Calretinin-immunoreactive Cells Research Articles

Connexin-36 distribution and layer-specific topography in the cat retina

Connexin-36 (Cx36) is the major constituent of mammalian retinal gap junctions positioned in key signal pathways. Here, we examined the laminar and large-scale topographical distribution of Cx36 punctate immunolabels in the retina of the cat, a classical model of the mammalian visual system. Calretinin-immunoreactive (CaR-IR) cell populations served to outline the nuclear and plexiform layers and to stain specific neuronal populations. CaR-IR cells included horizontal cells in the outer retina, numerous amacrine cells, and scattered cells in the ganglion cell layer. Cx36-IR plaques were found among horizontal cell dendrites albeit without systematic colocalization of the two labels. Diffuse Cx36 immunoreactivity was found in the cytoplasm of AII amacrine cells, but no colocalization of Cx36 plaques was observed with either the perikarya or the long varicose dendrites of the CaR-IR non-AII amacrine cells. Cx36 puncta were seen throughout the entire inner plexiform layer showing their highest density in the ON sublamina. The densities of AII amacrine cell bodies and Cx36 plaques in the ON sublamina were strongly correlated across a wide range of eccentricities suggesting their anatomical association. However, the high number of plaques per AII cell suggests that a considerable fraction of Cx36 gap junctions in the ON sublamina is formed by other cell types than AII amacrine cells drawing attention to extensive but less studied electrically coupled networks.

Identification of AⅡ amacrine, displaced amacrine, and bistratified ganglion cell types in human retina with antibodies against calretinin.

Antibodies against calretinin are markers for one type of rod pathway interneuron (AⅡ amacrine cell) in the retina of some but not all mammalian species. The AⅡ cells play a crucial role in night-time (scotopic) vision and have been proposed as a target for optogenetic restoration of vision in retinal disease. In the present study we aimed to characterize the AⅡ cells in human retina. Postmortem human donor eyes were obtained with ethical approval and processed for calretinin immunofluorescence. Calretinin-positive somas in the inner nuclear and the ganglion cell layer were filled with the lipophilic dye DiI. The large majority (over 80%) of calretinin-immunoreactive cells is located in the inner nuclear layer, is immunopositive for glycine transporter 1, and shows the typical morphology of AⅡ amacrine cells. In addition, a small proportion of calretinin-positive cells in the inner nuclear layer and in the ganglion cell layer is glutamic acid decarboxylase-positive and shows the morphology of widefield amacrine cells (stellate, semilunar, and thorny amacrine cells). About half of the calretinin cells in the ganglion cell layer are bistratified ganglion cells resembling the small bistratified (presumed blue-ON/yellow-OFF) and the G17 ganglion cell previously described in primates. We conclude that in human retina, antibodies against calretinin can be used to identify AⅡ amacrine cells in the inner nuclear layer as well as widefield amacrine and small bistratified ganglion cells in the ganglion cell layer.

Enteroendocrine profile of α-transducin immunoreactive cells in the gastrointestinal tract of the European sea bass (Dicentrarchus labrax)

The fish sensory epithelium contains three types of sensory cells denominated ciliated, microvillous, and crypt neurons. Each one differs from the other in its morphological, ultrastructural and molecular features, as well as in their projections to the central nervous system. Crypt neurons are present in both bony and cartilaginous fish and can be identified on the basis of their morphology and the expression of some specific proteins and genes. In this study we have investigated the morphology of crypt neurons, as well as the occurrence and co-localization of S100 protein, calretinin and TRPV4, three proposed markers for crypt cells, in the olfactory epithelium of adult zebrafish (Danio rerio) using double immunofluorescence associated to laser confocal microscopy. A sparse population of superficial S100 protein positive cells was detected being identified as crypt neurons. The calretinin immunoreactive cells were more abundant, occasionally resembling the morphology of the crypt cells but never displaying co-localization of both proteins. The TRPV4 positive cells differed in morphology from crypt cells, thus excluding the occurrence of TRPV4 in those cells. These results demonstrate that only S100 protein immunoreactivity can be used to identify crypt cells. Because some calretinin positive cells showed localization and morphology similar to the crypt cells of the sensory epithelium, the occurrence of two subtypes of crypt cells, S100 protein and calretinin positive, cannot be excluded. The significance of these findings remains to be elucidated.

