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The basolateral amygdala (BLA) and ventral hippocampal CA1 (vCA1) are cellularly and functionally diverse along their anterior–posterior and superficial-deep axes. Here, we find that anterior BLA (aBLA) and posterior BLA (pBLA) innervate deep-layer calbindin1-negative (Calb1−) and superficial-layer calbindin1-positive neurons (Calb1+) in vCA1, respectively. Photostimulation of pBLA–vCA1 inputs has an anxiolytic effect in mice, promoting approach behaviours during conflict exploratory tasks. By contrast, stimulating aBLA–vCA1 inputs induces anxiety-like behaviour resulting in fewer approaches. During conflict stages of the elevated plus maze task vCA1 Calb1+ neurons are preferentially activated at the open-to-closed arm transition, and photostimulation of vCA1 Calb1+ neurons at decision-making zones promotes approach with fewer retreats. In the APP/PS1 mouse model of Alzheimer’s disease, which shows anxiety-like behaviour, photostimulating the pBLA–vCA1 Calb1+ circuit ameliorates the anxiety in a Calb1-dependent manner. These findings suggest the pBLA–vCA1 Calb1+ circuit from heterogeneous BLA–vCA1 connections drives approach behaviour to reduce anxiety-like behaviour. Projections from the anterior and posterior basolateral amygdala (pBLA) to the ventral hippocampus CA1 (vCA1) are heterogenous. Here the authors show that activating the pathway from pBLA to vCA1 calbindin 1 positive neurons has an anxiolytic effect in approach-avoidance tasks in mice.

Significance Ca ²⁺ ions serve as a key cellular signal and are tightly controlled. One mechanism to limit free Ca ²⁺ ions is buffering by Ca ²⁺-binding proteins, which are strongly expressed in sensory hair cells of the ear. Here we studied how genetic disruption of the Ca ²⁺-binding proteins parvalbumin-α, calbindin-D28k, and calretinin affects exocytosis and sound encoding at the synapses of mouse inner hair cells (IHCs) and spiral ganglion neurons (SGNs). Mutant IHCs showed increased exocytosis, but the sound-evoked spiking activity in SGNs was unaltered. Together with mathematical modeling, this finding indicates that a large fraction of exocytosis in mutant IHCs occurred outside synapses. We conclude that Ca ²⁺-binding proteins shape presynaptic Ca ²⁺ signals to restrict exocytosis to active zones, thus enabling metabolically efficient sound encoding. EF-hand Ca ²⁺-binding proteins are thought to shape the spatiotemporal properties of cellular Ca ²⁺ signaling and are prominently expressed in sensory hair cells in the ear. Here, we combined genetic disruption of parvalbumin-α, calbindin-D28k, and calretinin in mice with patch-clamp recording, in vivo physiology, and mathematical modeling to study their role in Ca ²⁺ signaling, exocytosis, and sound encoding at the synapses of inner hair cells (IHCs). IHCs lacking all three proteins showed excessive exocytosis during prolonged depolarizations, despite enhanced Ca ²⁺-dependent inactivation of their Ca ²⁺ current. Exocytosis of readily releasable vesicles remained unchanged, in accordance with the estimated tight spatial coupling of Ca ²⁺ channels and release sites (effective “coupling distance” of 17 nm). Substitution experiments with synthetic Ca ²⁺ chelators indicated the presence of endogenous Ca ²⁺ buffers equivalent to 1 mM synthetic Ca ²⁺-binding sites, approximately half of them with kinetics as fast as 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Synaptic sound encoding was largely unaltered, suggesting that excess exocytosis occurs extrasynaptically. We conclude that EF-hand Ca ²⁺ buffers regulate presynaptic IHC function for metabolically efficient sound coding.

Rabies virus (RABV) infection leads to a fatal neurological outcome in humans and animals and is associated with major alterations in cellular gene expression. In this study, we describe the effects of RABV infection on the mRNA expression levels of two genes, encoding the Ca2+-binding proteins (Ca-BPs) calbindin D-28K (Calb1) and calretinin (Calb2), in the brains of BALB/c mice. Sixty 4-week-old mice were divided into two test groups and one control group. Mice were inoculated intramuscularly with either a street rabies virus (SRV) strain or a challenge virus standard (CVS-11) strain and sacrificed at 3-day intervals up to day 18 postinfection. A direct fluorescent antibody test (DFAT) was used to verify the presence of RABV antigen in brain tissues, and real-time quantitative PCR (RT-PCR) was used to assess gene expression. Infection with both RABV strains resulted in significant (p < 0.05) increases in Calb1 and Calb2 expression in the test animals when compared with the controls at various time points in the study. Correlation analysis indicated very weak insignificant (p > 0.05) negative and positive relationships, respectively, between Calb1 expression (r = -0.04) and Calb2 expression (r = 0.08) with viral load (CVS-11 strain). Insignificant (p > 0.05) relationships were also observed Calb1 expression (r = -0.28) and Calb2 expression (r = 0.06) and viral load for the SRV strain.The observed alterations in Calb1 and Calb2 expression in this study indicate possible impairments in neuronal Ca2+ buffering and Ca2+ homeostasis as a result of RABV infection and, consequently, possible involvement of calbindin-D28K and calretinin in the neuropathogenesis of rabies.

