Calcium-binding Proteins Calretinin Research Articles

Parvalbumin, calbindin, and calretinin immunostaining of 10 neural structures within the cerebellar cortex of 143 mammal species

In the current study we employed immunohistochemical techniques to investigate the localisation of three calcium-binding proteins (parvalbumin, calbindin, and calretinin) in 10 neuronal structures of the cerebellar cortex (stellate cells, basket cells, parallel fibres, climbing fibres, Purkinje cells, granule cells, Golgi type II cells, Lugaro neurons, unipolar brush neurons, and mossy fibres) in 143 species from across the mammalian radiation. Most often, parvalbumin was localised in the neural structures of the molecular and Purkinje cell layers but was absent in the granule cell layer. Calbindin was most often immunolocalised in the neural structures of the Purkinje cell layer and mossy fibres, whereas calretinin was most often immunolocalised in the climbing fibres of the molecular layer and all neural structures of the granule cell layer. Despite this general consistency, variations in the localisation of these three calcium-binding proteins were found in every lineage, and almost every species, the one exception being the western tree hyrax that showed the full suite of most often observed calcium-binding protein chemoarchitecture for the mammalian cerebellar cortex. These consistencies and variances in the calcium-binding protein chemoarchitecture of the cerebellar cortex of mammals may play significant roles in the species-specific learning and refining of motor, perceptual, and cognitive skills and capacities required to survive in the environments they inhabit.

Characterization of GABAergic marker expression in prefrontal cortex in dexamethasone induced depression/anxiety model.

The pivotal responsibility of GABAergic interneurons is inhibitory neurotransmission; in this way, their significance lies in regulating the maintenance of excitation/inhibition (E/I) balance in cortical circuits. An abundance of glucocorticoids (GCs) exposure results in a disorder of GABAergic interneurons in the prefrontal cortex (PFC); the relationship between this status and an enhanced vulnerability to neuropsychiatric ailments, like depression and anxiety, has been identified, but this connection is still poorly understood because systematic and comprehensive research is lacking. Here, we aim to investigate the impact of dexamethasone (DEX, a GC receptor agonist) on GABAergic interneurons in the PFC of eight-week-old adult male mice. A double-blind study was conducted where thirty-two mice were treated subcutaneously either saline or DEX (0.2 mg/10 ml per kg of body weight) dissolved in saline daily for 21 days. Weight measurements were taken at five-day intervals to assess the emotional changes in mice as well as the response to DEX treatment. Following the 21-day regimen of DEX injections, mice underwent examinations for depression/anxiety-like behaviours and GABAergic marker expression in PFC. In a depression/anxiety model generated by chronic DEX treatment, we found that our DEX procedure did trigger depression/anxiety-like behaviors in mice. Furthermore, DEX treatment reduced the expression levels of a GABA-synthesizing enzyme (GAD67), Reelin, calcium-binding proteins (parvalbumin and calretinin) and neuropeptides co-expressed in GABAergic neurons (somatostatin, neuropeptide Y and vasoactive intestinal peptide) in the PFC were reduced after 21 days of DEX treatment; these reductions were accompanied by decreases in brain size and cerebral cortex thickness. Our results indicate that a reduction in the number of GABAergic interneurons may result in deficiencies in cortical inhibitory neurotransmission, potentially causing an E/I imbalance in the PFC; this insight suggests a potential breakthrough strategy for the treatment of depression and anxiety.

Deletion of transcription factor Satb1 leads to disturbance in mice behavior through changes in the expression of genes-regulators of the excitive component of neurotransmission