Inhibitory interneuron classes express complementary AMPA-receptor patterns in macaque primary visual cortex.

Glutamate receptors mediate excitatory neurotransmission. A very prevalent type of glutamate receptor in the neocortex is the AMPA receptor (AMPAR). AMPARs mediate fast synaptic transmission and their functionality depends on the subunit composition. In primary visual cortex (area V1), the density and subunit composition of AMPARs differ among cortical layers and among cell types. The AMPARs expressed by the different types of inhibitory interneurons, which are crucial for network function, have not yet been characterized systematically. We investigated the distribution of AMPAR subunits in macaque V1 for three distinct subpopulations of inhibitory interneurons: parvalbumin-immunoreactive (PV-IR) interneurons, calbindin-immunoreactive (CB-IR) interneurons, and calretinin-immunoreactive (CR-IR) interneurons. We found that PV-IR cells, which have previously been identified as fast spiking, show high expression of the GluA2 and GluA3 subunits. In contrast, CB-IR and CR-IR cells, which tend to be intermediate spiking, show high expression of the GluA1 and GluA4 subunits. Thus, our data demonstrate that the expression of AMPARs divides inhibitory interneurons in macaque V1 into two categories that are compatible with existing classification methods based on calcium-binding proteins and firing behavior. Moreover, our findings suggest new approaches to target the different inhibitory interneuron classes pharmacologically in vivo.

Immunohistochemical characterization of the crypt neurons in the olfactory epithelium of adult zebrafish

The fish sensory epithelium contains three types of sensory cells denominated ciliated, microvillous, and crypt neurons. Each one differs from the other in its morphological, ultrastructural and molecular features, as well as in their projections to the central nervous system. Crypt neurons are present in both bony and cartilaginous fish and can be identified on the basis of their morphology and the expression of some specific proteins and genes. In this study we have investigated the morphology of crypt neurons, as well as the occurrence and co-localization of S100 protein, calretinin and TRPV4, three proposed markers for crypt cells, in the olfactory epithelium of adult zebrafish (Danio rerio) using double immunofluorescence associated to laser confocal microscopy. A sparse population of superficial S100 protein positive cells was detected being identified as crypt neurons. The calretinin immunoreactive cells were more abundant, occasionally resembling the morphology of the crypt cells but never displaying co-localization of both proteins. The TRPV4 positive cells differed in morphology from crypt cells, thus excluding the occurrence of TRPV4 in those cells. These results demonstrate that only S100 protein immunoreactivity can be used to identify crypt cells. Because some calretinin positive cells showed localization and morphology similar to the crypt cells of the sensory epithelium, the occurrence of two subtypes of crypt cells, S100 protein and calretinin positive, cannot be excluded. The significance of these findings remains to be elucidated.

Males but not females show differences in calbindin immunoreactivity in the dorsal thalamus of the mouse model of fragile X syndrome

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by the loss of the Fmr1 gene product, fragile X mental retardation protein. Here we analyze the immunohistochemical expression of calcium-binding proteins in the dorsal thalamus of Fmr1 knockout mice of both sexes and compare it with that of wildtype littermates. The spatial distribution pattern of calbindin-immunoreactive cells in the dorsal thalamus was similar in wildtype and knockout mice but there was a notable reduction in calbindin-immunoreactive cells in midline/intralaminar/posterior dorsal thalamic nuclei of male Fmr1 knockout mice. We counted the number of calbindin-immunoreactive cells in 18 distinct nuclei of the dorsal thalamus. Knockout male mice showed a significant reduction in calbindin-immunoreactive cells (range: 36-67% lower), whereas female knockout mice did not show significant differences (in any dorsal thalamic nucleus) when compared with their wildtype littermates. No variation in the calretinin expression pattern was observed throughout the dorsal thalamus. The number of calretinin-immunoreactive cells was similar for all experimental groups as well. Parvalbumin immunoreactivity was restricted to fibers and neuropil in the analyzed dorsal thalamic nuclei, and presented no differences between genotypes. Midline/intralaminar/posterior dorsal thalamic nuclei are involved in forebrain circuits related to memory, nociception, social fear, and auditory sensory integration; therefore, we suggest that downregulation of calbindin protein expression in the dorsal thalamus of male knockout mice should be taken into account when analyzing behavioral studies in the mouse model of FXS.

