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White adipose tissue (WAT) requires extracellular Ca2+ influx for lipolysis, differentiation, and expansion. This partly occurs via plasma membrane Ca2+ voltage-dependent channels (CaVs). However, WFA exists in different depots whose function varies with age, sex, and location. To explore whether their CaV expression profiles also differ we used RNAseq and qPCR on gonadal, mesenteric, retroperitoneal, and inguinal subcutaneous fat depots from rats of different ages and sex. CaV expression was found dependent on age, sex, and WFA location. In the gonadal depots of both sexes a significantly lower expression of CaV1.2 and CaV1.3 was seen for adults compared to pre-pubescent juveniles. A lower level of expression was also seen for CaV3.1 in adult male but not female gonadal WFA, the latter of whose expression remained unchanged with age. Relatively little expression of CaV3.2 and 3.2 was observed. In post-pubescent inguinal subcutaneous fat, where the third and fourth mammary glands are located, CaV3.1 was decreased in males but increased in females - thus suggesting that this channel is associated with mammogenesis; however, no difference in intracellular Ca2+ levels or adipocyte size were noted. For all adult depots, CaV3.1 expression was larger in females than males - a difference not seen in pre-pubescent rats. These observations are consistent with the changes of CaV3.1 expression seen in 3T3-L1 cell differentiation and the ability of selective CaV3.1 antagonists to inhibit adipogensis. Our results show that changes in CaV expression patterns occur in fat depots related to sexual dimorphism: reproductive tracts and mammogenesis.

Aims/hypothesis: Reduced occupancy of junctional occludin is a feature of human placental vessels in the diabetic milieu. However, the functional consequence of this and whether this loss is due to differential expression of occludin splice variants is not known. Our study aimed to investigate the effects of gestational diabetes mellitus (GDM), and its treatment, on endothelial junctional integrity, gene and protein expression of occludin splice variants, and potential regulation of expression by microRNAs (miRNAs)., Methods: Term placentas were obtained from normal pregnancies (n = 21), and pregnancies complicated by GDM where glucose levels were controlled by diet (n = 11) or metformin (n = 6). Gene and microRNA (miRNA) expression were determined by quantitative real-time PCR; protein expression by immunoblotting; endothelial junctional occupancy by fluorescence microscopy and systematic sampling; and paracellular leakage by perfusion of placental microvascular beds with 76 M r dextran. Transfection studies of miRNAs that target OCLN were performed in HUVECs, and the trans-endothelial electrical resistance and tracer permeability of the HUVECs were measured., Results: All three predicted OCLN gene splice variants and two occludin protein isoforms were found in human placental samples. In placental samples from diet-controlled GDM (d-GDM) pregnancies we found a lower percentage of conduit vessels showing occludin immunoreactivity (12%, p < 0.01), decreased levels of the fully functional occludin isoform-A protein (29%), and differential gene expression of OCLN variant 2 (33% decrease), variant 3 (3.3-fold increase). These changes were not seen in samples from the group with metformin-controlled GDM. In d-GDM placentas, increased numbers of conduit microvessels demonstrated extravasation of 76 M r dextran (2.0-fold). In d-GDM expression of one of the five potential miRNAs targeting OCLN, miR-181a-5p, expression was 2.1-fold that in normal pregnancies. Experimental overexpression of miR-181a-5p in HUVECs from normal pregnancies resulted in a highly significant downregulation of OCLN variant 1 (69%) and variant 2 (46%) gene expression, with decreased trans-endothelial resistance (78%) and increase in tracer permeability (1.3-fold)., Conclusions/interpretation: Downregulation of expression of OCLN variant 2 and the fully functional occludin isoform-A protein are a feature of placentas in d-GDM pregnancies. These may be behind the loss of junctional occludin and the increased extravasation of exogenous dextran observed. miR-181a-5p was in part responsible for the downregulation of occludin in placentas from d-GDM pregnancies. Induced overexpression of miR-181a-5p compromised the integrity of the endothelial barrier. Our data suggest that, despite good glucose control, the adoption of lifestyle changes alone during a GDM pregnancy may not be enough to prevent an alteration in the expression of occludin and the subsequent functional consequences in placentas and impaired vascular barrier function in offspring. Graphical abstract.