The transcription factor Satb1 exhibits widespread expression in multiple tissues and different regions of the brain. Notable levels of expression are observed in the neocortex, the nucleus of the diagonal band, the amygdala, and the tegmental area. In the ventral midbrain, Satb1-positive neurons are only found in a small portion of the substantia nigra, the ventral tegmental area, and the retrorubral field, and none were detected in the inferior colliculus. The transcription factor Satb1 is highly expressed in interneurons labeled as SST+, CR+, and NPY+, but not VIP+ interneurons. In mice with the Satb1 mutation, incomplete eye opening and a constriction reflex were observed. Behavioral tests revealed that mice lacking Satb1 exhibited deficits in motor coordination, characterized by reduced mobility on flat bars and decreased grip strength on metal wires. In addition, these mice exhibited elevated levels of motor activity in novel environments and heightened anxiety in the light-dark test. Satb1-deficient males demonstrated proficiency in passive avoidance tests, while their female counterparts exhibited no significant difference in entrance time to the dark compartment between the reproduction and learning stages, indicating an impairment in the process of conditioned response formation. To investigate the expression patterns of genes encoding vital protein kinases and proteins associated with neurotransmission, we extracted total RNA from the cerebral cortex of adult males with an incomplete deletion of Satb1. Real-time PCR analysis was conducted, which revealed a higher expression level of the pik3ca, pik3cb, and pic3cg genes responsible for phosphoinositide-3-kinase isoforms in Satb1-deficient mice compared to wild-type mice. The expression levels of genes encoding protein kinase C (Prkce and Prkcg) as well as Ca2+/calmodulin-dependent protein kinase II (Camk2) were lower in comparison to wild-type mice. Satb1-deficient mice displayed notably higher expression levels of genes encoding NMDA receptor subunits (Grin1, Grin2a, and Grin2b), whereas the expression of Gria1 (encoding the Glua2 subunit of AMPA receptors responsible for Ca2+ receptor conductance) decreased. The influence on excitatory glutamate receptor expression can cause hyperexcitability in animals, particularly when inhibitory neurotransmission is suppressed due to decreased Gabra1 and Gad65/67 expression encoding the GABA(A) receptor and glutamate decarboxylase. In addition, the levels of expression for the Calb1, Calb2, and Pvalb genes responsible for encoding the calcium-binding proteins calbindin, calretinin, and parvalbumin decreased in comparison to the wild-type mice. Thus, a correlation was found between behavioral disturbances and expression of genes that encode proteins related to brain development and neurotransmission after deleting the Satb1 transcription factor. Satb1-deleted mice exhibited hyperexcitation and impaired motor activity due to impaired gene expression.

Shades of gray in human white matter.

Anatomists have long expressed interest in neurons of the white matter, which is by definition supposed to be free of neurons. Hypotheses regarding their biochemical signature and physiological function are mainly derived from animal models. Here, we investigated 15 whole-brain human postmortem specimens, including cognitively normal cases and those with pathologic Alzheimer's disease (AD). Quantitative and qualitative methods were used to investigate differences in neuronal size and density, and the relationship between neuronal processes and vasculature. Double staining was used to evaluate colocalization of neurochemicals. Two topographically distinct populations of neurons emerged: one appearing to arise from developmental subplate neurons and the other embedded within deep, subcortical white matter. Both populations appeared to be neurochemically heterogeneous, showing positive reactivity to acetylcholinesterase (AChE) [but not choline acetyltransferase (ChAT)], neuronal nuclei (NeuN), nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), microtubule-associated protein 2 (MAP-2), somatostatin (SOM), nonphosphorylated neurofilament protein (SMI-32), and calcium-binding proteins calbindin-D28K (CB), calretinin (CRT), and parvalbumin (PV). PV was more richly expressed in superficial as opposed to deep white matter neurons (WMNs); subplate neurons were also significantly larger than their deeper counterparts. NADPH-d, a surrogate for nitric oxide synthase, allowed for the striking morphological visualization of subcortical WMNs. NADPH-d-positive subcortical neurons tended to embrace the outer walls of microvessels, suggesting a functional role in vasodilation. The presence of AChE positivity in these neurons, but not ChAT, suggests that they are cholinoceptive but noncholinergic. WMNs were also significantly smaller in AD compared to control cases. These observations provide a landscape for future systematic investigations.

Neuronal Calcium Sensor-1 Protects Cortical Neurons from Hyperexcitation and Ca2+ Overload during Ischemia by Protecting the Population of GABAergic Neurons.