Prenatal dexamethasone selectively decreases calretinin expression in the adult female lateral amygdala

Exposure to high levels of glucocorticoids (GCs) during early development results in lasting disturbances in emotional behavior in rodents. Inhibitory GABAergic neurons, classified by their expression of calcium binding proteins (CBPs), also contribute to stress-related behaviors and may be GC sensitive during development. Therefore, in the present study we investigated the effects of prenatal treatment with the glucocorticoid receptor agonist dexamethasone (DEX) on expression of calbindin and calretinin in brain areas critical to emotional regulation (basolateral/lateral amygdala and hippocampal CA1 and CA3 regions). Late gestational treatment with DEX (gestational days 18–22) significantly decreased the density of calretinin immunoreactive cells in the lateral amygdala of adult female offspring with no differences in the basolateral amygdala, hippocampal CA1, or CA3 regions. Moreover, there were no effects of gestational DEX treatment on calretinin expression in males. Calbindin expression in adulthood was unaltered within either amygdala or hippocampal subregion of either sex following prenatal DEX treatment. Together these findings indicate that late gestational DEX treatment causes a targeted reduction of calretinin within the lateral amygdala of females and this may be one mechanism through which developmental glucocorticoid exposure contributes to lasting alterations in emotional behavior.

Age-related changes in calbindin and calretinin immunoreactivity in the central auditory system of the rat

Age-related changes in the levels of major intracellular calcium buffers are known to occur in different parts of the mammalian brain, including the central auditory pathway. In the present study, we evaluate with immunohistochemistry and the western blot technique the effect that aging has on the calbindin- and calretinin-expressing system of neurons in the higher structures of the central auditory pathway, in the inferior colliculus (IC), medial geniculate body (MGB) and auditory cortex (AC) of two rat strains, the slowly aging Long-Evans and the fast aging Fischer 344. Interestingly, the age-related changes demonstrated a similar character regardless of the rat strain. In the IC of young animals, the majority of calbindin and calretinin immuno-reactive (CB and CR-ir) cells were found in the dorsal and external cortices and only sparse positive cells were present in the central nucleus of the IC. With aging, the number of CB-ir and CR-ir neurons decreased significantly in both the dorsal and external cortices. Furthermore, these declines were accompanied by an age-related reduction in the mean volumes of CB- and CR-ir neuronal somas. In the MGB of young rats, CB-ir neurons were present in abundant numbers in both the dorsal and ventral subdivisions, while CR-ir neurons were practically absent in this structure. With aging, the number and mean volume of CB-ir cells in the ventral subdivision of the MGB were significantly decreased. In comparison with the IC and MGB, age-related numerical and volumetric declines of both CB-ir and CR-ir neurons in the AC were less pronounced. Western blot protein analysis revealed a pronounced age-related decline in the levels of calbindin in both strains and in all examined brain regions. In contrast, the decline in calretinin levels with aging was less prominent, with a significant decline only in the IC of both strains. The observed age-related changes in the calbindin- and calretinin-expressing systems may contribute significantly to the deterioration of hearing function known as central presbycusis.

Effects of in utero endotoxemia on the ovine fetal brain A model for schizophrenia

Infections during pregnancy can adversely affect the development of the fetal brain. This may contribute to disease processes such as schizophrenia in later life. Changes in the (cyto-) architecture of the anterior cingulate cortex (ACC), particularly in GABA-ergic interneurons, play a role in the pathogenesis of schizophrenia. We hypothesized that exposure to infection during pregnancy could result in cyto-architectural changes in the fetal ACC, similar to the pathogenesis seen in schizophrenia. Fetal sheep of 110 days GA (term=150 days GA) received an intravenous injection of 100 ng or 500 ng lipopolysaccharide (LPS) or saline as control. After delivery at 113 days GA, the cyto-architecture of the cingulate cortex (CC) was examined by immunohistochemistry. High dose LPS exposure resulted in a decreased density of GFAP-, calbindin D-28K- and parvalbumin-immunoreactive cells in the CC. In addition, these cells and calretinin-immunoreactive cells showed a changed morphology with reduced cell processes. This study provides further evidence that intra-uterine endotoxemia can induce changes in the fetal brain which correspond with changes seen in schizophrenia.