The function of 5-hydroxymethylcytosine (5hmC) is poorly understood. 5hmC is an epigenetic modification of DNA, resulting from the oxidation of 5-methylcytosine (5mC) by the Fe2 + , and 2-oxoglutarate-dependent, 10-11 translocation methylcytosine dioxygenases (TET1, TET2, and TET3). Recent evidence suggests that, in addition to being an intermediate in active demethylation, 5hmC may also have an epigenetic role. 5hmC is enriched in the adult brain, where it has been implicated in regulating neurogenesis. The rate of adult neurogenesis decreases with age, however physical exercise has been shown to counteract this deficit. Here, we investigated the impact of voluntary exercise on the age-related changes of TET1, TET2 , expression and 5hmC content in the hippocampus and hypothalamus. For this purpose, we used voluntary exercise in young adult (3 months) and aged (18 months) mice as a rodent model of healthy brain aging. We measured the levels of hippocampal and hypothalamic TET1, TET2 mRNA, and 5hmC and memory [Object Location (OL) test] in mice that either exercised for 1 month or remained sedentary. While aging was associated with decreased TET1 and TET2 expression, voluntary exercise counteracted the decline in expression. Moreover, aged mice that exercised had higher hippocampal 5hmC content in the promoter region of miR-137 , an miRNA involved in adult neurogenesis. Exercise improved memory in aged mice, and there was a positive correlation between 5hmC miR-137 levels and performance in the OL test. In the hypothalamus neither exercise nor aging affected TET1 or TET2 expression. These results suggest that exercise partially restores the age-related decrease in hippocampal TET1 and TET2 expression, which may be linked to the improvement in memory. Future studies should further determine the specific genes where changes in 5hmC levels may mediate the exercise-induced improvements in memory and neurogenesis in aged animals.

Background: Dysfunction of dopaminergic, GABAergic, and glutamatergic function underlies many core symptoms of schizophrenia. Combined neonatal injection of the N-methyl-D-aspartate (NMDA) receptor antagonist, phencyclidine (PCP), and post-weaning social isolation of rats produces a behavioral syndrome with translational relevance to several core symptoms of schizophrenia. This study uses DNA microarray to characterize alterations in hippocampal neurotransmitter-related gene expression and examines the ability of the sodium channel blocker, lamotrigine, to reverse behavioral changes in this model., Methods: Fifty-four male Lister-hooded rat pups either received phencyclidine (PCP, 10mg/kg, s.c.) on post-natal days (PND) 7, 9, and 11 before being weaned on PND 23 into separate cages (isolation; PCP-SI; n = 31) or received vehicle injection and group-housing (2-4 per cage; V-GH; n = 23) from weaning. The effect of lamotrigine on locomotor activity, novel object recognition, and prepulse inhibition of acoustic startle was examined (PND 60-75) and drug-free hippocampal gene expression on PND 70., Results: Acute lamotrigine (10-15mg/kg i.p.) reversed the hyperactivity and novel object recognition impairment induced by PCP-SI but had no effect on the prepulse inhibition deficit. Microarray revealed small but significant down-regulation of hippocampal genes involved in glutamate metabolism, dopamine neurotransmission, and GABA receptor signaling and in specific schizophrenia-linked genes, including parvalbumin (PVALB) and GAD67, in PCP-SI rats, which resemble changes reported in schizophrenia., Conclusions: Findings indicate that alterations in dopamine neurotransmission, glutamate metabolism, and GABA signaling may contribute to some of the behavioral deficits observed following PCP-SI, and that lamotrigine may have some utility as an adjunctive therapy to improve certain cognitive deficits symptoms in schizophrenia., (© The Author 2016. Published by Oxford University Press on behalf of CINP.)

