Literature context: 984147 rat 1:500 RRID:AB_2255365 ImmunoStar Rabbit 1:4.000
In cortical structures, principal cell activity is tightly regulated by different GABAergic interneurons (INs). In particular, vasoactive intestinal polypeptide-expressing (VIP+) INs innervate preferentially other INs, providing a structural basis for temporal disinhibition of principal cells. However, relatively little is known about VIP+ INs in the amygdaloid basolateral complex (BLA). In this study, we report that VIP+ INs have a variable density in the distinct subdivisions of the mouse BLA. Based on different anatomical, neurochemical and electrophysiological criteria, VIP+ INs could be identified as interneuron-selective INs and basket cells expressing CB1 cannabinoid receptors. Whole-cell recordings of VIP+ interneuron-selective INs revealed 3 different spiking patterns, which did not associate with the expression of calretinin. Genetic targeting combined with optogenetics and in vitro recordings allowed us to identify several types of BLA INs innervated by VIP+ INs, including other interneuron-selective INs, basket and neurogliaform cells. Moreover, light stimulation of VIP+ basket cell axon terminals, characterized by CB1 sensitivity, evoked IPSPs in ∼20% of principal neurons. Finally, we show that VIP+ INs receive a dense innervation from both GABAergic, although only 10% from other VIP+ INs, and distinct glutamatergic inputs, identified by their expression of different vesicular glutamate transporters.In conclusion, our study provides a wide-range analysis of single-cell properties of VIP+ INs in the mouse BLA and of their intrinsic and extrinsic connectivity. Our results reinforce the knowledge that VIP+ INs are structurally and functionally heterogeneous and that this heterogeneity could mediate different roles in amygdala-dependent functions.Significance statement:We provide the first comprehensive analysis of the distribution of VIP+ interneurons across the entire mouse BLA, as well as of their morphological and physiological properties. VIP+ interneurons in the neocortex preferentially target other interneurons to form a disinhibitory network that facilitates principal cell firing. Our study is the first to demonstrate the presence of such a disinhibitory circuitry in the BLA. We observed structural and functional heterogeneity of these INs and characterized their input/output connectivity. We also identified several types of BLA interneurons postsynaptic to VIP+ INs, whose inhibition may provide a temporal window for principal cell firing and facilitate associative plasticity, e.g. in fear learning. Disinhibition, thus, is emerging as a general mechanism, not limited to the neocortex.
Literature context: AB354, Millipore; RRID:AB_2255365), rabbit antibody to NPY (1:200
The mammalian hippocampus, comprised of serially connected subfields, participates in diverse behavioral and cognitive functions. It has been postulated that parallel circuitry embedded within hippocampal subfields may underlie such functional diversity. We sought to identify, delineate, and manipulate this putatively parallel architecture in the dorsal subiculum, the primary output subfield of the dorsal hippocampus. Population and single-cell RNA-seq revealed that the subiculum can be divided into two spatially adjacent subregions associated with prominent differences in pyramidal cell gene expression. Pyramidal cells occupying these two regions differed in their long-range inputs, local wiring, projection targets, and electrophysiological properties. Leveraging gene-expression differences across these regions, we use genetically restricted neuronal silencing to show that these regions differentially contribute to spatial working memory. This work provides a coherent molecular-, cellular-, circuit-, and behavioral-level demonstration that the hippocampus embeds structurally and functionally dissociable streams within its serial architecture.
Literature context: tostatin Chemicon Cat# MAB354; RRID:AB_2255365 Rabbit polyclonal antibody to T
Every animal species has a signature blood glucose level or glycemic set point. These set points are different, and the normal glycemic levels (normoglycemia) of one species would be life threatening for other species. Mouse normoglycemia can be considered diabetic for humans. The biological determinants of the glycemic set point remain unclear. Here we show that the pancreatic islet imposes its glycemic set point on the organism, making it the bona fide glucostat in the body. Moreover, and in contrast to rodent islets, glucagon input from the alpha cell to the insulin-secreting beta cell is necessary to fine-tune the distinctive human set point. These findings affect transplantation and regenerative approaches to treat diabetes because restoring normoglycemia may require more than replacing only the beta cells. Furthermore, therapeutic strategies using glucagon receptor antagonists as hypoglycemic agents need to be reassessed, as they may reset the overall glucostat in the organism.
