a specific distribution. Forty-seven percent were located within the upper 200 microns of SC, 40% within the deep superficial gray layer, 11% in the optic layer, and only 2% below that layer. Almost all ENK-labeled cells were small (mean area of 117 microns2). Some of these had horizontal fusiform cell bodies and horizontally oriented dendrites. Others had small round somata and thin, obliquely oriented dendrites. In double-labeling experiments, 18% of anti-ENK-labeled cells were also immunoreactive for GABA. Four distinct types of ENK-labeled profile were identified with the electron microscope. Presynaptic dendrites (PSD) with loose accumulations of synaptic vesicles were densely labeled with the antiserum. Conventional dendrites were also labeled. Both types of labeled profile received input from unlabeled synaptic terminals, including those from the retina that contained pale mitochondria and round synaptic vesicles and formed asymmetric synaptic contacts. Retinal terminals were never labeled with the antisera. However, some axon terminals with round synaptic vesicles, dark mitochondria, and symmetric synaptic densities were labeled by the antisera, as were some thinly myelinated axons. These results show that there is a small population of enkephalinergic neurons in the cat SC, some of which also contain GABA. Because not all cells with identical morphologies were double labeled, it appears that neurons of like morphology are chemically heterogeneous.

Animals↗

PubMed1989

Origin, distribution, and morphology of serotonergic afferents to the cat superior colliculus: a light and electron microscope immunocytochemistry study.

We have studied the serotonergic (5-HT) projection to the cat superior colliculus (SC) using serotonin antibody immunocytochemistry and retrograde transport of peroxidase-conjugated wheatgerm agglutinin (WGA-HRP). In 3 experiments, the two labels were combined in order to double label cells with both anti-5-HT and WGA-HRP. In the remaining experiments, the two labels were examined separately. Serotonin-like immunoreactive fibers were found throughout all layers of SC, but were most densely distributed within the zonal and upper superficial gray layers. Most 5-HT fibers were thin and had characteristic varicosities and terminal swellings. At the EM level, immunoreactive terminals and varicosities were found to contain small agranular vesicles and occasionally large granular vesicles (LGVs). Conventional synaptic densities were only rarely observed. Injections of WGA-HRP into SC resulted in labeling of neurons throughout the dorsal raphe nucleus and surrounding ventrolateral periaqueductal gray. Only a few cells were found in the raphe medianus and raphe pontis and none within the raphe magnus or other medullary raphe nuclei. Cells in the dorsal raphe giving rise to the SC projection varied in shape, size, and morphology and must represent more than one cell type. The morphology of these cells was indistinguishable from that of cells in the dorsal raphe which were double labeled by anti-5-HT and WGA-HRP. We conclude that the 5-HT innervation of the superior colliculus varies in density in different laminae, arises from several different cell types, and originates primarily from the dorsal raphe nucleus with minor projections from raphe medianus and raphe pontis.

Animals↗

PubMed1989

The neurons of the substantia nigra and zona incerta which project to the cat superior colliculus are GABA immunoreactive: a double-label study using GABA immunocytochemistry and lectin retrograde transport.

The neurotransmitter cytochemistry of neurons in the substantia nigra and zona incerta which project to the cat superior colliculus was examined. Neurons in both structures were double-labeled with an antibody to the transmitter GABA and a retrograde tracer, [3H]n-acetylated wheat germ agglutinin, injected into the superior colliculus. All cells in the zona incerta and substantia nigra which projected to the superior colliculus were labeled by the GABA antiserum. Most other neurons within the zona incerta and virtually all within the substantia nigra pars reticulata and pars lateralis were also labeled by the GABA antibody. By contrast, neurons in the substantia nigra pars compacta were not labeled by either the GABA antibody or wheat germ agglutinin. Nigrotectal cells in the substantia nigra were of medium to large size and most had stellate-shaped cell bodies. Zona incerta cells projecting to the superior colliculus were also of medium to large size, but most had horizontal fusiform cell bodies. This study demonstrates two new findings: (1) that all nigrotectal neurons in cat are immunoreactive to a GABA antibody and probably contain the neurotransmitter GABA; and (2) that these GABA immunoreactive neurons in cat are found not only in the substantia nigra pars reticulata but also within the pars lateralis. Zona incerta cells projecting to the superior colliculus have a different morphology but are also apparently GABAergic. These data provide an anatomical substrate for the known inhibitory action of the nigrotectal pathway on superior colliculus neurons.

Animals↗

PubMed1989

Cat-301 antibody selectively labels neurons in the Y-innervated laminae of the cat superior colliculus.