We present a first-draft digital reconstruction of the microcircuitry of somatosensory cortex of juvenile rat. The reconstruction uses cellular and synaptic organizing principles to algorithmically reconstruct detailed anatomy and physiology from sparse experimental data. An objective anatomical method defines a neocortical volume of 0.29 ± 0.01 mm(3) containing ~31,000 neurons, and patch-clamp studies identify 55 layer-specific morphological and 207 morpho-electrical neuron subtypes. When digitally reconstructed neurons are positioned in the volume and synapse formation is restricted to biological bouton densities and numbers of synapses per connection, their overlapping arbors form ~8 million connections with ~37 million synapses. Simulations reproduce an array of in vitro and in vivo experiments without parameter tuning. Additionally, we find a spectrum of network states with a sharp transition from synchronous to asynchronous activity, modulated by physiological mechanisms. The spectrum of network states, dynamically reconfigured around this transition, supports diverse information processing strategies., Paperclip: VIDEO ABSTRACT., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Lack of physical activity and increased levels of stress contribute to the development of multiple physical and mental disorders. An increasing number of studies relate voluntary exercise with greater resilience to psychological stress, a process that is highly regulated by the hypothalamic-pituitary-adrenal (HPA) axis. However, the molecular mechanisms underlying the beneficial effects of exercise on stress resilience are still poorly understood. Here we have studied the impact of long term exercise and housing conditions on: a) hippocampal expression of glucocorticoid receptor (Nr3c1), b) epigenetic regulation of Nr3c1 (DNA methylation at the Nr3c1-1F promoter and miR-124 expression), c) anxiety (elevated plus maze, EPM), and d) adrenal gland weight and adrenocorticotropic hormone receptor (Mc2r) expression., Results: Exercise increased Nr3c1 and Nr3c1-1F expression and decreased miR-124 levels in the hippocampus in single-housed mice, suggesting enhanced resilience to stress. The opposite was found for pair-housed animals. Bisulfite sequencing showed virtually no DNA methylation in the Nr3c1-1F promoter region. Single-housing increased the time spent on stretch attend postures. Exercise decreased the time spent at the open arms of the EPM, however, the mobility of the exercise groups was significantly lower. Exercise had opposite effects on the adrenal gland weight of single and pair-housed mice, while it had no effect on adrenal Mc2r expression., Conclusions: These results suggest that exercise exerts a positive impact on stress resilience in single-housed mice that could be mediated by decreasing miR-124 and increasing Nr3c1 expression in the hippocampus. However, pair-housing reverses these effects possibly due to stress from dominance disputes between pairs.

The patch-clamp technique has allowed for detailed studies on the electrical properties of neurons. Dye loading through patch pipettes enabled characterizing the morphological properties of the neurons. In addition, the patch-clamp technique also allows for harvesting mRNA from single cells to study gene expression at the single cell level (known as single-cell RT-PCR). The combination of these three approaches makes possible the study of the GEM profile of neurons (gene expression, electrophysiology, and morphology) using a single patch pipette and patch-clamp recording. This combination provides a powerful technique to investigate and correlate the neuron's gene expression with its phenotype (electrical behavior and morphology). The harvesting and amplification of single cell mRNA for gene expression studies is a challenging task, especially for researchers with sparse or no training in molecular biology (see Notes 1,2 and 5). Here we describe in detail the GEM profiling approach with special attention to the gene expression profiling.