Literature context: ti-somatostatin (Merck, MAB354, RRID:AB_2255365, directed against synthetic pep
Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre-dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)-BAC, named VGAT-stop-tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron-specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration-related genomic disruption. Robust and inhibitory neuron-specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT-stop-tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4-CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X-chromosome and will be used to map X-chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well-characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse.
Literature context: hetic 1-14 cyclic SST, MAB 354; RRID:AB_2255365 EMD Millipore, Germany; 2h at r
The signaling diversity of GABAergic interneurons to post-synaptic neurons is crucial to generate the functional heterogeneity that characterizes brain circuits. Whether this diversity applies to other brain cells, such as the glial cells astrocytes, remains unexplored. Using optogenetics and two-photon functional imaging in the adult mouse neocortex, we here reveal that parvalbumin- and somatostatin-expressing interneurons, two key interneuron classes in the brain, differentially signal to astrocytes inducing weak and robust GABAB receptor-mediated Ca2+ elevations, respectively. Furthermore, the astrocyte response depresses upon parvalbumin interneuron repetitive stimulations and potentiates upon somatostatin interneuron repetitive stimulations, revealing a distinguished astrocyte plasticity. Remarkably, the potentiated response crucially depends on the neuropeptide somatostatin, released by somatostatin interneurons, which activates somatostatin receptors at astrocytic processes. Our study unveils, in the living brain, a hitherto unidentified signaling specificity between interneuron subtypes and astrocytes opening a new perspective into the role of astrocytes as non-neuronal components of inhibitory circuits.
Literature context: statin (clone YC7)MilliporeCat# MAB354goat anti-somatostatin (D-20)San
Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting.
Literature context: Myelin Basic Protein Millipore RRID:AB_2255365 MILLIPORE:AB_980
Focal cortical stroke often leads to persistent motor deficits, prompting the need for more effective interventions. The efficacy of rehabilitation can be increased by 'plasticity-stimulating' treatments that enhance experience-dependent modifications in spared areas. Transcallosal pathways represent a promising therapeutic target, but their role in post-stroke recovery remains controversial. Here, we demonstrate that the contralesional cortex exerts an enhanced interhemispheric inhibition over the perilesional tissue after focal cortical stroke in mouse forelimb motor cortex. Accordingly, we designed a rehabilitation protocol combining intensive, repeatable exercises on a robotic platform with reversible inactivation of the contralesional cortex. This treatment promoted recovery in general motor tests and in manual dexterity with remarkable restoration of pre-lesion movement patterns, evaluated by kinematic analysis. Recovery was accompanied by a reduction of transcallosal inhibition and 'plasticity brakes' over the perilesional tissue. Our data support the use of combinatorial clinical therapies exploiting robotic devices and modulation of interhemispheric connectivity.
Literature context: n antibody Millipore RRID:AB_2255365 Rabbit polyclonal anti-VIP anti
The basic architecture of the mammalian neocortex is remarkably similar across species. Pallial structures in the reptilian brain are considered amniote precursors of mammalian neocortex, whereas pallia of anamniotes ("lower" vertebrates) have been deemed largely insignificant with respect to homology. Here, we examine the cytoarchitecture of the lateral pallium in the lamprey, the phylogenetically oldest group of extant vertebrates. We reveal a three-layered structure with similar excitatory cell types as in the mammalian cortex and GABAergic interneurons. The ventral parts are sensory areas receiving monosynaptic thalamic input that can be activated from the optic nerve, whereas the dorsal parts contain motor areas with efferent projections to the brainstem, receiving oligosynaptic thalamic input. Both regions receive monosynaptic olfactory input. This three-layered "primordial" lamprey lateral pallium has evolved most features of the three-layered reptilian cortices and is thereby a precursor of the six-layered "neo" cortex with a long-standing evolutionary precedent (some 500 million years ago).
