Robust expression of the TRPC1 channel associated with photoreceptor loss in the rat retina.

Baseline intracellular calcium levels are significantly higher in neuronal and glial cells of rat retinas with retinitis pigmentosa (RP). Although this situation could initiate multiple detrimental pathways that lead to cell death, we considered the possibility of TRPC1 being involved in maintaining calcium homeostasis in the retina by acting as a component of store-operated calcium (SOC) channels with special relevance during photoreceptor degeneration. In this study, we examined by Western blot the expression of TRPC1 in healthy control rat retinas (Sprague-Dawley, SD) and retinas with RP (P23H-1 rats). We also analyzed its specific cellular distribution by immunofluorescence to recognize changes during neurodegeneration and to determine whether its presence is consistent with high basal calcium levels and cellular survival in degenerating retinas. We found that TRPC1 immunostaining was widely distributed across the retina in both rat strains, SD and P23H, and its expression levels significantly increased in the retinas with advanced degeneration compared to the age-control SD rats. In the outer retina, TRPC1 immunoreactivity was distributed in pigment epithelium cells, the photoreceptor inner segments of older animals, and the outer plexiform layer. In the inner retina, TRPC1 labeling was detected in horizontal cells, specific somata of bipolar and amacrine cells, and cellular processes in all the strata of the inner plexiform layer. Somata and processes were also highly immunoreactive in the ganglion cell layer and astrocytes in the nerve fiber layer in all animals. In the P23H rat retinas, the TRPC1 distribution pattern changed according to advancing photoreceptor degeneration and the gliosis reaction, with TRPC1 immunoreactive Müller cells mainly in advanced stages of disease. The cellular TRPC1 immunoreactivity found in this work suggests different mechanisms of activation of these channels depending on the cell type. Furthermore, the results support the idea that photoreceptor loss due to RP is associated with robust TRPC1 protein expression in the rat inner retina and raise the possibility of TRPC1 channels contributing to maintain high basal calcium levels during neurodegeneration and/or maintenance processes of the inner retina.

Pre- and postsynaptic alterations in the visual cortex of the P23H-1 retinal degeneration rat model.

P23H rats express a variant of rhodopsin with a mutation that leads to loss of visual function with similar properties as human autosomal dominant retinitis pigmentosa (RP). The advances made in different therapeutic strategies to recover visual system functionality reveal the need to know whether progressive retina degeneration affects the visual cortex structure. Here we are interested in detecting cortical alterations in young rats with moderate retinal degeneration, and in adulthood when degeneration is severer. For this purpose, we studied the synaptic architecture of the primary visual cortex (V1) by analyzing a series of pre- and postsynaptic elements related to excitatory glutamatergic transmission. Visual cortices from control Sprague Dawley (SD) and P23H rats at postnatal days 30 (P30) and P230 were used to evaluate the distribution of vesicular glutamate transporters VGLUT1 and VGLUT2 by immunofluorescence, and to analyze the expression of postsynaptic density protein-95 (PSD-95) by Western blot. The amount and dendritic spine distribution along the apical shafts of the layer V pyramidal neurons, stained by the Golgi-Cox method, were also studied. We observed that at P30, RP does not significantly affect any of the studied markers and structures, which suggests in young P23H rats that visual cortex connectivity seems preserved. However, in adult rats, although VGLUT1 immunoreactivity and PSD-95 expression were similar between both groups, a narrower and stronger VGLUT2-immunoreactive band in layer IV was observed in the P23H rats. Furthermore, RP significantly decreased the density of dendritic spines and altered their distribution along the apical shafts of pyramidal neurons, which remained in a more immature state compared to the P230 SD rats. Our results indicate that the most notable changes in the visual cortex structure take place after a prolonged retinal degeneration period that affected the presynaptic thalamocortical VGLUT2-immunoreactive terminals and postsynaptic dendritic spines from layer V pyramidal cells. Although plasticity is more limited at these ages, future studies will determine how reversible these changes are and to what extent they can affect the visual system's functionality.

TRPC1 Channels Are Expressed in Pyramidal Neurons and in a Subset of Somatostatin Interneurons in the Rat Neocortex.