The retinoic acid derivative fenretinide (FR) is capable of transdifferentiating cultured retinal pigment epithelial (RPE) cells towards a neuronal-like phenotype, but the underlying mechanisms are not understood. To identify genes involved in this process we performed a microarray analysis of RPE cells pre- and post-FR treatment, and observed a marked down-regulation of AnnexinA8 (AnxA8) in transdifferentiated cells. To determine whether AnxA8 plays a role in maintaining RPE cell phenotype we directly manipulated AnxA8 expression in cultured and primary RPE cells using siRNA-mediated gene suppression, and over-expression of AnxA8-GFP in conjunction with exposure to FR. Treatment of RPE cells with AnxA8 siRNA recapitulated exposure to FR, with cell cycle arrest, neuronal transdifferentiation, and concomitant up-regulation of the neuronal markers calretinin and calbindin, as assessed by real-time PCR and immunofluorescence. In contrast, AnxA8 transient over-expression in ARPE-19 cells prevented FR-induced differentiation. Ectopic expression of AnxA8 in AnxA8-depleted cells led to decreased neuronal marker staining, and normal cell growth as judged by phosphohistone H3 staining, cell counting and cleaved caspase-3 levels. These data show that down-regulation of AnxA8 is both necessary and sufficient for neuronal transdifferentiation of RPE cells and reveal an essential role for AnxA8 as a key regulator of RPE phenotype.

The incidence rates of light-induced retinopathies have increased significantly in the last decades because of continuous exposure to light from different electronic devices. Recent studies showed that exposure to blue light had been related to the pathogenesis of light-induced retinopathies. However, the pathophysiological mechanisms underlying changes induced by light exposure are not fully known yet. In the present study, the effects of exposure to light at different wavelengths with emission peaks in the blue light range (400–500 nm) on the localization of Calretinin-N18 (CaR-N18) and Calbindin-D28K (CaB-D28K) in adult zebrafish retina are studied using double immunofluorescence with confocal laser microscopy. CaB-D28K and CaR-N18 are two homologous cytosolic calcium-binding proteins (CaBPs) implicated in essential process regulation in central and peripheral nervous systems. CaB-D28K and CaR-N18 distributions are investigated to elucidate their potential role in maintaining retinal homeostasis under distinct light conditions and darkness. The results showed that light influences CaB-D28K and CaR-N18 distribution in the retina of adult zebrafish, suggesting that these CaBPs could be involved in the pathophysiology of retinal damage induced by the short-wavelength visible light spectrum.

Neuronal calcium-binding protein 1 and -2 (NECAB1/2) localize to multiple excitatory neuron populations in the mouse spinal cord. Here, we analyzed rat and human spinal cord, combining in situ hybridization and immunohistochemistry, complementing newly collated data on mouse spinal cord for direct comparisons. Necab1/2 mRNA transcripts showed complementary distribution in rodent’s spinal cord. Multiple-labeling fluorescence histochemistry with neuronal phenotypic markers localized NECAB1 to a dense fiber plexus in the dorsal horn, to neurons mainly in superficial layers and to commissural interneurons in both rodent species. NECAB1-positive (+) motor neurons were only found in mice. NECAB1 distribution in the human spinal cord was similar with the addition of NECAB1-like immunoreactivity surrounding myelinated axons. NECAB2 was mainly present in excitatory synaptic boutons in the dorsal horn of all three species, and often in calbindin-D28k + neuronal somata. Rodent ependymal cells expressed calbindin-D28k. In humans, they instead were NECAB2 + and/or calretinin + . Our results reveal that the association of NECAB2 to excitatory neuronal circuits in the spinal cord is evolutionarily conserved across the mammalian species investigated so far. In contrast, NECAB1 expression is more heterogeneous. Thus, our study suggests that the phenotypic segregation of NECAB1 and -2 to respective excitatory and inhibitory spinal systems can underpin functional modalities in determining the fidelity of synaptic neurotransmission and neuronal responsiveness, and might bear translational relevance to humans.