Prenatal exposure to maternal cigarette smoking in humans or nicotine in experimental animals is associated with elevated blood pressure in the offspring. This effect may be limited to genetically vulnerable individuals and related to alterations in the kidneys. Here we investigated whether prenatal exposure to nicotine (PEN) alters kidney morphology and gene expression, and whether these effects differ between two genetically distant strains, i.e. spontaneously hypertensive (SHR) and Brown Norway (BN) rats. The results showed that, in SHR but not in BN offspring, PEN decreases kidney glomerular mass and increases renal expression of the angiotensin II type 1b receptor gene; the latter is not mediated through changes in DNA methylation of the proximal promoter of this gene. The results also showed that PEN alters expression of multiple genes involved in the kidney nervous system function, with mostly opposite effects being seen in SHR and BN. These results suggest that, in genetically vulnerable individuals, PEN leads to morphological and molecular changes in the kidneys that may contribute to fetal programming of hypertension., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Adolescence is characterized by major developmental changes that may render the individual vulnerable to stress and the development of psychopathologies in a sex-specific manner. Earlier we reported lower anxiety-like behavior and higher risk-taking and novelty seeking in rats previously exposed to peri-pubertal stress. Here we studied whether peri-pubertal stress affected the acquisition and extinction of fear memories and/or the associated functional engagement of various brain regions, as assessed with 2-deoxyglucose. We showed that while peri-pubertal stress reduced freezing during the acquisition of fear memories (training) in both sexes, it had a sex-specific effect on extinction of these memories. Moreover hippocampus, basal amygdala and cingulate and motor cortices showed higher metabolic rates during extinction in rats exposed to peri-pubertal stress. Interestingly, activation of the infralimbic cortex was negatively correlated with freezing during extinction only in control males, while only males stressed during puberty showed a significant correlation between behavior during extinction and metabolic activation of hippocampus, amygdala and paraventricular nucleus. No correlations between brain activation and behavior during extinction were observed in females (control or stress). These results indicate that exposure to peri-pubertal stress affects behavior and brain metabolism when the individual is exposed to an additional stressful challenge. Some of these effects are sex-specific., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Adolescence is a period of major physical, hormonal, and psychological change. It is also characterized by a significant increase in the incidence of psychopathologies and this increase is gender-specific. Likewise, stress during adolescence is associated with the development of psychiatric disorders later in life. Previously, using a rat model of psychogenic stress (exposure to predator odor followed by placement on an elevated platform) during the pre-pubertal period (postnatal days 28-30), we reported sex-specific effects on auditory and contextual fear conditioning. Here, we study the short-term impact of psychogenic stress before and during puberty (postnatal days 28-42) on behavior (novelty seeking, risk taking, anxiety, and depression) and hypothalamus-pituitary-adrenocortical (HPA) axis activation during late adolescence (postnatal days 45-51). Peri-pubertal stress decreased anxiety-like behavior and increased risk taking and novelty seeking behaviors during late adolescence (measured with the elevated plus maze, open field and exposure to novel object tests and intake of chocopop pellets before or immediate after stress). Finally neither depressive-like behavior (measured at the forced-swim test) nor HPA response to stress (blood corticosterone and glucose) were affected by peri-pubertal stress. Nevertheless, when controlling for the basal anxiety of the mothers, animals exposed to peri-pubertal stress showed a significant decrease in corticosterone levels immediate after an acute stressor. The results from this study suggest that exposure to mild stressors during the peri-pubertal period induces a broad spectrum of behavioral changes in late adolescence, which may exacerbate the independence-building behaviors naturally happening during this transitional period (increase in curiosity, sensation-seeking, and risk-taking behaviors).

The thick-tufted layer V pyramidal (TTL5) neuron is a key neuron providing output from the neocortex. Although it has been extensively studied, principles governing its dendritic and axonal arborization during development are still not fully quantified. Using 3-D model neurons reconstructed from biocytin-labeled cells in the rat somatosensory cortex, this study provides a detailed morphological analysis of TTL5 cells at postnatal day (P) 7, 14, 21, 36, and 60. Three developmental periods were revealed, which were characterized by distinct growing rates and properties of alterations in different compartments. From P7 to P14, almost all compartments grew fast, and filopodia-like segments along apical dendrite disappeared; From P14 to P21, the growth was localized on specified segments of each compartment, and the densities of spines and boutons were significantly increased; From P21 to P60, the number of basal dendritic segments was significantly increased at specified branch orders, and some basal and oblique dendritic segments were lengthened or thickened. Development changes were therefore seen in two modes: the fast overall growth during the first period and the slow localized growth (thickening mainly on intermediates or lengthening mainly on terminals) at the subsequent stages. The lengthening may be accompanied by the retraction on different segments. These results reveal a differential regulation in the arborization of neuronal compartments during development, supporting the notion of functional compartmental development. This quantification provides new insight into the potential value of the TTL5 morphology for information processing, and for other purposes as well.