Disturbances in calcium homeostasis due to canonical transient receptor potential (TRPC) and/or store-operated calcium (SOC) channels can play a key role in a large number of brain disorders. TRPC channels are plasma membrane cation channels included in the transient receptor potential (TRP) superfamily. The most widely distributed member of the TRPC subfamily in the brain is TRPC1, which is frequently linked to group I metabotropic glutamate receptors (mGluRs) and to the components of SOC channels. Proposing TRPC/SOC channels as a therapeutic target in neurological diseases previously requires a detailed knowledge of the distribution of such molecules in the brain. The aim of our study was to analyze the neuroanatomical distribution of TRPC1 in the rat neocortex. By double- and triple-labeling and confocal microscopy, we tested the presence of TRPC1 by using a series of specific neurochemical markers. TRPC1 was abundant in SMI 32-positive pyramidal neurons, and in some glutamic acid decarboxylase 67 (GAD67) interneurons, but was lacking in glial fibrillary acidic protein (GFAP)-positive glial cells. In neurons it colocalized with postsynaptic marker MAP2 in cell bodies and apical dendritic trunks and it was virtually absent in synaptophysin-immunoreactive terminals. By using a panel of antibodies to classify interneurons, we identified the GABAergic interneurons that contained TRPC1. TRPC1 was lacking in basket and chandelier parvalbumin (PVALB) cells, and a very low percentage of calretinin (CALR) or calbindin (CALB) interneurons expressed TRPC1. Moreover, 63% of somatostatin (SST) expressing-cells and 37% of reelin-positive cells expressed TRPC1. All the SST/TRPC1 double-labeled cells, many of which were presumptive Martinotti cells (MC), were positive for reelin. The presence of TRPC1 in the somata and apical dendritic trunks of neocortical pyramidal cells suggests a role for this channel in sensory processing and synaptic plasticity. Conversely in SST/reelin interneurons, TRPC1 could modulate GABAergic transmission, which is responsible for shaping the coordinated activity of the pyramidal cells in the cortical network. In future studies, it would be relevant to investigate whether TRPC1 could be involved in the expression or processing of reelin in SST inhibitory interneurons.

TRPC1 and metabotropic glutamate receptor expression in rat auditory midbrain neurons.

Canonical transient receptor potential (TRPC) channels are plasma membrane cation channels included in the TRP superfamily. TRPC1 is expressed widely in the central nervous system and is linked to group I metabotropic glutamate receptors (mGluRs). In the auditory brainstem, TRPC1 expression has never been described, although group I mGluRs are present. In the central nucleus of the inferior colliculus (CIC), activation of group I mGluRs induces an extracellular Ca(2+) influx after store depletion. Therefore, this study examines whether TRPC1 is expressed in this region to establish a correlation with mGluRs. By quantitative reverse transcription-polymerase chain reaction and Western blotting, this study assesses the presence of TRPC1 along with both group I mGluR subtypes mGluR1 and mGluR5 in the rat inferior colliculus (IC). All these molecules present a robust expression in the IC. By confocal double immunofluorescence, this study also demonstrates that TRPC1 colocalizes with parvalbumin, a CIC neuronal marker, in many cells. Conversely, TRPC1 was lacking in glial fibrillary acidic protein-positive glial cells. All the glutamate acid decarboxylase 67 (GAD67)-immunoreactive neurons and many GAD67-negative neurons were positive to TRPC1, which indicates the presence of TRPC1 in γ-aminobutyric acid (GABA)-ergic and non-GABAeregic neurons. With regard to subcellular distribution, TRPC1 was absent in synaptophysin-immunoreactive axonic terminals but colocalized with postsynaptic marker microtubule-associated protein 2 in cell bodies and dendrites. TRPC1 totally overlapped group I mGluRs, which supports the involvement of TRPC1 in the mGluR pathway and, likely, in auditory signal processing at the midbrain level. .

Relationship between rat retinal degeneration and potassium channel KCNQ5 expression.

KCNQ5/Kv7.5 is a low-threshold non-inactivating voltage-gated potassium channel preferentially targeted to excitatory endings in brain neurons. The M-type current is mediated by KCNQ5 channel subunits in monkey retinal pigment epithelium cells and in brain neurons. This study was undertaken to analyze KCNQ5 expression and the interaction signals of KCNQ5 with other proteins in normal rat retina and during photoreceptor degeneration. The KCNQ5 expression pattern was studied by immunocytochemistry and Western blot in normal rat retinas (Sprague-Dawley, SD) and P23H-1 rats as a retinitis pigmentosa model. The physical interactions of KCNQ5 with calmodulin (CaM), vesicular glutamate transporter 1 (VGluT1) and glial fibrillary acidic protein (GFAP) were analyzed by in situ proximity ligation assays and were supported by calcium recording. KCNQ5 expression was found in the plexiform layers, ganglion cell layer and basal membrane of the retinal pigment epithelium. The physical interactions among KCNQ5 and CaM, VGluT1 and GFAP changed with age and during retinal degeneration. The maximal level of KCNQ5/CaM interaction was found when photoreceptors had almost completely disappeared; the KCNQ5/VGluT1 interaction signal decreased and the KCNQ5/GFAP interaction increased in the inner retina, while degeneration progressed. The basal calcium levels in the astrocytes and neurons of P23H-1 were higher than in the control SD retinas. This study demonstrates that KCNQ5 is present in the rat retina where its activity may be moderated by CaM. Retinal degeneration progression in P23H-1 rats can be followed by an interaction between KCNQ5 with CaM in an in situ system. The relationship between KCNQ5 and VGluT1 or GFAP needs to be more cautiously interpreted.