Calcium-binding proteins and GFAP immunoreactivity alterations in murine hippocampus after 1 month of exposure to 835 MHz radiofrequency at SAR values of 1.6 and 4.0 W/kg

Widespread use of wireless mobile communication has raised concerns of adverse effect to the brain owing to the proximity during use due to the electromagnetic field emitted by mobile phones. Changes in calcium ion concentrations via binding proteins can disturb calcium homeostasis; however, the correlation between calcium-binding protein (CaBP) immunoreactivity (IR) and glial cells has not been determined with different SAR values. Different SAR values [1.6 (E1.6 group) and 4.0 (E4 group) W/kg] were applied to determine the distribution of calbindin D28-k (CB), calretinin (CR), and glial fibrillary acidic protein (GFAP) IR in murine hippocampus. Compared with sham control group, decreased CB and CR IRs, loss of CB and CR immunoreactive cells and increased GFAP IR exhibiting hypertrophic cytoplasmic processes were noted in both experimental groups. E4 group showed a prominent decrement in CB and CR IR than the E1.6 group due to down-regulation of CaBP proteins and neuronal loss. GFAP IR was more prominent in the E4 group than the E1.6 group. Decrement in the CaBPs can affect the calcium-buffering capacity leading to cell death, while increased GFAP IR and changes in astrocyte morphology, may mediate brain injury due to radiofrequency exposure.

Specific amacrine cell changes in an induced mouse model of glaucoma

To investigate retinal cell population changes under chronic elevated intraocular pressure in an inducible mouse model of glaucoma. Chronic unilateral ocular hypertension was induced in 40 C57BL6/J mice by ablation of the limbal episcleral veins. After 5, 20, 40 and 60 days of elevated intraocular pressure, specific retinal cell types were identified and/or quantified by immunohistochemistry for protein kinase C α, glial fibrillary acidic protein, parvalbumin and calretinin. Apoptotic cells were identified by TUNEL and cleaved caspase-3 immunohistochemistry. Elevations in intraocular pressure in the range 22-30 mmHg were developed and sustained in mice for up to 60 days. Protein kinase C α immunoreactivity localized to bipolar cells was unchanged. We observed a rapid increase in glial fibrillary acidic protein expression in Müller cells and a progressive loss of parvalbumin-labelled ganglion cells. After 60 days of elevated intraocular pressure, calretinin-immunoreactive cell counts declined by 55.4% and 46.4% in the inner nuclear and ganglion cell layers, respectively. However, at all time points examined, the markers of cell death were only observed in the ganglion cell layer, not in the inner nuclear layer. In addition to ganglion cell death and reactive Müller cell changes, chronic experimental elevation of intraocular pressure alters calcium-binding protein immunohistochemistry in amacrine cells. However, these changes are not indicative of amacrine cell loss but may represent early indicators of cellular distress that precede physiological dysfunction or cell death.

Expression of podoplanin and calretinin in meningioma: an immunohistochemical study

Meningioma is derived from arachnoid cells covering the surface of the brain and spinal cord, and it has characteristics shared by mesothelioma arising from the pleura and peritoneum in that it takes an epithelioid appearance despite its mesenchymal origin. We examined the immunohistochemical expression of podoplanin and calretinin, both of which are well-known markers of mesothelioma, in 24 surgical cases of meningioma of various types. In most cases, a linear immunoreactivity for podoplanin was found along the cell surface of most neoplastic cells. An intracytoplasmic, finely granular, or diffuse immunoreactivity was also noted in some cells. These findings corresponded well to immunoreactivity for epithelial membrane antigen (EMA), but immunoreactivity for podoplanin was more crisp or sharply delineated and clear compared with that for EMA. These findings indicate that podoplanin can be used as an immunohistochemical marker that is equivalent to EMA in the differential diagnosis of meningioma. However, most meningiomas did not contain calretinin-immunoreactive cells, a finding that differs from the diffuse immunoreactivity seen in mesothelioma.