A defection of blood circulation in the brain leads to ischemia, damage, and the death of nerve cells. It is known that individual populations of GABAergic neurons are the least resistant to the damaging factors of ischemia and therefore they die first of all, which leads to impaired inhibition in neuronal networks. To date, the neuroprotective properties of a number of calcium-binding proteins (calbindin, calretinin, and parvalbumin), which are markers of GABAergic neurons, are known. Neuronal calcium sensor-1 (NCS-1) is a signaling protein that is expressed in all types of neurons and is involved in the regulation of neurotransmission. The role of NCS-1 in the protection of neurons and especially their individual populations from ischemia and hyperexcitation has not been practically studied. In this work, using the methods of fluorescence microscopy, vitality tests, immunocytochemistry, and PCR analysis, the molecular mechanisms of the protective action of NCS-1 in ischemia/reoxygenation and hyperammonemia were established. Since NCS-1 is most expressed in GABAergic neurons, the knockdown of this protein with siRNA led to the most pronounced consequences in GABAergic neurons. The knockdown of NCS-1 (NCS-1-KD) suppressed the basic expression of protective proteins without significantly reducing cell viability. However, ischemia-like conditions (oxygen-glucose deprivation, OGD) and subsequent 24-h reoxygenation led to a more massive activation of apoptosis and necrosis in neurons with NCS-1-KD, compared to control cells. The mass death of NCS-1-KD cells during OGD and hyperammonemia has been associated with the induction of a more pronounced network hyperexcitation symptom, especially in the population of GABAergic neurons, leading to a global increase in cytosolic calcium ([Ca2+]i). The symptom of hyperexcitation of neurons with NCS-1-KD correlated with a decrease in the level of expression of the calcium-binding protein-parvalbumin. This was accompanied by an increase in the expression of excitatory ionotropic glutamate receptors, N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (NMDAR and AMPAR) against the background of suppression of the expression of glutamate decarboxylase (synthesis of γ-aminobutyric acid).

The retrosplenial cortex of Carollia perspicillata, Seba's short‐tailed fruit bat

Retrosplenial cortex (RSC) is a brain region involved in critical cognitive functions including memory, planning, and spatial navigation and is commonly affected in neurodegenerative diseases. Subregions of RSC are typically described as Brodmann areas 29 and 30, which are defined by cytoarchitectural features. Using immunofluorescence, we studied the distributions of neurons immunoreactive for NeuN, latexin, and calcium binding proteins (calbindin, calretinin, and parvalbumin) in RSC of Carollia perspicillata, Seba's short-tailed fruit bat. We observed that latexin was specifically present in areas 29a and 29b but not 29c and 30. We further identified distribution patterns of calcium binding proteins that group areas 29a and 29b separately from areas 29c and 30. We conclude first that latexin is a useful marker to classify subregions of RSC and second that these subregions contain distinct patterns of neuronal immunoreactivity for calcium binding proteins. Given the long lifespan of Carollia, bat RSC may be a useful model in studying age-related neurodegeneration.

Morphological Diversity of Calretinin Interneurons Generated From Adult Mouse Olfactory Bulb Core Neural Stem Cells.

Neural stem cells (NSCs) in the olfactory bulb (OB) core can generate mature interneurons in the adult mice brain. The vast majority of these adult generated cells express the calcium-binding protein Calretinin (CalR), and they migrate towards different OB layers. However, these cells have yet to be fully characterized and hence, to achieve this we injected retroviral particles expressing GFP into the OB core of adult animals and found that the CalR+ neurons generated from NSCs mainly migrate to the granule cell layer (GCL) and glomerular layer (GL) in similar proportions. In addition, since morphology and function are closely related, we used three-dimensional imaging techniques to analyze the morphology of these adult born cells, describing new subtypes of CalR+ interneurons based on their dendritic arborizations and projections, as well as their localization in the GCL or GL. We also show that the migration and morphology of these newly generated neurons can be altered by misexpressing the transcription factor Tbr1 in the OB core. Therefore, the morphology acquired by neurons located in a specific OB layer is the result of a combination of both extrinsic (e.g., layer allocation) and intrinsic mechanisms (e.g., transcription factors). Defining the cellular processes and molecular mechanisms that govern adult neurogenesis might help better understand brain circuit formation and plasticity, as well as eventually opening the way to develop strategies for brain repair.