Prenatal exposure to maternal cigarette smoking (PEMCS) is associated with variations in brain and behavior in adolescence. Epigenetic mechanisms may mediate some of the consequences of PEMCS through methylation of deoxyribonucleic acid (DNA) in genes important for brain development, such as the brain-derived neurotrophic factor (BDNF). In the current study, we used bisulfite sequencing to assess DNA methylation of the BDNF promoter in the blood of adolescents whose mothers smoked during pregnancy. We demonstrate that PEMCS is associated with higher rates of DNA methylation in the BDNF-6 exon. These results suggest that PEMCS may lead to long-term down-regulation of BDNF expression via the increase of DNA methylation in its promoter region. Such mechanisms could, in turn, lead to modifications in both development and plasticity of the brain exposed in utero to maternal cigarette smoking.

We review here the development of Hodgkin-Huxley (HH) type models of cerebral cortex and thalamic neurons for network simulations. The intrinsic electrophysiological properties of cortical neurons were analyzed from several preparations, and we selected the four most prominent electrophysiological classes of neurons. These four classes are "fast spiking", "regular spiking", "intrinsically bursting" and "low-threshold spike" cells. For each class, we fit "minimal" HH type models to experimental data. The models contain the minimal set of voltage-dependent currents to account for the data. To obtain models as generic as possible, we used data from different preparations in vivo and in vitro, such as rat somatosensory cortex and thalamus, guinea-pig visual and frontal cortex, ferret visual cortex, cat visual cortex and cat association cortex. For two cell classes, we used automatic fitting procedures applied to several cells, which revealed substantial cell-to-cell variability within each class. The selection of such cellular models constitutes a necessary step towards building network simulations of the thalamocortical system with realistic cellular dynamical properties.

Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project.

Adolescence is a period of major physical, hormonal, and psychological changes. It is also characterized by a significant increase in the incidence of psychopathologies and this increase is gender-specific. Stress during adolescence is associated with the development of psychiatric disorders later in life. In this study, we evaluated the impact of psychogenic stress (exposure to predator odor followed by placement on an elevated platform) experienced before puberty (days 28-30) on fear memories and hormonal response of male and female rats during adolescence and early adulthood. Stress before puberty impacted in a sex- and age-specific way on the responses to auditory and contextual fear conditioning in adolescence and adulthood: (a) increased conditioned fear to the tone in males during adolescence but not during adulthood; (b) impaired extinction to the tone in adult males; and (c) reduced freezing responses to the context in adolescent females. Stress before puberty did not influence the corticosterone levels 30 minutes after an additional stressor given in adulthood. These results indicate that stress experienced prior to puberty can exert a sex-related differential impact on fear-related behaviors displayed by individuals during late adolescence and early adulthood.

The patch-clamp technique has allowed detailed studies on the electrical properties of neurons. Dye loading through patch pipettes has allowed characterizing the morphological properties of the neurons. In addition, the patch-clamp technique also allows harvesting mRNA from single cells to study gene expression at the single-cell level (known as single-cell reverse transcription-polymerase chain reaction [RT-PCR] [1-3]). The combination of these three approaches allows determination of the Gene expression, Electrophysiology and Morphology (GEM) profile of neurons (gene expression, electrophysiology, and morphology) using a single patch pipette and patch-clamp recording. This combination provides a powerful technique to study and correlate the neuron's gene expression with its phenotype (electrical behavior and morphology) ( 4 - 7 ). The harvesting and amplification of single-cell mRNA for gene expression studies is a challenging task, especially for researchers with sparse or no training in molecular biology (see Notes 1 and 2). Here, we describe in detail the GEM profiling approach with special attention to the gene expression profiling.

