In situ and transcriptomic identification of microglia in synapse-rich regions of the developing zebrafish brain.

Microglia are brain resident macrophages that play vital roles in central nervous system (CNS) development, homeostasis, and pathology. Microglia both remodel synapses and engulf apoptotic cell corpses during development, but whether unique molecular programs regulate these distinct phagocytic functions is unknown. Here we identify a molecularly distinct microglial subset in the synapse rich regions of the zebrafish (Danio rerio) brain. We found that ramified microglia increased in synaptic regions of the midbrain and hindbrain between 7 and 28 days post fertilization. In contrast, microglia in the optic tectum were ameboid and clustered around neurogenic zones. Using single-cell mRNA sequencing combined with metadata from regional bulk sequencing, we identified synaptic-region associated microglia (SAMs) that were highly enriched in the hindbrain and expressed multiple candidate synapse modulating genes, including genes in the complement pathway. In contrast, neurogenic associated microglia (NAMs) were enriched in the optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish the zebrafish hindbrain as a model for investigating microglial-synapse interactions.

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.

Somatostatin-like immunoreactivity in the midbrain of the cat.

Somatostatin (SS) immunoreactivity was localized in cat brain sections with an immunoperoxidase technique. Cell bodies in the midbrain containing SS immunoreactivity were found in the superficial and intermediate gray layers of the superior colliculus, the interpeduncular nucleus, the raphe, the inferior colliculus and nucleus of its brachium, the nucleus of the optic tract, and the lateral tegmental field. Additional positive neurons were seen in the parabigeminal nucleus and in the dorsal periaqueductal gray in kitten material. Immunoreactive fibers were observed in the periaqueductal gray and in the midbrain tegmentum, with particularly dense labeling just dorsal to the substantia nigra and in the parabrachial nuclei. This is the first report of the distribution of SS immunoreactivity in the midbrain of the cat. It is concluded that somatostatin has a distribution compatible with a role as a major neurotransmitter/neuromodulator within certain midbrain nuclei, especially the interpeduncular nucleus and the superior colliculus.

Laminar organization of peptide-like immunoreactivity in the anuran optic tectum.

Peptide, 5-hydroxytryptamine (5-HT)-, tyrosine hydroxylase (TOH)-, and glial fibrillary acidic protein (GFAP)-like immunoreactivity was studied in the optic tectum of Rana pipiens. Peroxidase-antiperoxidase and indirect immunofluorescence single- and double-labeling methods were used to compare differential laminar distribution of each of these substances. Substance P (SP), leucine-enkephalin (LENK), cholecystokinin octapeptide (CCK8), bombesin (BOM), avian pancreatic polypeptide (APP), and possibly neurotensin display unique individual patterns of laminar distribution of processes and cell bodies throughout the tectum. A correlative analysis of the topographical distribution of SP, LENK, BOM, and APP on the basis of double-labeled sections shows a precise laminar segregation of these substances. Vasoactive intestinal peptide-, beta-endorphin-, and ranatensinlike immunoreactivity is consistently absent from our material. 5HT- and TOH-like immunoreactivity discloses a reticular array of fibers without clear evidence of laminar organization. This peptide-like laminar organization is particularly elaborate throughout the superficial neuropil of the optic tectum, the major retinorecipient zone. The pattern of lamination demonstrated in the present study differs in several important features from that previously described on the basis of several histological methods. The cells of origin of processes (axons and/or dendrites) in the superficial tectal neuropil may be either intrinsic or extrinsic to the tectum. Special reference is made to conflicting evidence regarding the possibility of a retinal contribution to peptide-like tectal lamination.

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.

Non-uniform distribution of cellular phenotypes in the optic tectum of the leopard frog.

Regional specialization in the retina have been described in a number of species. We have investigated whether such specializations can be found in the optic tectum, an area of the brain responsible for the processing of visual information. Using the tectum of Rana pipiens, we have examined the distribution of three different cell types defined on the basis of their immunoreactivity to somatostatin, substance P, and serotonin antibodies. These three immunoreactive cell populations had differing, nonuniform distributions in the optic tectum. Somatostatin-line immunoreactive cells were largely restricted to the caudal one-third of the tectum, whereas both substance P-like immunoreactive (SP-ir) and serotonin-like immunoreactive (5-HT-ir) cells were found unequally represented throughout the tectum. The percentage of SP-ir cells decreased significantly in both the posterior and medial directions from its high in the anterior lateral tectum. Although serotonin-like immunoreactivity was also greatest in the lateral tectum and decreased significantly medially, it was largely constant in the anterior-to-posterior dimension. The populations of SP-ir and 5-HT-ir cells were nonoverlapping. Our results suggest that information may be processed differently in different regions of the optic tectum.