Development of neurochemical labeling in the intermediolateral nucleus of cats' spinal cord.

NeuN is a neuron-specific nuclear protein expressed in most mature neuronal cell types, with some exceptions. These exceptions are known mainly for the brain but not for the spinal cord or the spinal visceral networks for which only scarce information is available. One of the most defined visceral structures in the spinal cord is the sympathetic intermediolateral nucleus located within the thoracolumbar segments. We investigated the NeuN staining in the intermediolateral nucleus and compared it with the staining for two neurochemical markers of visceral neurons: nitric oxide synthase and calcium-binding protein calretinin in adult cats and in kittens aged 0, 14, and 35 days. A clear NeuN-immunonegativity was obtained for intermediolateral neurons labeled for nitric oxide synthase for both adult cats and kittens. In contrast, a matched immunopositivity for the NeuN and calretinin was obtained, showing an age-dependent degree of this colocalization, which was high in newborn kittens, decreased on postnatal 14 and 35 days and persisted at a moderate level up to adulthood. Perhaps our data displayed a heterogeneity of the intermediolateral neurons.

Localization of BDNF and Calretinin in Olfactory Epithelium and Taste Buds of Zebrafish (Danio rerio)

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and it is involved in several fundamental functions in the central and peripheral nervous systems, and in sensory organs. BDNF regulates the chemosensory systems of mammals and is consistently expressed in those organs. In zebrafish, the key role of BDNF in the biology of the hair cells of the inner ear and lateral line system has recently been demonstrated. However, only some information is available about its occurrence in the olfactory epithelium, taste buds, and cutaneous isolated chemosensory cells. Therefore, this study was undertaken to analyze the involvement of BDNF in the chemosensory organs of zebrafish during the larval and adult stages. To identify cells displaying BDNF, we compared the cellular pattern of BDNF-displaying cells with those immunoreactive for calretinin and S100 protein. Our results demonstrate the localization of BDNF in the sensory part of the olfactory epithelium, mainly in the ciliated olfactory sensory neurons in larvae and adult zebrafish. Intense immunoreaction for BDNF was also observed in the chemosensory cells of oral and cutaneous taste buds. Moreover, a subpopulation of olfactory sensory neurons and chemosensory cells of olfactory rosette and taste bud, respectively, showed marked immunopositivity for calcium-binding protein S100 and calretinin. These results demonstrate the possible role of BDNF in the development and maintenance of olfactory sensory neurons and sensory cells in the olfactory epithelium and taste organs of zebrafish during all stages of development.

This Week in The Journal

Chuangeng Zhang, Meijian Wang, Shengyin Lin, and Ruili Xie (see pages [2729–2742][1]) Auditory nerve fibers are categorized by their spontaneous spike rate and auditory threshold, and they differ in their expression of the calcium binding protein calretinin (CR). Whereas low-threshold/high-

Dual medial prefrontal cortex and hippocampus projecting neurons in the paraventricular nucleus of the thalamus.

The paraventricular nucleus (PVT) of the midline thalamus is a critical higher-order cortico-thalamo-cortical integration site that plays a critical role in various behaviors including reward seeking, cue saliency, and emotional memory. Anatomical studies have shown that PVT projects to both medial prefrontal cortex (mPFC) and hippocampus (HC). However, dual mPFC-HC projecting neurons which could serve a role in synchronizing mPFC and HC activity during PVT-dependent behaviors, have not been explored. Here we used a dual retrograde adenoassociated virus (AAV) tracing approach to characterize the location and proportion of different projection populations that send collaterals to mPFC and/or ventral hippocampus (vHC) in rats. Additionally, we examined the distribution of calcium binding proteins calretinin (CR) and calbindin (CB) with respect to these projection populations in PVT. We found that PVT contains separate populations of cells that project to mPFC, vHC, and those that innervate both regions. Interestingly, dual mPFC-HC projecting cells expressed neither CR nor CB. Topographically, CB+ and CR+ containingcells clustered around dual projecting neurons in PVT. These results are consistent with the features of dual mPFC-vHC projecting cells in the nucleus reuniens (RE) and suggestive of a functional mPFC-PVT-vHC system that may support mPFC-vHC interactions in PVT-dependent behaviors.