Alteration of inhibitory circuits in the somatosensory cortex of Ts65Dn mice, a model for Down’s syndrome

Down's syndrome (DS), with an incidence of one in 800 live births, is the most common genetic disorder associated with mental retardation. This trisomy on chromosome 21 induces a variable phenotype in which the only common feature is the presence of mental retardation. The neural mechanisms underlying mental retardation might include defects in the formation of neuronal networks and neural plasticity. DS patients have alterations in the morphology, the density and the distribution of dendritic spines in the pyramidal neurons of the cortex. Our hypothesis is that the deficits in dendritic arborization observed in the principal neurons of DS patients and Ts65Dn mice (a model for DS that mimics most of the structural alterations observed in humans) may be mediated to some extent by changes in their inhibitory inputs. Different types of interneurons control different types of inhibition. Therefore, to understand well the changes in inhibition in DS, it is necessary to study the different types of interneurons separately. We have studied the expression of synaptophysin, Glutamic acid decarboxylase-67 (GAD-67) and calcium-binding protein-expressing cells in the primary somatosensory cortex of 4-5 month old Ts65Dn mice. We have observed an increment of GAD67 immunoreactivity that is related mainly to an increment of calretinin-immunoreactive cells and among them the ones with bipolar morphology. Since there is a propensity for epilepsy in DS patients, this increase in interneurons might reflect an attempt by the system to block overexcitation rather than an increment in total inhibition and could explain the deficit in interneurons and principal cells observed in elderly DS patients.

Developmental changes in the distribution of calretinin-immunoreactive cells in human fetal nasal epithelium

Immunoreactivity of the calcium binging protein calretinin is often used as a marker of olfactory neurons. Although the immunoreactivity and density of olfactory neurons are known to change between developmental stages in the human fetus, previous descriptions have been limited to the olfactory epithelium and/or the nasal septum and have not included the entire nasal cavity. Using horizontal semi serial sections of heads of six mid-term fetuses (9-15 weeks of gestation), we examined the topographical anatomy of calretinin-positive olfactory neurons. By 9 weeks of gestation, the distribution of calretinin-positive cells reached levels inferior to the developing inferior meatus. By 12 weeks, concentrations in the inferior end had reached the level of the inferior meatus and the middle meatus carried abundant positive cells. However, by 15 weeks, calretinin positive cells were restricted to levels superior to the middle meatus and in the vomeronasal organ. Placode-derived cells are initially distributed antero-inferiorly along the nasal epithelium, but most lose their calretinin immunoreactivity. They might differentiate into the neuroendocrine cells embedded between nasal respiratory epithelial cells. The final differentiation of calretinin-positive cells was likely to require connection to the olfactory bulb and accessory bulb.

Neocortical neuron types in Xenarthra and Afrotheria: implications for brain evolution in mammals

Interpreting the evolution of neuronal types in the cerebral cortex of mammals requires information from a diversity of species. However, there is currently a paucity of data from the Xenarthra and Afrotheria, two major phylogenetic groups that diverged close to the base of the eutherian mammal adaptive radiation. In this study, we used immunohistochemistry to examine the distribution and morphology of neocortical neurons stained for nonphosphorylated neurofilament protein, calbindin, calretinin, parvalbumin, and neuropeptide Y in three xenarthran species-the giant anteater (Myrmecophaga tridactyla), the lesser anteater (Tamandua tetradactyla), and the two-toed sloth (Choloepus didactylus)-and two afrotherian species-the rock hyrax (Procavia capensis) and the black and rufous giant elephant shrew (Rhynchocyon petersi). We also studied the distribution and morphology of astrocytes using glial fibrillary acidic protein as a marker. In all of these species, nonphosphorylated neurofilament protein-immunoreactive neurons predominated in layer V. These neurons exhibited diverse morphologies with regional variation. Specifically, high proportions of atypical neurofilament-enriched neuron classes were observed, including extraverted neurons, inverted pyramidal neurons, fusiform neurons, and other multipolar types. In addition, many projection neurons in layers II-III were found to contain calbindin. Among interneurons, parvalbumin- and calbindin-expressing cells were generally denser compared to calretinin-immunoreactive cells. We traced the evolution of certain cortical architectural traits using phylogenetic analysis. Based on our reconstruction of character evolution, we found that the living xenarthrans and afrotherians show many similarities to the stem eutherian mammal, whereas other eutherian lineages display a greater number of derived traits.