Distribution of galanin-like immunoreactivity in the brain of Rana esculenta and Xenopus laevis.

The immunocytochemical distribution of galanin-containing perikarya and nerve terminals in the brain of Rana esculenta and Xenopus laevis was determined with antisera directed toward either porcine or rat galanin. The pattern of galanin-like immunoreactivity appeared to be identical with antisera directed toward either target antigen. The distribution of galanin-like immunoreactivity was similar in Rana esculenta and Xenopus laevis except for the absence of a distinct laminar distribution of immunoreactivity in the optic tectum of Xenopus laevis. Galanin-containing perikarya were located in all major subdivisions of the brain except the metencephalon. In the telencephalon, immunoreactive perikarya were detected in the pars medialis of the amygdala and the preoptic area. In the diencephalon, immunoreactive perikarya were detected in the caudal half of the suprachiasmatic nucleus, the nucleus of the periventricular organ, the ventral hypothalamus, and the median eminence. In the mesencephalon, immunoreactive perikarya were detected near the midline of the rostroventral tegmentum, in the torus semicircularis and, occasionally, in lamina A and layer 6 of the optic tectum. In the myelencephalon, labelled perikarya were detected only in the caudal half of the nucleus of the solitary tract. Immunoreactive nerve fibers of varying density were observed in all subdivisions of the brain with the densest accumulations of fibers occurring in the pars lateralis of the amygdala and the preoptic area. Dense accumulations of nerve fibers were also found in the lateral septum, the medial forebrain bundle, the periventricular region of the diencephalon, the ventral hypothalamus, the median eminence, the mesencephalic central gray, the laminar nucleus of the torus semicircularis, several laminae of the optic tectum, the interpeduncular nucleus, the isthmic nucleus, the central gray of the rhombencephalon, and the dorsolateral caudal medulla. The extensive system of galanin-containing perikarya and nerve fibers in the brain of representatives of two families of anurans showed many similarities to the distribution of galanin-containing perikarya and nerve fibers previously described for the mammalian brain.

Cluster-and-sheet pattern of enkephalin-like immunoreactivity in the superior colliculus of the cat.

The distribution of enkephalin-like immunoreactivity in the superior colliculus has been studied in the cat with the peroxidase-antiperoxidase method. Two striking patterns of immunoreactivity were observed. In the superficial layers there is a thin, dense horizontal band of immunoreactivity in the neuropil of the most dorsal tier of the superficial gray layer (sublamina 1). Because this sublayer corresponds to the zone of densest contralateral retinotectal projection, an intraocular injection of horseradish peroxidase was made in one cat to allow direct comparison of the distributions of opiate-like immunoreactivity and transported tracer in the contralateral superior colliculus. There was a detailed similarity between the two, including the presence of a gap in both at the presumptive site of the optic disc representation. The presence of enkephalin-like immunoreactivity in neural perikarya in and near sublamina 1 of the superficial gray layer, however, raised the possibility that the immunoreactive band is part of an intrinsic opiate system. Deeper in the superficial gray layer there was appreciable but weaker immunoreactivity in the neuropil and fewer immunoreactive neurons. In the intermediate gray layer and, especially medially, even deeper in the superior colliculus, enkephalin-like immunoreactivity was organized into small (100-300 micron wide) patches. In the intermediate gray layer these tended to be arranged periodically, five-seven patches being spaced at 200-600 micron intervals in caudal transverse sections. In some sections adjoining patches appeared to be fused. The patches were absent or difficult to detect in rostral sections. Caudally, they sometimes were adjacent to blood vessels penetrating the intermediate gray layer, but other times were not. Serial section reconstructions suggested that the patches observed in individual sections are part of larger arrays which have the form of anastomotic bands running in longitudinal directions somewhat oblique to the sagittal plane. It is concluded that an opiate mechanism may play a part in controlling the effects of incoming retinal information in the superficial gray layer, directly or indirectly, and that opiate peptides may also act in modulating one or more afferent or efferent systems of the deep collicular layers. Accordingly, from the functional standpoint, enkephalin-like peptides may influence both visual and sensory motor processing in the superior colliculus.

Distribution and regulation of substance P-related peptide in the frog visual system.

Modulation of visual signal activity has consequences for both signal processing and for activity-dependent structuring mechanisms. Among the neuromodulatory agents found in visual areas are substance P (SP)-related peptides. This article reviews what is known about these substances in the amphibian retina and optic tectum with special emphasis on the leopard frog, Rana pipiens. It is found that the distribution of these SP-related peptides is remarkably similar to that seen in mammals. This suggests that study of model amphibian systems may significantly enhance our understanding of how neuropeptides contribute to visual system function and organization.