Cat-301 is a monoclonal antibody which recognizes a cell surface associated antigen of selected neurons in the central nervous system (CNS). In the visual system, cat-301 selectively labels Y-like cells in several visual structures, including portions of the lateral geniculate nucleus complex and visual cortex. The cat superior colliculus (SC) also receives Y input and contains cells driven by Y input which are selectively distributed in the deep superficial gray and deeper laminae. If cat-301 is selective to the Y-cell system in SC, labeled cells should be restricted to those laminae. To test this hypothesis, we have examined quantitatively the laminar distribution, percentage, size, and morphology of cells in SC labeled by the cat-301 antibody. Cat-301 labeled a variety of cells in the cat SC. Labeled cells were found within the deep portion of the superficial gray layer (6.6%), optic layer (27.6%), intermediate gray layer (26.9%), and the deep gray and white layers (38.5%). By contrast, only 2 of 667 labeled cells (0.3%) were found within that part of the upper superficial gray layer innervated exclusively by W input and thought to contain only W-driven cells. When considered as a percentage of the total cell population, cat-301 labeled cells represented less than 3% of cells in the superficial gray layer and approximately 15% in the deeper layers. Neurons labeled by cat-301 were all of medium to large size (mean average diameter = 33.3 microns; range = 15-84 microns) and included vertical fusiform and stellate cells in the upper layers and the very large neurons found in the intermediate gray and deeper layers. These results provide further evidence that the cat-301 antibody selectively recognizes the Y channel of the cat visual system.

Animals↗

PubMed1988

Immunocytochemical localization of gamma-aminobutyric acid (GABA) in the cat superior colliculus.

This paper reports the pattern of labeling in the cat superior colliculus produced by an antiserum raised against BSA-conjugated gamma aminobutyric acid (GABA) and visualized by light and electron microscope immunocytochemistry. Neuropil labeling was densest within the zonal and superficial gray layers but was also found in the deep layers. Neurons labeled by the GABA antibody were also most dense within the zonal and superficial gray layers, although many labeled neurons were also found in the deeper layers. The ratio of labeled to unlabeled cells varied from an average of 45% in the superficial subdivision and the intermediate gray layer to less than 30% in the deeper laminae. Almost all intensely labeled cells were small (mean area = 127 micron 2) and had varied morphologies. Several types of labeled cell were observed with the electron microscope. One type had a horizontal, fusiform cell body and a deeply invaginated nucleus. Another type had a small round or ovoid cell body with cytoplasm clumped at one end. Labeled cells with other morphologies were also occasionally seen. No labeled glial cells were found. Two types of vesicle-containing dendrite were stained by the GABA antibody. One type had loose accumulations of small synaptic vesicles and often received input from retinal terminals. Another type had spines also containing small synaptic vesicles. Labeled dendrites without synaptic vesicles were also seen frequently. Putative axon terminals labeled by the GABA antibody had densely packed synaptic vesicles and formed symmetric synaptic contacts. Labeled myelinated axons were also commonly found. These results confirm those using uptake of tritiated GABA (Mize et al.: J. Comp. Neurol. 202:385-396, '81, J. Comp. Neurol, 206:180-192, '82) in that two of the same classes of GABA neuron, horizontal I and granule I cells, were identified in the superficial laminae. However, the GABA antiserum used in this study also revealed a third class of GABA neuron with vesicle-containing spines. The antiserum also labeled a significant number of putative GABAergic neurons located in the deep subdivision of the cat superior colliculus which were not previously recognized by using transmitter autoradiography.

Animals↗

PubMed1988

Quantitative immunocytochemistry using an image analyzer. I. Hardware evaluation, image processing, and data analysis.

In this review we describe how video-based image analysis systems are used to measure immunocytochemically labeled tissue. The general principles underlying hardware and software procedures are emphasized. First, the characteristics of image analyzers are described, including the densitometric measure, spatial resolution, gray scale resolution, dynamic range, and acquisition and processing speed. The errors produced by these instruments are described and methods for correcting or reducing the errors are discussed. Methods for evaluating image analyzers are also presented, including spatial resolution, photometric transfer function, short- and long-term temporal variability, and measurement error. The procedures used to measure immunocytochemically labeled cells and fibers are then described. Immunoreactive profiles are imaged and enhanced using an edge sharpening operator and then extracted using segmentation, a procedure which captures all labeled profiles above a threshold gray level. Binary operators, including erosion and dilation, are applied to separate objects and to remove artifacts. The software then automatically measures the geometry and optical density of the extracted profiles. The procedures are rapid and efficient methods for measuring simultaneously the position, geometry, and labeling intensity of immunocytochemically labeled tissue, including cells, fibers, and whole fields. A companion paper describes non-biological standards we have developed to estimate antigen concentration from the optical density produced by antibody labeling (Nabors et al., 1988).

Animals↗

PubMed1988

Quantitative immunocytochemistry using an image analyzer. II. Concentration standards for transmitter immunocytochemistry.