Literature context: #MAB354, RRID:AB_2255365 NPAS4, rabbit Lin et al., 2008
Individuals suffering from substance-use disorders develop strong associations between the drug's rewarding effects and environmental cues, creating powerful, enduring triggers for relapse. We found that dephosphorylated, nuclear histone deacetylase 5 (HDAC5) in the nucleus accumbens (NAc) reduced cocaine reward-context associations and relapse-like behaviors in a cocaine self-administration model. We also discovered that HDAC5 associates with an activity-sensitive enhancer of the Npas4 gene and negatively regulates NPAS4 expression. Exposure to cocaine and the test chamber induced rapid and transient NPAS4 expression in a small subpopulation of FOS-positive neurons in the NAc. Conditional deletion of Npas4 in the NAc significantly reduced cocaine conditioned place preference and delayed learning of the drug-reinforced action during cocaine self-administration, without affecting cue-induced reinstatement of drug seeking. These data suggest that HDAC5 and NPAS4 in the NAc are critically involved in reward-relevant learning and memory processes and that nuclear HDAC5 limits reinstatement of drug seeking independent of NPAS4.
Literature context: 354; RRID:AB_2255365 Rabbit polyclonal anti-Calretin
Appropriate growth and synaptic integration of GABAergic inhibitory interneurons are essential for functional neural circuits in the brain. Here, we demonstrate that disruption of primary cilia function following the selective loss of ciliary GTPase Arl13b in interneurons impairs interneuronal morphology and synaptic connectivity, leading to altered excitatory/inhibitory activity balance. The altered morphology and connectivity of cilia mutant interneurons and the functional deficits are rescued by either chemogenetic activation of ciliary G-protein-coupled receptor (GPCR) signaling or the selective induction of Sstr3, a ciliary GPCR, in Arl13b-deficient cilia. Our results thus define a specific requirement for primary cilia-mediated GPCR signaling in interneuronal connectivity and inhibitory circuit formation.
Literature context: t anti-SST Milipore Cat#MAB354, RRID:AB_2255365 Mouse anti-PV Sigma Cat#P3088,
GABAergic interneurons play important roles in cortical circuit development. However, there are multiple populations of interneurons and their respective developmental contributions remain poorly explored. Neuregulin 1 (NRG1) and its interneuron-specific receptor ERBB4 are critical genes for interneuron maturation. Using a conditional ErbB4 deletion, we tested the role of vasoactive intestinal peptide (VIP)-expressing interneurons in the postnatal maturation of cortical circuits in vivo. ErbB4 removal from VIP interneurons during development leads to changes in their activity, along with severe dysregulation of cortical temporal organization and state dependence. These alterations emerge during adolescence, and mature animals in which VIP interneurons lack ErbB4 exhibit reduced cortical responses to sensory stimuli and impaired sensory learning. Our data support a key role for VIP interneurons in cortical circuit development and suggest a possible contribution to pathophysiology in neurodevelopmental disorders. These findings provide a new perspective on the role of GABAergic interneuron diversity in cortical development. VIDEO ABSTRACT.
Literature context: n (1:300; RRID:AB_2255365; catalog n
It has been proposed that the deficit in β-cell mass in type 1 diabetes (T1D) may be due, in part, to β-cell degranulation to chromogranin-positive hormone-negative (CPHN) cells. The frequency and distribution of pancreatic CPHN cells were investigated in 19 children with T1D compared with 14 non-diabetic (ND) children. We further evaluated these cells for replication and expression of endocrine lineage markers Nkx6.1 and Nkx2.2, and compared these frequencies with those previously reported in CPHN cells in adults with T1D. In contrast to adults' cells, pancreatic CPHN cells were comparably abundant (percentage of endocrine cells ± standard error of the mean, 1.4 ± 0.2 vs 1.0 ± 0.2 in patients with T1D vs ND subjects, respectively; P = not significant) and comparably distributed in children with T1D vs ND donors. Replication of CPHN cells was detected but unchanged in children with T1D vs ND children, as was the percentage of CPHN cells expressing Nkx6.1 or NKx2.2. In children with T1D, the frequency of pancreatic CPHN cells was not increased, and this differed from adults with T1D.