Differential distribution of inhibitory neuron types in subregions of claustrum and dorsal endopiriform nucleus of the short-tailed fruit bat.

Few brain regions have such wide-ranging inputs and outputs as the claustrum does, and fewer have posed equivalent challenges in defining their structural boundaries. We studied the distributions of three calcium-binding proteins-calretinin, parvalbumin, and calbindin-in the claustrum and dorsal endopiriform nucleus of the fruit bat, Carollia perspicillata. The proportionately large sizes of claustrum and dorsal endopiriform nucleus in Carollia brain afford unique access to these structures' intrinsic anatomy. Latexin immunoreactivity permits a separation of claustrum into core and shell subregions and an equivalent separation of dorsal endopiriform nucleus. Using latexin labeling, we found that the claustral shell in Carollia brain can be further subdivided into at least four distinct subregions. Calretinin and parvalbumin immunoreactivity reinforced the boundaries of the claustral core and its shell subregions with diametrically opposite distribution patterns. Calretinin, parvalbumin, and calbindin all colocalized with GAD67, indicating that these proteins label inhibitory neurons in both claustrum and dorsal endopiriform nucleus. Calretinin, however, also colocalized with latexin in a subset of neurons. Confocal microscopy revealed appositions that suggest synaptic contacts between cells labeled for each of the three calcium-binding proteins and latexin-immunoreactive somata in claustrum and dorsal endopiriform nucleus. Our results indicate significant subregional differences in the intrinsic inhibitory connectivity within and between claustrum and dorsal endopiriform nucleus. We conclude that the claustrum is structurally more complex than previously appreciated and that claustral and dorsal endopiriform nucleus subregions are differentially modulated by multiple inhibitory systems. These findings can also account for the excitability differences between claustrum and dorsal endopiriform nucleus described previously.

Identifying Types of Neurons in the Human Colonic Enteric Nervous System.

Distinguishing and characterising the different classes of neurons that make up a neural circuit has been a long-term goal for many neuroscientists. The enteric nervous system is a large but moderately simple part of the nervous system. Enteric neurons in laboratory animals have been extensively characterised morphologically, electrophysiologically, by projections and immunohistochemically. However, studies of human enteric nervous system are less advanced despite the potential availability of tissue from elective surgery (with appropriate ethics permits). Recent studies using single cell sequencing have confirmed and extended the classification of enteric neurons in mice and human, but it is not clear whether an encompassing classification has been achieved. We present preliminary data on a means to distinguish classes of myenteric neurons in specimens of human colon combining immunohistochemical, morphological, projection and size data on single cells. A method to apply multiple layers of antisera to specimens was developed, allowing up to 12 markers to be characterised in individual neurons. Applied to multi-axonal Dogiel type II neurons, this approach demonstrated that they constitute fewer than 5% of myenteric neurons, are nearly all immunoreactive for choline acetyltransferase and tachykinins. Many express the calcium-binding proteins calbindin and calretinin and they are larger than average myenteric cells. This methodology provides a complementary approach to single-cell mRNA profiling to provide a comprehensive account of the types of myenteric neurons in the human colon.

Pathways for Memory, Cognition and Emotional Context: Hippocampal, Subgenual Area 25, and Amygdalar Axons Show Unique Interactions in the Primate Thalamic Reuniens Nucleus.