Molecules and cells are the signalling elements in microcircuits. Recent studies have uncovered bewildering diversity in postsynaptic signalling properties in all areas of the vertebrate nervous system. Major effort is now being invested in establishing the specialized signalling properties at the cellular and molecular levels in microcircuits in specific brain regions. This review is part of the TINS Microcircuits Special Feature.

Neocortical neurones can be classified according to several independent criteria: morphological, physiological, and molecular expression (neuropeptides (NPs) and/or calcium-binding proteins (CaBPs)). While it has been suggested that particular NPs and CaBPs characterize certain anatomical subtypes of neurones, there is also considerable overlap in their expression, and little is known about simultaneous expression of multiple NPs and CaBPs in morphologically characterized neocortical neurones. Here we determined the gene expression profiles of calbindin (CB), parvalbumin (PV), calretinin (CR), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), somatostatin (SOM) and cholecystokinin (CCK) in 268 morphologically identified neurones located in layers 2-6 in the juvenile rat somatosensory neocortex. We used patch-clamp electrodes to label neurones with biocytin and harvest the cytoplasm to perform single-cell RT-multiplex PCR. Quality threshold clustering, an unsupervised algorithm that clustered neurones according to their entire profile of expressed genes, revealed seven distinct clusters. Surprisingly, each cluster preferentially contained one anatomical class. Artificial neural networks using softmax regression predicted anatomical types at nearly optimal statistical levels. Classification tree-splitting (CART), a simple binary neuropeptide decision tree algorithm, revealed the manner in which expression of the multiple mRNAs relates to different anatomical classes. Pruning the CART tree revealed the key predictors of anatomical class (in order of importance: SOM, PV, VIP, and NPY). We reveal here, for the first time, a strong relationship between specific combinations of NP and CaBP gene expressions and the anatomical class of neocortical neurones.

Whole-cell patch-clamp recordings followed by histochemical staining and single-cell RT-PCR were obtained from 180 Martinotti interneurones located in layers II to VI of the somatosensory cortex of Wistar rats (P13-P16) in order to examine their anatomical, electrophysiological and molecular properties. Martinotti cells (MCs) mostly displayed ovoid-shaped somata, bitufted dendritic morphologies, and axons with characteristic spiny boutons projecting to layer I and spreading horizontally across neighbouring columns more than 1 mm. Electron microscopic examination of MC boutons revealed that all synapses were symmetrical and most synapses (71%) were formed onto dendritic shafts. MCs were found to contact tuft, apical and basal dendrites in multiple neocortical layers: layer II/III MCs targeted mostly layer I and to a lesser degree layer II/III; layer IV MCs targeted mostly layer IV and to a lesser degree layer I; layer V and VI MCs targeted mostly layer IV and layer I and to a lesser degree the layer in which their somata was located. MCs typically displayed spike train accommodation (90%; n = 127) in response to depolarizing somatic current injections, but some displayed non-accommodating (8%) and a few displayed irregular spiking responses (2%). Some accommodating and irregular spiking MCs also responded initially with bursts (17%). Accommodating responses were found in all layers, non-accommodating mostly in upper layers and bursting mostly in layer V. Single-cell multiplex RT-PCR performed on 63 MCs located throughout layers II-VI, revealed that all MCs were somatostatin (SOM) positive, and negative for parvalbumin (PV) as well as vasoactive intestinal peptide (VIP). Calbindin (CB), calretinin (CR), neuropeptide Y (NPY) and cholecystokinin (CCK) were co- expressed with SOM in some MCs. Some layer-specific trends seem to exist. Finally, 24 accommodating MCs were examined for the expression of 26 ion channel genes. The ion channels with the highest expression in these MCs were (from highest to lowest); Cabeta1, Kv3.3, HCN4, Cabeta4, Kv3.2, Kv3.1, Kv2.1, HCN3, Caalpha1G, Kv3.4, Kv4.2, Kv1.1 and HCN2. In summary, this study provides the first detailed analysis of the anatomical, electrophysiological and molecular properties of Martinotti cells located in different neocortical layers. It is proposed that MCs are crucial interneurones for feedback inhibition in and between neocortical layers and columns.