Literature context: tostatin (RRID:AB_2255365, 1:200 rat
Large scale transitions between active (up) and silent (down) states during quiet wakefulness or NREM sleep regulate fundamental cortical functions and are known to involve both excitatory and inhibitory cells. However, if and how inhibition regulates these activity transitions is unclear. Using fluorescence-targeted electrophysiological recording and cell-specific optogenetic manipulation in both anesthetized and non-anesthetized mice, we found that two major classes of interneurons, the parvalbumin and the somatostatin positive cells, tightly control both up-to-down and down-to-up state transitions. Inhibitory regulation of state transition was observed under both natural and optogenetically-evoked conditions. Moreover, perturbative optogenetic experiments revealed that the inhibitory control of state transition was interneuron-type specific. Finally, local manipulation of small ensembles of interneurons affected cortical populations millimetres away from the modulated region. Together, these results demonstrate that inhibition potently gates transitions between cortical activity states, and reveal the cellular mechanisms by which local inhibitory microcircuits regulate state transitions at the mesoscale.
Literature context: ratMilliporeCat# MAB354; RRID: AB2255365Alexa 488, mouseInvitrogenCat# A
Neurexins are recognized as key organizers of synapses that are essential for normal brain function. However, it is unclear whether neurexins are fundamental building blocks of all synapses with similar overall functions or context-dependent specifiers of synapse properties. To address this question, we produced triple cKO (conditional knockout) mice that allow ablating all neurexin expression in mice. Using neuron-specific manipulations combined with immunocytochemistry, paired recordings, and two-photon Ca2+ imaging, we analyzed excitatory synapses formed by climbing fibers on Purkinje cells in cerebellum and inhibitory synapses formed by parvalbumin- or somatostatin-positive interneurons on pyramidal layer 5 neurons in the medial prefrontal cortex. After pan-neurexin deletions, we observed in these synapses severe but dramatically different synaptic phenotypes that ranged from major impairments in their distribution and function (climbing-fiber synapses) to large decreases in synapse numbers (parvalbumin-positive synapses) and severe alterations in action potential-induced presynaptic Ca2+ transients (somatostatin-positive synapses). Thus, neurexins function primarily as context-dependent specifiers of synapses.
Literature context: # MAB354; RRID:AB_2255365 Rabbit pol
The basal ganglia (BG) integrate inputs from diverse sensorimotor, limbic, and associative regions to guide action-selection and goal-directed behaviors. The entopeduncular nucleus (EP) is a major BG output nucleus and has been suggested to channel signals from distinct BG nuclei to target regions involved in diverse functions. Here we use single-cell transcriptional and molecular analyses to demonstrate that the EP contains at least three classes of projection neurons-glutamate/GABA co-releasing somatostatin neurons, glutamatergic parvalbumin neurons, and GABAergic parvalbumin neurons. These classes comprise functionally and anatomically distinct output pathways that differentially affect EP target regions, such as the lateral habenula (LHb) and thalamus. Furthermore, LHb- and thalamic-projecting EP neurons are differentially innervated by subclasses of striatal and pallidal neurons. Therefore, we identify previously unknown subdivisions within the EP and reveal the existence of cascading, molecularly distinct projections through striatum and globus pallidus to EP targets within epithalamus and thalamus.
Literature context: -somatostatin (1:100; Millipore MAB354,Â Billerica,Â MA), rabbit anti-cf
The medial subnucleus of the amygdala (MeA) plays a central role in processing sensory cues required for innate behaviors. However, whether there is a link between developmental programs and the emergence of inborn behaviors remains unknown. Our previous studies revealed that the telencephalic preoptic area (POA) embryonic niche is a novel source of MeA destined progenitors. Here, we show that the POA is comprised of distinct progenitor pools complementarily marked by the transcription factors Dbx1 and Foxp2. As determined by molecular and electrophysiological criteria this embryonic parcellation predicts postnatal MeA inhibitory neuronal subtype identity. We further find that Dbx1-derived and Foxp2+ cells in the MeA are differentially activated in response to innate behavioral cues in a sex-specific manner. Thus, developmental transcription factor expression is predictive of MeA neuronal identity and sex-specific neuronal responses, providing a potential developmental logic for how innate behaviors could be processed by different MeA neuronal subtypes.