Interaction of monoclonal antibodies to electroplaque acetylcholine receptors with the alpha-bungarotoxin binding site of goldfish brain.

Polyclonal and monoclonal antibodies raised against acetylcholine receptors from Torpedo californica and Electrophorus electricus electroplaque were tested for interaction with the [125I]alpha-bungarotoxin binding protein of goldfish brain. A subset of monoclonal antibodies which recognize the main immunogenic region of the alpha subunit of the Electrophorus acetylcholine receptor interacted at high affinity with the [125I]alpha-bungarotoxin binding protein. Using immunofluorescence, these antibodies were shown to label the same layers of the optic tectum as [125I]alpha-bungarotoxin.

A monoclonal antibody which binds to the surface of chick brain cells and myotubes: cell selectivity and properties of the antigen.

A monoclonal antibody has been obtained which binds to the cell surface of cultured chick myotubes and retinal and tectal neurons but not fibroblasts, myoblasts and embryonic liver cells. Indirect immunocytochemistry reveals that antigen is present in all layers of the chick neural retina. The antibody therefore recognizes an antigen common to most, if not all, chick neurons. The antigen has been identified by staining SDS gels with [125I]monoclonal antibody and appears to be a polydisperse collection of polypeptides with molecular weights centered about 250 kdalton.

Monoclonal antibodies specific for glia in the chick nervous system.

Two monoclonal antibodies that bind to previously undescribed glial antigens are discussed. One antibody, 3A7, binds to Müller cells in the chick retina and radial glia in the optic tectum. The other, 7G4, also binds to Müller cells, However, in the tectum it binds to a small cell type in the stratum opticum. The developmental appearance of the antigens and their localization in cells in tissue culture are also presented.

Degeneration of optic axons induces the expression of major histocompatibility antigens.

Eye removal from adult rats causes induction of major histocompatibility complex (MHC) antigen expression by cells within areas of optic terminal degeneration. Class I MHC antigen expression occurs through the degenerating optic terminal areas, while class II MHC antigen expression is found within areas of degenerating myelinated axons. This could be an important step in the process of immunological recognition and subsequent rejection of neural transplants that are located in the region of the degenerating fibers. It could also be a precipitating factor in certain neurodegenerative diseases of the central nervous system.

Inflammatory responses in the rat superior colliculus after eye enucleation.

Ocular enucleation induces profound morphological alterations in central visual areas. However, little is known about the response of glial cells and possible inflammatory processes in visual brain areas resulting from eye enucleation. In this study, immunoblotting and immunostaining assays revealed increased expression of astrocyte and microglia markers in the rat superior colliculus (SC) between 1 and 15 days after contralateral enucleation. A transient increase of neuronal COX-2 protein expression was also found in the SC. To evaluate the role of an anti-inflammatory drug in attenuating both COX-2 and glial cell activation, the synthetic glucocorticoid dexamethasone (DEX) was administered (1 mg/kg i.p., for 3 days) to enucleated rats. Immunoblotting data revealed that DEX treatment significantly inhibited COX-2 protein expression. Postlesion immunostaining for astrocyte and microglia markers was also significantly reduced by DEX treatment. These findings suggest that the removal of retinal ganglion cell input generates inflammatory responses in central retinorecipient structures.

Specific cell surface labels in the visual centers of Xenopus laevis tadpole identified using monoclonal antibodies.

Monoclonal antibodies (MAbs) against the optic tectum of Xenopus tadpoles were generated and screened by the immunofluorescent staining of frozen sections of tadpole brains. MAb-A5 stains the 8th and 9th plexiform layers of the optic tectum, whereas MAb-B2 stains all but the eighth and ninth plexiform layers of the optic tectum. MAb-A5 antigen is also detectable in the nucleus of Belonci, the corpus geniculatum thalamicum, the pretectal area, and the basal optic nucleus, all targets of the optic nerve, but is not detectable in the optic nerve or the optic tract. On the other hand, MAb-B2 does not stain any of these visual centers, though many fibers surrounding them are stained. Eye-enucleation experiments showed that MAb-A5 antigen is expressed in the optic tectum even when it is not innervated by optic nerves. Staining of viable brains with these MAbs indicates that these antigens are cell surface molecules. Immunoadsorption followed by SDS-PAGE suggests that proteins are constituents of these antigens. The MAb-A5 antigen in the diencephalon and the mesencephalon is not detectable at stage 35/36, but is detectable at stage 39 when the optic nerves begin to innervate the optic tectum. The spatial as well as the temporal patterns of the expression of the MAb-A5 antigen suggest that this molecule may be involved in the target recognition of optic nerve fibers.