Voluntary exercise increases the new cell formation in the hippocampus of ovariectomized mice

Voluntary exercise, such as running, can induce dramatic increases in adult hippocampal neurogenesis and improve learning and memory function. A recent report showed that exercise also improved memory problems in postmenopausal women. In this study, we examined whether voluntary running exercise could increase new cell formation in the hippocampus under menopausal conditions, modeled with ovariectomized (OVX) mice. Voluntary running exercise for 1 week significantly increased the number of bromodeoxyuridine (BrdU)- and Ki-67-immunoreactive cells in the hippocampus of mice 2 weeks after ovariectomy. In addition, 1 week of voluntary running exercise after 2 weeks in OVX mice increased the numbers of doublecortin- and calretinin-immunoreactive cells in the hippocampus. These data demonstrate that exercise may increase the birth of new cells in the hippocampus under estrogen-deprived conditions, suggesting that exercise may be helpful in improving brain function in climacteric women.

Distribution of calretinin during development of the olfactory system in the brown trout, Salmo trutta fario: Comparison with other immunohistochemical markers

Immunocytochemical techniques were used to investigate the appearance and distribution of calretinin in the olfactory system of developing and adult brown trout ( Salmo trutta fario L.). The earliest calretinin-immunoreactive (CR-ir) cells were detected in the olfactory placode of 5-mm embryos. In 8-mm embryos, a CR-ir olfactory nerve was observed. The number of CR-ir olfactory receptor cells increased rapidly, and in fry and adults they were characterized by light and electron microscopy as pertaining to three morphological types of receptor cell, called microvillous, ciliated and rod-like cells or crypt cells. Comparisons of the cells labeled with CR and with more general olfactory markers (acetylated tubulin and keyhole limpet haemocyanin) in alevins and fry revealed that CR-ir cells represent only a subpopulation of olfactory receptor cells. Large cells located in the primordial mitral cell layer were the first CR-ir neuronal population of the olfactory bulbs and were observed in 7-mm embryos. These cells express high HuC/D immunoreactivity and were negative for glutamic acid decarboxylase and tyrosine hydroxylase. CR immunoreactivity diminished with development and most large cells of the mitral cell layer were CR-negative in fry. In later embryos and in alevins, CR-ir granule-like cells were observed in the olfactory bulbs. Comparisons of the terminal fields of primary olfactory fibers labeled with CR and with a more general olfactory marker in the olfactory bulbs of fry and adults revealed significant differences, with most glomeruli of the dorsomedial field receiving CR-negative olfactory fibers. These results suggest new criteria for understanding the organization of the olfactory system of the trout, and hence of teleosts. Our results also suggest that CR is involved in specific functions in the olfactory system during development.

Loss of GABAergic neurons in the subiculum and its functional implications in temporal lobe epilepsy