The reuniens nucleus (RE) is situated at the most ventral position of the midline thalamus. In rats and mice RE is distinguished by bidirectional connections with the hippocampus and medial prefrontal cortex (mPFC) and a role in memory and cognition. In primates, many foundational questions pertaining to RE remain unresolved. We addressed these issues by investigating the composition of the rhesus monkey RE in both sexes by labeling for GABA, a marker of inhibitory neurons, and for the calcium-binding proteins parvalbumin (PV), calbindin (CB), and calretinin (CR), which label thalamic excitatory neurons that project to cortex. As in rats and mice, the macaque RE was mostly populated by CB and CR neurons, characteristic of matrix-dominant nuclei, and had bidirectional connections with hippocampus and mPFC area 25 (A25). Unlike rodents, we found GABAergic neurons in the monkey RE and a sparser but consistent population of core-associated thalamocortical PV neurons. RE had stronger connections with the basal amygdalar complex than in rats or mice. Amygdalar terminations were enriched with mitochondria and frequently formed successive synapses with the same postsynaptic structures, suggesting an active and robust pathway to RE. Significantly, hippocampal pathways formed multisynaptic complexes that uniquely involved excitatory projection neurons and dendrites of local inhibitory neurons in RE, extending this synaptic principle beyond sensory to high-order thalamic nuclei. Convergent pathways from hippocampus, A25, and amygdala in RE position it to flexibly coordinate activity for memory, cognition, and emotional context, which are disrupted in several psychiatric and neurologic diseases in humans.SIGNIFICANCE STATEMENT The primate RE is a central node for memory and cognition through connections with the hippocampus and mPFC. As in rats or mice, the primate RE is a matrix-dominant thalamic nucleus, suggesting signal traffic to the upper cortical layers. Unlike rats or mice, the primate RE contains inhibitory neurons, synaptic specializations with the hippocampal pathway, and robust connections with the amygdala, suggesting unique adaptations. Convergence of hippocampal, mPFC, and amygdalar pathways in RE may help unravel a circuit basis for binding diverse signals for conscious flexible behaviors and the synthesis of memory with affective significance in primates, whereas disruption of distinct circuit nodes may occur in psychiatric disorders in humans.

Immuno-histological detection of resistant columnar units and vulnerable networks in the rat retina after asphyxia-induced transient cardiac arrest.

Stroke-related loss of vision is one of the residual impairments, restricting the quality of life. However, studies of the ocular manifestations of asphyxia cardiac arrest/resuscitation (ACA/R) have reported very heterogeneous results. We aimed to evaluate the ACA/R-induced degeneration pattern of the different retinal cell populations in rats using different immuno-histological stainings. The staining pattern of toluidine blue and the ganglion cell markers β-III-tubulin and NeuN; the calcium-binding protein parvalbumin, indicating ganglion, amacrine, and horizontal cells; calretinin D28k, indicating ganglion and amacrine cells; calbindin, indicating horizontal cells; Chx 10, indicating cone bipolar cells; PKCα, indicating ON-type rod bipolar cells; arrestin, indicating cones; and rhodopsin, a marker of rods, as well as the glial cell markers GFAP (indicating astroglia and Müller cells) and IBA1 (indicating microglia), were evaluated after survival times of 7 and 21 days in an ACA/R rat model. Moreover, quantitative morphological analysis of the optic nerve was performed. The ACA/R specimens were compared with those from sham-operated and completely naïve rats. ACA/R-induced effects were: (i) a significant reduction of retinal thickness after long-term survival; (ii) ganglion cell degeneration, including their fiber network in the inner plexiform layer; (iii) degeneration of amacrine and cone bipolar cells; (iv) degeneration of cone photoreceptors; (v) enhanced resistance to ACA/R by rod photoreceptors, ON-type rod bipolar and horizontal cells, possibly caused by the strong upregulation of the calcium-binding proteins calretinin, parvalbumin, and calbindin, counteracting the detrimental calcium overload; (vi) significant activation of Müller cells as further element of retinal anti-stress self-defense mechanisms; and (vii) morphological alterations of the optic nerve in form of deformed fibers. Regardless of the many defects, the surviving neuronal structures seemed to be able to maintain retinal functionality, which can be additionally improved by regenerative processes true to the "use it or lose it" dogma.

The density of calretinin striatal interneurons is decreased in 6-OHDA-lesioned mice.