In rodent models of Alzheimer's disease (AD) and epilepsy, seizure-dependent induction of ΔFosB results in epigenetic silencing of calbindin. Hippocampal inhibition of ΔFosB or elevation of calbindin rescues spatial memory deficits in mouse models of AD. The calcium-binding protein calbindin-D28k is critical for hippocampal function and cognition 1 , 2 , 3 , but its expression is markedly decreased in various neurological disorders associated with epileptiform activity and seizures 4 , 5 , 6 , 7 . In Alzheimer's disease (AD) and epilepsy, both of which are accompanied by recurrent seizures 8 , the severity of cognitive deficits reflects the degree of calbindin reduction in the hippocampal dentate gyrus (DG) 4 , 9 , 10 . However, despite the importance of calbindin in both neuronal physiology and pathology, the regulatory mechanisms that control its expression in the hippocampus are poorly understood. Here we report an epigenetic mechanism through which seizures chronically suppress hippocampal calbindin expression and impair cognition. We demonstrate that ΔFosB, a highly stable transcription factor, is induced in the hippocampus in mouse models of AD and seizures, in which it binds and triggers histone deacetylation at the promoter of the calbindin gene ( Calb1 ) and downregulates Calb1 transcription. Notably, increasing DG calbindin levels, either by direct virus-mediated expression or inhibition of ΔFosB signaling, improves spatial memory in a mouse model of AD. Moreover, levels of ΔFosB and calbindin expression are inversely related in the DG of individuals with temporal lobe epilepsy (TLE) or AD and correlate with performance on the Mini-Mental State Examination (MMSE). We propose that chronic suppression of calbindin by ΔFosB is one mechanism through which intermittent seizures drive persistent cognitive deficits in conditions accompanied by recurrent seizures.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by core symptoms including deficits in social interaction, repetitive and stereotyped behaviors, along with higher levels of anxiety and cognitive impairments. Previous studies demonstrate pronounced reduced density of calretinin (CR)-expressing GABAergic interneurons in both ASD patients and animal models. The object of the current study was to determine the role of CR in ASD-relevant behavioral aberrations. Herein, the mRNA and protein levels of CR in the prefrontal cortex (PFC) of mouse model of ASD based on prenatal exposure to valproic acid (VPA) were determined by qRT-PCR and Western blot analysis, respectively. Moreover, the behavioral abnormalities in naive mice with CR deficiency mediated by recombinant adeno-associated virus (rAAV) were evaluated in a comprehensive testing battery including social interaction, marble burying, self-grooming, open-field, elevated plus maze and novel object recognition tests. Furthermore, the action potential changes caused by CR deficiency were examined in neurons within the PFC in naive mouse. The results show that the mRNA and protein levels of PFC CR of VPA-induced mouse ASD model were reduced. Concomitantly, mice with CR knockdown displayed ASD-like behavioral aberrations, such as social impairments, elevated stereotypes, anxiety and memory defects. Intriguingly, patch-clamp recordings revealed that CR knockdown provoked decreased neuronal excitability by increasing action potential discharge frequencies together with decreased action potential threshold and rheobase. Our findings support a notion that CR knockdown might contribute to ASD-like phenotypes, with the pathogenesis most likely stemming from increased neuronal excitability.

The development of therapeutic approaches to improve the life quality of people suffering from different types of body paralysis is a current major medical challenge. Brain-machine interface (BMI) can potentially help reestablishing lost sensory and motor functions, allowing patients to use their own brain activity to restore sensorimotor control of paralyzed body parts. Chronic implants of multielectrodes, employed to record neural activity directly from the brain parenchyma, constitute the fundamental component of a BMI. However, before this technique may be effectively available to human clinical trials, it is essential to characterize its long-term impact on the nervous tissue in animal models. In the present study we evaluated how chronic implanted tungsten microelectrode arrays impact the distribution and morphology of interneurons reactive to calcium-binding proteins calbindin (CB), calretinin (CR) and parvalbumin (PV) across the rat's motor cortex. Our results revealed that chronic microelectrode arrays were well tolerated by the nervous tissue, with recordings remaining viable for up to 6 months after implantation. Furthermore, neither the morphology nor the distribution of inhibitory neurons were broadly impacted. Moreover, restricted microglial activation was observed on the implanted sites. On the whole, our results confirm and expand the notion that tungsten multielectrodes can be deemed as a feasible candidate to future human BMI studies.

Histological morphology alone is not sufficient for the pathological diagnosis of malignant mesothelioma. Positive and negative immunohistochemical markers are necessary to differentiate it from lung adenocarcinoma. As calretinin and D2-40, the recognized positive markers of mesothelioma, are expressed in lung adenocarcinoma to some extent, novel markers with high specificity are desirable. In this study, we investigated the applicability of glypican-1 immunohistochemistry to differentiate epithelioid mesothelioma from lung adenocarcinoma. We investigated 82 cases of epithelioid mesothelioma and 97 cases of lung adenocarcinoma for glypican-1 expression by immunohistochemistry using a commercially available antibody. All 82 cases of epithelioid mesothelioma showed glypican-1 expression, most with diffuse and strong reactivity. In contrast, only three cases of lung adenocarcinoma showed focal glypican-1 expression. Glypican-1 expression showed 100 sensitivity, 97% specificity, and a 98% accuracy rate to differentiate epithelioid mesothelioma from lung adenocarcinoma. The sensitivity of glypican -1 immunohistochemistry is as high as that of calretinin and D2-40, and its specificity is far better than that of calretinin and D2-40. Therefore, we recommend including glypican -1 immunohistochemistry as a positive marker of epithelioid mesothelioma.