Developmentally regulated and spatially restricted antigens of radial glial cells.

Radial glial cells, present in many parts of the embryonic vertebrate central nervous system (CNS), have been implicated in the guidance of neuroblasts from the ventricular zone to their laminar destinations. Moreover, radial glial cells may be progenitors of some CNS neurons and glia. To gain new insight into the structure and development of these cells, we have generated and characterized a panel of monoclonal antibodies that recognize radial glial cells of the chick optic tectum. Mice were immunized with homogenates of embryonic day (E) 10 tectum, and antibodies were analyzed by immunofluorescence and immunoblotting. We describe here three pairs of antibodies. 1) H5 and a previously generated antibody, R5 (Dräger et al., J. Neurosci. 4:2025, 1984), stain the whole extent of the radial glial cell from E7 to E20. In cultures prepared from E10 tecta, both stain a filamentous meshwork in glial cells but not in neurons. On immunoblots, both recognize a protein of approximately 52 kD that is closely related (or identical) to vimentin. 2) H28 and H29 stain radial glia between E7 and E14, but not later. Moreover, H28 and H29 staining is markedly more intense in the ventricular and intermediate zones than in the laminae of the tectal plate. Both of these antibodies recognize an intracellular epitope in cultured glial cells and a protein of approximately 35 kD on immunoblots. 3) H2 and H27 recognize antigens concentrated in the most superficial processes and endfeet of radial glia in late (E16-E20) embryos. They stain distinct structures in cultured glia, suggesting that they recognize distinct antigens. H27 recognizes a protein of approximately 29 kD on immunoblots. Thus antibodies H5 and R5 are good markers of radial glial cells at all stages, whereas the others define antigens that are developmentally regulated and localized to discrete domains. Together, these antibodies can be used to study temporal and spatial specializations of radial glia.

An aberrant retinal pathway and visual centers in Xenopus tadpoles share a common cell surface molecule, A5 antigen.

A monoclonal antibody A5 (MAb-A5), which was raised against Xenopus tadpole tectal cells, recognizes a cell surface-related protein molecule (A5 antigen) expressed on the visual centers of Xenopus tadpoles (S. Takagi, T. Tsuji, T. Amagai, T. Takamatsu, and H. Fujisawa, 1987, Dev. Biol. 122, 90-100). The present immunohistochemistry using MAb-A5 indicated that, in addition to the visual centers, A5 antigen was expressed on the general somatic sensory tract in the medulla and spinal cord of Xenopus tadpoles. As the general somatic sensory tract has been shown to be a pathway for ectopically transplanted retinal axons (M. Constantine-Paton and R. R. Capranica, 1976, J. Comp. Neurol. 170, 17-32; M. J. Katz and R. J. Lasek, 1979, J. Comp. Neurol. 183, 817-832), we examined whether retinal axons transplanted close to the spinal cord or medulla preferentially grow into the A5 antigen-positive general somatic sensory tract. We performed eye transplantation at embryonic stages and detected precise locations and trajectories of transplanted retinal axons within the medulla and spinal cord in tadpoles after filling retinal axons with horseradish peroxidase (HRP). HRP histochemistry in combination with MAb-A5 immunohistochemistry indicated that almost all HRP-filled transplanted retinal axons joined the A5 antigen-positive general somatic sensory tract. These findings suggest the involvement of A5 antigen in specific cell-cell recognition between retinal axons and their targets.

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.

GABA-like and glutamate-like immunoreactivity in the pretecto-olivary pathway in the rat.

The identities of neurotransmitters of the pretecto-olivary projection neurons and of the nerve terminals contacting them were investigated using a double-label method with retrograde labelling in combination with gamma-aminobutyric acid (GABA) and glutamate immunocytochemistry in the nucleus of the optic tract and the dorsal terminal nucleus of the accessory optic system in the rat both light and electron microscopically. At the light microscopic level, the somata of all projection neurons identified by a label of horseradish peroxidase reaction product were stained moderately but reliably for glutamate immunoreactivity. In no case, any retrogradely labelled neuron was found to be stained for GABA immunoreactivity. However, the somata and proximal dendrites of these cells were surrounded with many intensively stained puncta, indicating strong reactions with the anti-GABA antibodies. In contrast, no immunostaining with anti-glycine or anti-taurine antibodies was obtained. Electron microscopic investigations demonstrated that immunogold-positive axosomatic or axodendritic synapses on the retrogradely labelled neurons corresponded to some of the GABA-positive puncta in semithin sections. The results suggest that the projection neurons receive a strong inhibitory input mediated by GABA and send their directionally selective information to the inferior olive by glutamatergic projections.