Interneurons play a significant role in the functional organization of the striatum and some of them display marked plastic changes in dopamine-depleted conditions. Here, we applied immunohistochemistry on brain sections from 6-hydroxydopamine (6-OHDA) mouse model of Parkinson's disease and sham animals to characterize the regional distribution and the morphological and neurochemical changes of striatal interneurons expressing the calcium-binding protein calretinin (CR). Two morphological subtypes of calretinin-immunostained (CR +) interneurons referred, respectively, as small- and medium-sized CR + interneurons were detected in 6-OHDA- and sham-lesioned animals. The small cells (9-12µm) prevail in the anterior and dorsal striatal regions; they stain intensely for CR and display a single slightly varicose and moderately arborized process. The medium-sized CR + interneurons (15-20µm) are more numerous than the small CR + cells and rather uniformly distributed within the striatum; they stain weakly for CR and display 2-3 long, slightly varicose and poorly branched dendrites. The density of medium CR + interneurons is significantly decreased in the dopamine-depleted striatum (158 ± 15neurons/mm3), when compared to sham animals (370 ± 41neurons/mm3), whereas that of the small-sized CR + interneurons is unchanged (174 ± 46neurons/mm3 in 6-OHDA-lesioned striatum and 164 ± 22neurons/mm3 in sham-lesioned striatum). The nucleus accumbens is populated only by medium-sized CR + interneurons, which are distributed equally among the core and shell compartments and whose density is unaltered after dopamine denervation. Our results provide the first evidence that the medium-sized striatal interneurons expressing low level of CR are specifically targeted by dopamine denervation, while the small and intensely immunoreactive CR + cells remain unaffected. These findings suggest that high expression of the calcium-binding protein CR might protect striatal interneurons against an increase in intracellular calcium level that is believed to arise from altered glutamate corticostriatal transmission in Parkinson's disease.

Characterization of basal forebrain glutamate neurons suggests a role in control of arousal and avoidance behavior.

The basal forebrain (BF) is involved in arousal, attention, and reward processing but the role of individual BF neuronal subtypes is still being uncovered. Glutamatergic neurons are the least well-understood of the three main BF neurotransmitter phenotypes. Here we analyzed the distribution, size, calcium-binding protein content and projections of the major group of BF glutamatergic neurons expressing the vesicular glutamate transporter subtype 2 (vGluT2) and tested the functional effect of activating them. Mice expressing Cre recombinase under the control of the vGluT2 promoter were crossed with a reporter strain expressing the red fluorescent protein, tdTomato, to generate vGluT2-cre-tdTomato mice. Immunohistochemical staining for choline acetyltransferase and a cross with mice expressing green fluorescent protein selectively in GABAergic neurons confirmed that cholinergic, GABAergic and vGluT2+ neurons represent distinct BF subpopulations. Subsets of BF vGluT2+ neurons expressed the calcium-binding proteins calbindin or calretinin, suggesting that multiple subtypes of BF vGluT2+ neurons exist. Anterograde tracing using adeno-associated viral vectors expressing channelrhodopsin2-enhanced yellow fluorescent fusion proteins revealed major projections of BF vGluT2+ neurons to neighboring BF cholinergic and parvalbumin neurons, as well as to extra-BF areas involved in the control of arousal or aversive/rewarding behavior such as the lateral habenula and ventral tegmental area. Optogenetic activation of BF vGluT2+ neurons elicited a striking avoidance of the area where stimulation was given, whereas stimulation of BF parvalbumin or cholinergic neurons did not. Together with previous optogenetic findings suggesting an arousal-promoting role, our findings suggest that BF vGluT2 neurons play a dual role in promoting wakefulness and avoidance behavior.