Hearing impairment in the P23H-1 retinal degeneration rat model.

The transgenic P23H line 1 (P23H-1) rat expresses a variant of rhodopsin with a mutation that leads to loss of visual function. This rat strain is an experimental model usually employed to study photoreceptor degeneration. Although the mutated protein should not interfere with other sensory functions, observing severe loss of auditory reflexes in response to natural sounds led us to study auditory brain response (ABR) recording. Animals were separated into different hearing levels following the response to natural stimuli (hand clapping and kissing sounds). Of all the analyzed animals, 25.9% presented auditory loss before 50 days of age (P50) and 45% were totally deaf by P200. ABR recordings showed that all the rats had a higher hearing threshold than the control Sprague-Dawley (SD) rats, which was also higher than any other rat strains. The integrity of the central and peripheral auditory pathway was analyzed by histology and immunocytochemistry. In the cochlear nucleus (CN), statistical differences were found between SD and P23H-1 rats in VGluT1 distribution, but none were found when labeling all the CN synapses with anti-Syntaxin. This finding suggests anatomical and/or molecular abnormalities in the auditory downstream pathway. The inner ear of the hypoacusic P23H-1 rats showed several anatomical defects, including loss and disruption of hair cells and spiral ganglion neurons. All these results can explain, at least in part, how hearing impairment can occur in a high percentage of P23H-1 rats. P23H-1 rats may be considered an experimental model with visual and auditory dysfunctions in future research.

Expression of calcium-binding proteins in layer 1 reelin-immunoreactive cells during rat and mouse neocortical development.

Cajal-Retzius cells in layer 1 of the developing cerebral cortex and their product of secretion, reelin, an extracellular matrix protein, play a crucial role in establishing the correct lamination pattern in this tissue. As many studies into reelin signaling routes and pathological alterations are conducted in murine models, we used double-labeling and confocal microscopy to compare the distribution of the cell-specific markers, calretinin and calbindin, in reelin-immunoreactive cells during postnatal rat and mouse neocortical development. In the rat, neither calretinin nor calbindin colocalized with reelin in Cajal-Retzius cells at P0-P2. From P5 to P14, the colocalization of reelin and calretinin was commonly found in presumptive rat subpial piriform cells. These cells progressively lacked calretinin expression and persisted into adulthood as part of the pool of layer 1 reelin-positive interneurons. Conversely, in the mouse, reelin-immunoreactive Cajal-Retzius cells colocalized with calretinin and/or calbindin. Subpial piriform cells containing reelin and calretinin were identified at P5-P7, but lacked calretinin expression at P14. In adult mice, as in the rat, reelin-immunoreactive cells did not colocalize with calcium-binding proteins. Our results reveal a complex neurochemical profile of layer 1 cells in the rat neocortex, which makes using a single calcium-binding protein as a marker of rodent reelin-immunoreactive cells difficult.

Signalling routes and developmental regulation of group I metabotropic glutamate receptors in rat auditory midbrain neurons.

Group I metabotropic glutamate receptors (mGluRs) are linked to intracellular Ca(2+) signalling and play important roles related to synaptic plasticity and development. In neurons from the central nucleus of the inferior colliculus (CIC), the activation of these receptors evokes large [Ca(2+) ](i) responses. By using optical imaging of the fluorescent Ca(2+) -sensitive dye Fura-2, we have explored which [Ca(2+) ](i) routes are triggered by group I mGluR activation in young CIC neurons and whether mGluR-induced [Ca(2+) ](i) responses are regulated during postnatal development. In addition, real-time quantitative RT-PCR was used to study the developmental expression of both group I mGluR subtypes, mGluR1 and mGluR5. Application of DHPG, a specific agonist of group I mGluRs, was used on CIC slices from young rats to elicit [Ca(2+) ](i) responses. A majority of responses consisted of an initial thapsigargin-sensitive Ca(2+) peak, related to store depletion, followed by a plateau phase, sensitive to the store-operated Ca(2+) entry blocker 2-APB. During postnatal development, from P6 to P17, DHPG-induced [Ca(2+) ](i) responses changed. The largest Ca(2+) responses were reached at P6, whereas lower peak and plateau responses were found after hearing onset, at P13-P14 and P17. qRT-PCR analysis also revealed important differences in the expression of both mGluR1 and mGluR5 subtypes during development, with the highest levels of both subtypes at P7 and a developmental decrease of both transcripts. Our results suggest both intra- and extracellular routes for [Ca(2+) ](i) increases linked to group I mGluRs in CIC neurons and a regulation of group I mGluR activity and expression during auditory development.