Image analyzers can measure both the optical density and geometry of immunocytochemically labeled cells and fibers, as reviewed in a companion paper (Mize et al., 1988). In this paper, we report a procedure which allows us to estimate the concentration of a neurotransmitter based upon the optical density of antibody labeling produced by immunocytochemistry. To accomplish this, we developed a standard which binds conjugated neurotransmitters. Several artificial media for the standard were compared, including agar, gelatin, and agar-gelatin. A 3% agar matrix was found to be most suitable because it cut well and was nearly transparent. The agar sections were activated with cyanogen bromide/acetonitrile to promote coupling to the antigen. To test the standard, we used gamma-aminobutyric acid (GABA) conjugated to bovine serum albumin (BSA) as the antigen. The antibody was directed against this conjugate. Activated agar sections were incubated in serial dilutions of the tritium-labeled GABA/BSA conjugate. The radioactivity of some of these sections was measured to estimate the amount of coupled antigen. The remaining sections were incubated in the GABA antibody and processed for immunocytochemistry. The optical density of these sections was measured with an image analyzer. A linear relationship was found between GABA concentration and optical density over a range of at least 0.01 to 1 nmol/mg of agar. These results show that the concentration of bound GABA can be estimated from the optical density of sections labeled by antibody immunocytochemistry. The applicability of this technique to fixed brain tissue is discussed.

Image Processing, Computer-Assisted↗

PubMed1988

Laminar organization and ultrastructure of GABA-immunoreactive neurons and processes in the dorsal lateral geniculate nucleus of the tree shrew (Tupaia belangeri).

The distribution and ultrastructure of neurons and neuropil labeled by an antiserum to gamma-aminobutyric acid (GABA) were examined in the lateral geniculate nucleus (LGN) of the tree shrew (Tupaia belangeri). The LGN of this species segregates center type and cell class into three distinct pairs of laminae: a medial pair (laminae 1 and 2) containing ON-center cells, a more lateral pair (4, 5) containing OFF-center cells, and 2 laminae (3, 6) containing W-like cells. The relationship between this laminar segregation and the distribution of GABA immunoreactivity was investigated in the present study. GABA-immunoreactive neurons and neuropil were present in all six of the laminae. However, both the density of labeled cells (adjusted for neuronal density across laminae) and the density of labeled neuropil showed a medial-to-lateral gradient. The adjusted density of labeled cells was higher laterally than medially, and the density of labeled neuropil was significantly greater in the more lateral OFF-center laminae and W-like laminae than in the medial two ON-center laminae. Thus, inhibitory, GABAergic influences may modulate to different degrees the visual signals in the ON, OFF, and W pathways. Labeled cells had a mean cross-sectional area (107 microns 2) approximately one-half that of unlabeled cells (216 microns 2). They constitute 16-34% of the neurons in the LGN. At the electron microscope level, three different kinds of labeled profile were observed. Vesicle containing profiles like the F2 profiles of cat were postsynaptic to retinal terminals and presynaptic to conventional dendrites. F1 axon terminals with dense clusters of vesicles were also labeled as were some myelinated axons. Another labeled profile, which we suggest should be called an F3 process, was a large dendrite of irregular caliber with punctate groups of vesicles near the synapse. Our results suggest that GABAergic circuitry is an important part of the functional organization in the LGN of the tree shrew.

Animals↗

PubMed1987

The innervation density of serotonergic (5-HT) fibers varies in different subdivisions of the cat lateral geniculate nucleus complex.

The innervation density of serotonin (5-HT)-immunoreactive fibers, identified using an antibody to 5-HT, was found to differ in the 4 subdivisions of the cat lateral geniculate nucleus complex (LGN). The mean density (fiber length per unit area) of anti-5-HT-stained fibers was highest in the ventral LGN (0.062 micron per micron 2), moderate in the medial interlaminar nucleus (MIN) and the parvicellular C laminae of the dorsal LGN (0.039-0.040 per micron 2), and lowest in the A and magnocellular C laminae of the dorsal LGN (0.020 per micron 2). The fiber density in MIN was particularly dense along the medial edge of the nucleus, a region called the geniculate wing. The heaviest serotonin innervation is thus found in geniculate structures receiving input from W-type retinal ganglion cells and lightest in structures receiving X and Y input.

Animals↗

PubMed1986

Quantitative comparison of retinal synapses in the dorsal and ventral (parvicellular) C laminae of the cat dorsal lateral geniculate nucleus.

Three physiological classes of retinal ganglion cell project to the cat dorsal lateral geniculate nucleus (DLGN). The dorsal laminae A, A1, and magnocellular C receive X and Y retinal input, whereas the ventral parvicellular laminae C1 and C2 receive predominantly W input. We have compared quantitatively the retinal synaptic terminals of the dorsal and ventral laminae to determine whether there are morphological differences in the terminals that correspond to their different response properties. Anterogradely labeled retinal synaptic terminals in all laminae contained pale mitochondria and large, round synaptic vesicles. However, retinal terminals with pale mitochondria varied in size and synaptic organization in different laminae. The terminals in the A laminae were, on average, quite large and made numerous contacts with conventional dendritic profiles and with profiles that themselves contained synaptic vesicles (F2 profiles). The terminals in lamina C that contained pale mitochondria had a smaller overall mean area. Terminals with pale mitochondria in C1 and C2 were almost all small and synapsed with F2 profiles less frequently than did terminals in the A laminae or in lamina C. These results provide quantitative evidence that visual areas receiving W-type retinal input contain smaller retinal terminals and have a different synaptic organization from that of laminae receiving X and Y input.

Animals↗