Neonatal exposure to monosodium glutamate results in impaired auditory brainstem structure and function

Excitotoxic injury during the neonatal period has been shown to result in neurodegenerative changes in several different brain regions. Exposure to monosodium glutamate (MSG) during the first two postnatal weeks results in glutamate neurotoxicity in the cochlea and has been shown to result in damage to cochlear hair cells and fewer neurons in the spiral ganglion. Further, we have shown that such exposure results in fewer neurons in the cochlear nucleus and superior olivary complex and abnormal expression of the calcium binding proteins calbindin and calretinin. Based on these findings, we hypothesized that neonatal MSG exposure would result in loss of neurons at more rostral levels in the auditory brainstem, and this exposure would result in abnormal brainstem auditory evoked potentials. We identified a significantly lower density of neurons in the spiral ganglion, heterogenous loss of neurons in the globular bushy cell-trapezoid body circuit, and fewer neurons in the nuclei of the lateral lemniscus and central nucleus of the inferior colliculus. The most severe loss of neurons was found in the inferior colliculus. Click-evoked auditory brainstem responses revealed significantly higher thresholds and longer latency responses, but these did not deteriorate with age. These results, together with our previous findings, indicate that neonatal exposure to MSG results in fewer neurons throughout the entire auditory brainstem and results in abnormal auditory brainstem responses.

Neonatal proinflammatory challenge evokes a microglial response and affects the ratio between subtypes of GABAergic interneurons in the hippocampus of juvenile rats: sex-dependent and sex-independent effects.

Neonatal proinflammatory challenge (NPC) may contribute to the development of psychiatric disorders in adults. A double exposure of neonatal rats to lipopolysaccharide, a component of cellular wall of gram-negative bacteria, on postnatal days 3 and 5 provokes the development of depressive- and anxiety-like behaviors. NPC impairs neuroplasticity and cognition in adult animals, significant modifications of neuroplasticity being evident even in adolescence. We studied effects of NPC on microglia and GABAergic neuronal population of the dorsal hippocampus in juvenile male and female rats using immunofluorescent histochemistry. The expression of glutamic acid decarboxylase-67 (GAD67) and calcium-binding proteins calretinin, calbindin, and parvalbumin were used as quantitative markers of GABAergic interneurons and their specific subpopulations, respectively. NPC induced changes of microglial morphology indicating inflammatory activation mostly expressed in CA3 field; the effect was similar in males and females. The number of GAD67 expressing neurons was similar in the dorsal hippocampus of females and males independently on the NPC. The portion of calbindin-immunoreactive GAD67-positive neurons significantly increased while the portion of calretinin-immunoreactive GAD67-positive neurons significantly decreased in the CA1 field of rats exposed to NPC independently on their sex. NPC did not affect the parvalbumin-positive subpopulation of GABAergic neurons in the hippocampus of rats of either sex. These data suggest that NPC-induced modification of GABAergic neuronal population composition under the proinflammatory conditions is involved in the maintenance of excitation/inhibition homeostatic balance in the hippocampus.

Calretinin immunoreactivity in the inferior lobe of the hypothalamus and associated nuclei of the firemouth cichlid, Thorichthys meeki

The distribution of the calcium-binding protein calretinin (CR) was examined by an immunohistochemical method using specific antibodies. CR is involved in the visual system, and the inferior lobe of the hypothalamus represents a multisensory integration center in cichlids. The focus of the present study was to analyze the distribution of CR immunoreactivity in a cichlid fish, the firemouth cichlid, Thorichthys meeki, for the hypothalamic inferior lobe and for the torus lateralis, nucleus glomerulosus, nucleus posterior tuberis, and corpus mamillare as associated nuclei of the hypothalamus. CR-immunoreactive (CR-ir) cell bodies were visualized in the lateral and medial part of the diffuse nucleus of the inferior lobe, ventral portion of the central nucleus of the inferior lobe, torus lateralis, nucleus glomerulosus, and nucleus posterior tuberis. CR-ir fibers could be detected in the dorsal portion of the central nucleus of the inferior lobe and corpus mamillare. The strongest labeling of CR-ir neuropil was observed in the lateral part of the diffuse nucleus of the inferior lobe, outer zone of the periventricular nucleus of the inferior lobe, torus lateralis, nucleus glomerulosus, and nucleus posterior tuberis. CR is abundantly present in the inferior lobe of the hypothalamus and associated nuclei. The role of CR in highly active processes in the inferior lobe of cichlids will be discussed.