Mammals adapt to a rapidly changing world because of the sophisticated cognitive functions that are supported by the neocortex. The neocortex, which forms almost 80% of the human brain, seems to have arisen from repeated duplication of a stereotypical microcircuit template with subtle specializations for different brain regions and species. The quest to unravel the blueprint of this template started more than a century ago and has revealed an immensely intricate design. The largest obstacle is the daunting variety of inhibitory interneurons that are found in the circuit. This review focuses on the organizing principles that govern the diversity of inhibitory interneurons and their circuits.

Cell morphogenesis requires dynamic reorganization of the actin cytoskeleton, a process that is tightly regulated by the Rho family of small GTPases. These GTPases act as molecular switches by shuttling between their inactive GDP-bound and active GTP-bound forms. Here we show that Nir2, a novel protein related to Drosophila retinal degeneration B (RdgB), markedly affects cell morphology through a novel Rho-inhibitory domain (Rid) which resides in its N-terminal region. Rid exhibits sequence homology with the Rho-binding site of formin-homology (FH) proteins and leads to an apparent loss of F-actin staining when ectopically expressed in mammalian cells. We also show that Rid inhibits Rho-mediated stress fiber formation and lysophosphatidic acid-induced RhoA activation. Biochemical studies demonstrated that Nir2, via Rid, preferentially binds to the inactive GDP-bound form of the small GTPase Rho. Microinjection of antibodies against Nir2 into neuronal cells markedly attenuates neurite extension, whereas overexpression of Nir2 in these cells attenuates Rho-mediated neurite retraction. These results implicate Nir2 as a novel regulator of the small GTPase Rho in actin cytoskeleton reorganization and cell morphogenesis.

Anatomical, electrophysiological and molecular diversity of basket cell-like interneurons in layers II-IV of rat somatosensory cortex were studied using patch-clamp electrodes filled with biocytin. This multiparametric study shows that neocortical basket cells (BCs) are composed of three distinct subclasses: classical large (LBC) and small (SBC) basket cells and a third subclass, the nest basket cell (NBC). Anatomically, NBCs were distinct from LBCs and SBCs in that they formed simpler dendritic arbors and an axonal plexus of inter-mediate density, composed of a few long, smooth axonal branches. Electrophysiologically, NBCs exhibited diverse discharge responses to depolarizing current injections including accommodation, non-accommodation and stuttering. Single-cell multiplex RT-PCR revealed distinct mRNA expression patterns for the calcium binding proteins parvalbumin (PV), calbindin (CB) and calretinin (CR), and the neuropeptides somatostatin (SOM), vasoactive intestinal peptide (VIP), cholecystokinin (CCK) and neuropeptide Y (NPY) for each BC-subclass. SBCs lacked NPY expression but invariably expressed VIP, whereas neither VIP, CR nor SOM expression was detected in LBCs, and VIP and CR expression was absent in NBCs. Electro-physiologically distinct types of NBCs formed GABAergic synapses with specific dynamics onto pyramidal cells (PCs) and received either strongly facilitating or depressing synaptic inputs from PCs. Finally, NBCs were found to be the most common basket cell in layers II/III, while LBCs were the most common in layer IV. These data provide multiparametric distinguishing features of three major subclasses of basket cells and indicate that NBCs are powerful interneurons that provide most of the (peri-)somatic inhibition in the supragranular layers.