Differences in glutamate-mediated calcium responses in the ventral cochlear nucleus and inferior colliculus of the developing rat.

Using optical recordings of neuronal [Ca(2+)]i in brain slices from young rats (P9-P11), we report distinct regulation of Ca(2+) signalling mediated by the activity of glutamate receptors in the ventral cochlear nucleus (VCN) and the central nucleus of the inferior colliculus (CIC) in the midbrain. [Ca(2+)]i increases were recorded after bath-stimulation of slices with glutamate agonists of both ionotropic (AMPA/kainate or NMDA) and group I metabotropic glutamate receptors (mGluRs). NMDA-induced [Ca(2+)]i responses were similar in both auditory nuclei. Kainate-induced [Ca(2+)]i increases recorded in the VCN were over two-fold larger than those in the CIC. Blockade of kainate-induced [Ca(2+)]i responses in VCN neurons with 1-naphtylacetyl spermine (NAS) demonstrated that Ca(2+)-permeable AMPA receptors predominated in the VCN. In contrast, abundant Ca(2+)-impermeable AMPA receptors were found in the CIC. Both mGluR1 and mGluR5 subtypes of group I mGluRs were present in the CIC and the VCN. However, Group I mGluR [Ca(2+)]i responses elicited by DHPG were two fold higher in CIC than in VCN neurons. Therefore, our findings suggest that Ca(2+) signalling in auditory neurons may be differentially regulated at different levels of the auditory pathway through preferential activation of different classes of glutamate receptors, which may have implications for hierarchical auditory signal processing and plasticity, at least during the early developmental stages of hearing.

Development of glutamate receptors in auditory neurons from long-term organotypic cultures of the embryonic chick hindbrain.

We used long-range organotypic cultures of auditory nuclei in the chick hindbrain to test the development of glutamate receptor activity in auditory neurons growing in a tissue environment that includes early deprivation of peripheral glutamatergic input, subsequent to removal of the otocyst. Cultures started at embryonic day (E)5, and lasted from 6 h to 15 days. Neuronal migration, clustering and axonal extension from the nucleus magnocellularis (NM) to the nucleus laminaris (NL) partially resembled events in vivo. However, the distinctive laminar organization of the NL was not observed. Glutamate receptor (GluR) activity was tested with optical recordings of intracellular Ca2+ in the NM. alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)/kainate receptors had Ca2+ responses with a time course similar to that in control slices. Peak amplitude, however, was significantly lower. N-methyl-D-aspartate (NMDA)-mediated Ca2+ responses were higher in 2-day cultures (E5 + 2d) than in E7 explant controls, returning later to control values. Metabotropic GluRs did not elicit Ca2+ responses at standard agonist doses. Blocking NMDA or AMPA/kainate receptors with specific antagonists for 10 days in culture did not limit neuronal survival. Blocking metabotropic GluRs resulted in complete neuronal loss. Thus, ionotropic GluRs are not required for NM neuronal survival. However, their activity during development is affected when neurons grow in an in vitro environment that includes prevention of arrival of peripheral glutamatergic input.

Expression and developmental regulation of the K+-Cl- cotransporter KCC2 in the cochlear nucleus.

KCC2 is a neuron-specific Cl- transporter whose role in adult central neurons is to maintain low intracellular Cl- concentrations and, therefore, generate an inward-directed electrochemical gradient for Cl- needed for the hyperpolarizing responses to the inhibitory amino acids GABA and glycine. We report that the KCC2 protein is intensely expressed in CN neurons and preferentially associated with plasma membrane domains, consistent with GABA and glycinergic-mediated inhibition in this auditory nucleus. Postnatal KCC2 expression and distribution patterns are similar in developing and adult CN neurons and do not match the time course of GABergic or glycinergic synaptogenesis. Therefore, in the CN, neither KCC2 protein upregulation nor progressive integration in the plasma membrane seem to be involved in KCC2 developmental regulation. Considering that GABA and glycine are depolarizing during early postnatal development, it is conceivable that KCC2 is in place but inactive during early postnatal development in the CN and becomes active as inhibitory synaptogenesis proceeds. This notion is supported by the finding that the phosphorylation state of KCC2 differs from developing to adult CN, with the phosphorylated form predominating in the latter.