Clinical and experimental evidence suggest that the subiculum plays an important role in the maintenance of temporal lobe seizures. Using the pilocarpine-model of temporal lobe epilepsy (TLE), the present study examines the vulnerability of GABAergic subicular interneurons to recurrent seizures and determines its functional implications. In the subiculum of pilocarpine-treated animals, the density of glutamic acid decarboxylase (GAD) mRNA-positive cells was reduced in all layers. Our data indicate a substantial loss of parvalbumin-immunoreactive neurons in the pyramidal cell and molecular layer whereas calretinin-immunoreactive cells were predominantly reduced in the molecular layer. Though the subiculum of pilocarpine-treated rats showed an increased intensity of GAD65 immunoreactivity, the density of GAD65 containing synaptic terminals in the pyramidal cell layer was decreased indicating an increase in the GAD65 intensity of surviving synaptic terminals. We observed a decrease in evoked inhibitory post-synaptic currents that mediate dendritic inhibition as well as a decline in the frequency of miniature inhibitory post-synaptic currents (mIPSCs) that are restricted to the perisomatic region. The decrease in mIPSC frequency (-30%) matched with the reduced number of perisomatic GAD-positive terminals (-28%) suggesting a decrease of pre-synaptic GABAergic input onto pyramidal cells in epileptic animals. Though cell loss in the subiculum has not been considered as a pathogenic factor in human and experimental TLE, our data suggest that the vulnerability of subicular GABAergic interneurons causes an input-specific disturbance of the subicular inhibitory system.

The Calretinin Immunoreactive Ganglion Cell Postsynaptic to the ON-Cholinergic Amacrine Cell in the Guinea Pig

Purpose: To demonstrate the characterization calretinin-immunoreactive displaced amacrine cells in the ganglion cell layer using immunohistochemistry and electron microscopy. Methods: For immunohistochemistry, sections from guinea pig retina were incubated with mouse monoclonal antibody directed against calretinin. For double label studies, sections were incuated in mixture of mouse monoclonal anti-calretinin or rabbit polyclonal anti-calretinin with following antibodies : goat polyclonal anti-ChAT, rabbit polyclonal anti-GABA, mouse monoclonal anti-GABAA receptor α1, β2/3. Sections were analyzed using Bio-rad Radiance Plus confocal scanning microscope. Stained sections from three guinea pig were observed with transmission electron microscope. Results: Calretinin immunoreactivity was present in displaced amacrine cells and ganglion cells gaving rise to processes ramified in the inner part of the inner plexiform layer in stratum 4. The same stratum was also occupied by the dendrites of ON-cholinergic amacrine cells. Double-labeling demonstrated that dendrites and cell bodies of displaced amacrine cells colocalized with ON-cholinergic amacrine cells and dendrites of ganglion cells directly overlapped with dendrites of ON-cholinergic amacrine cells. The synaptic connectivity was identified by electron microscopy. Ganglion cell dendrites received synaptic input from ON-cholinergic amacrine cell. GABAA receptor β2/3 subunit bands cofaciculates the dendrites of displaced amacrine cell and ganglion cell that are juxtapose to the α1 subunit of GABAA receptor. Conclusions: These results indicate that ON-cholinergic amacrine cells modulate calretinin-labeled ganglion cell via GABAA receptor β2/3 in the guinea pig retina.

Calbindin-D28k and calretinin as markers of retinal neurons in the anuran amphibian Rana perezi

In the present study we have analyzed the distribution of the calcium binding proteins calbindin-D28k (CB) and calretinin (CR) immunoreactive cells in the retina of the anuran Rana perezi using poly- and mono-clonal antibodies that were proven to be specific in the amphibian brain, without cross-reactivity. Double immunohistofluorescence techniques were used to demonstrate colocalization of both proteins in the same retinal cells. In addition, retrograde tracing experiments from the optic nerve were conducted to labeled ganglion cells and these were observed in combination with CB and/or CR immunohistochemistry. Cells containing CB were identified as all cones, scattered bipolar and amacrine cells together with cells in the ganglion cell layer. The pattern of CR immunoreactivity was strikingly different. Abundant cells contained CR in the inner retinal layers including horizontal, bipolar and amacrine cells, and cells in the ganglion cell layer. By means of double immunohistochemistry it was found that only subpopulations of amacrine cells and cells in the ganglion cell layer contained both CB and CR. Tracing from the optic nerve revealed retrogradely labeled ganglion cells with different morphologies and most of them contained CB and/or CR. All these data taken together suggest that in amphibians CB and CR are distinctly and precisely distributed in retinal neurons showing, however, peculiar features not observed previously in other vertebrates.