Literature context: # MAB354 RRID:AB_2255365 NKX2-1 Pro
GABAergic interneurons are essential for neural circuit function, and their loss or dysfunction is implicated in human neuropsychiatric disease. In vitro methods for interneuron generation hold promise for studying human cellular and functional properties and, ultimately, for therapeutic cell replacement. Here we describe a protocol for generating cortical interneurons from hESCs and analyze the properties and maturation time course of cell types using single-cell RNA-seq. We find that the cell types produced mimic in vivo temporal patterns of neuron and glial production, with immature progenitors and neurons observed early and mature cortical neurons and glial cell types produced late. By comparing the transcriptomes of immature interneurons to those of more mature neurons, we identified genes important for human interneuron differentiation. Many of these genes were previously implicated in neurodevelopmental and neuropsychiatric disorders.
Literature context: t# MAB354 RRID:AB_2255365 Mouse anti
Functional circuits in the visual cortex require the coordinated activity of excitatory and inhibitory neurons. Molecular genetic approaches in the mouse have led to the "local non-specific pooling principle" of inhibitory connectivity, in which inhibitory neurons are untuned for stimulus features due to the random pooling of local inputs. However, it remains unclear whether this principle generalizes to species with a columnar organization of feature selectivity such as carnivores, primates, and humans. Here we use virally mediated GABAergic-specific GCaMP6f expression to demonstrate that inhibitory neurons in ferret visual cortex respond robustly and selectively to oriented stimuli. We find that the tuning of inhibitory neurons is inconsistent with the local non-specific pooling of excitatory inputs and that inhibitory neurons exhibit orientation-specific noise correlations with local and distant excitatory neurons. These findings challenge the generality of the non-specific pooling principle for inhibitory neurons, suggesting different rules for functional excitatory-inhibitory interactions in non-murine species.
Literature context: # MAB354, RRID:AB_2255365 Experiment
The earliest stages of Alzheimer's disease (AD) are characterized by the formation of mature tangles in the entorhinal cortex and disorientation and confusion when navigating familiar places. The medial entorhinal cortex (MEC) contains specialized neurons called grid cells that form part of the spatial navigation system. Here we show in a transgenic mouse model expressing mutant human tau predominantly in the EC that the formation of mature tangles in old mice was associated with excitatory cell loss and deficits in grid cell function, including destabilized grid fields and reduced firing rates, as well as altered network activity. Overt tau pathology in the aged mice was accompanied by spatial memory deficits. Therefore, tau pathology initiated in the entorhinal cortex could lead to deficits in grid cell firing and underlie the deterioration of spatial cognition seen in human AD.
Literature context: t#MAB354, RRID:AB_2255365 Rabbit ant
The recent discovery that genetically modified α cells can regenerate and convert into β-like cells in vivo holds great promise for diabetes research. However, to eventually translate these findings to human, it is crucial to discover compounds with similar activities. Herein, we report the identification of GABA as an inducer of α-to-β-like cell conversion in vivo. This conversion induces α cell replacement mechanisms through the mobilization of duct-lining precursor cells that adopt an α cell identity prior to being converted into β-like cells, solely upon sustained GABA exposure. Importantly, these neo-generated β-like cells are functional and can repeatedly reverse chemically induced diabetes in vivo. Similarly, the treatment of transplanted human islets with GABA results in a loss of α cells and a concomitant increase in β-like cell counts, suggestive of α-to-β-like cell conversion processes also in humans. This newly discovered GABA-induced α cell-mediated β-like cell neogenesis could therefore represent an unprecedented hope toward improved therapies for diabetes.
Literature context: tin (RRID:AB_2255365);GFAP (RRI
Long-term diffuse traumatic brain injury (dTBI) causes neuronal hyperexcitation in supragranular layers in sensory cortex, likely through reduced inhibition. Other forms of TBI affect inhibitory interneurons in subcortical areas but it is unknown if this occurs in cortex, or in any brain area in dTBI. We investigated dTBI effects on inhibitory neurons and astrocytes in somatosensory and motor cortex, and hippocampus, 8 weeks post-TBI. Brains were labeled with antibodies against calbindin (CB), parvalbumin (PV), calretinin (CR) and neuropeptide Y (NPY), and somatostatin (SOM) and glial fibrillary acidic protein (GFAP), a marker for astrogliosis during neurodegeneration. Despite persistent behavioral deficits in rotarod performance up to the time of brain extraction (TBI = 73.13 ± 5.23% mean ± SEM, Sham = 92.29 ± 5.56%, P < 0.01), motor cortex showed only a significant increase, in NPY neurons in supragranular layers (mean cells/mm2 ± SEM, Sham = 16 ± 0.971, TBI = 25 ± 1.51, P = 0.001). In somatosensory cortex, only CR+ neurons showed changes, being decreased in supragranular (TBI = 19 ± 1.18, Sham = 25 ± 1.10, P < 0.01) and increased in infragranular (TBI = 28 ± 1.35, Sham = 24 ± 1.07, P < 0.05) layers. Heterogeneous changes were seen in hippocampal staining: CB+ decreased in dentate gyrus (TBI = 2 ± 0.382, Sham = 4 ± 0.383, P < 0.01), PV+ increased in CA1 (TBI = 39 ± 1.26, Sham = 33 ± 1.69, P < 0.05) and CA2/3 (TBI = 26 ± 2.10, Sham = 20 ± 1.49, P < 0.05), and CR+ decreased in CA1 (TBI = 10 ± 1.02, Sham = 14 ± 1.14, P < 0.05). Astrogliosis significantly increased in corpus callosum (TBI = 6.7 ± 0.69, Sham = 2.5 ± 0.38; P = 0.007). While dTBI effects on inhibitory neurons appear region- and type-specific, a common feature in all cases of decrease was that changes occurred in dendrite targeting interneurons involved in neuronal integration. J. Comp. Neurol. 524:3530-3560, 2016. © 2016 Wiley Periodicals, Inc.
Literature context: ipore, MAB354, rat, monoclonal, AB_22553651: 100CalretininRecombinant rat
GPR88 is a neuronal cerebral orphan G-protein-coupled receptor (GPCR) that has been linked to various psychiatric disorders. However, no extensive description of its localization has been provided so far. Here, we investigate the spatiotemporal expression of the GPR88 in prenatal and postnatal rat tissues by using in situ hybridization and immunohistochemistry. GPR88 protein was initially detected at embryonic day 16 (E16) in the striatal primordium. From E16-E20 to adulthood, the highest expression levels of both protein and mRNA were observed in striatum, olfactory tubercle, nucleus accumbens, amygdala, and neocortex, whereas in spinal cord, pons, and medulla GPR88 expression remains discrete. We observed an intracellular redistribution of GPR88 during cortical lamination. In the cortical plate of the developing cortex, GPR88 presents a classical GPCR plasma membrane/cytoplasmic localization that shifts, on the day of birth, to nuclei of neurons progressively settling in layers V to II. This intranuclear localization remains throughout adulthood and was also detected in monkey and human cortex as well as in the amygdala and hypothalamus of rats. Apart from the central nervous system, GPR88 was transiently expressed at high levels in peripheral tissues, including adrenal cortex (E16-E21) and cochlear ganglia (E19-P3), and also at moderate levels in retina (E18-E19) and spleen (E21-P7). The description of the GPR88 anatomical expression pattern may provide precious functional insights into this novel receptor. Furthermore, the GRP88 nuclear localization suggests nonclassical GPCR modes of action of the protein that could be relevant for cortical development and psychiatric disorders. J. Comp. Neurol. 524:2776-2802, 2016. © 2016 Wiley Periodicals, Inc.
Literature context: tibody; 1:300; Millipore MAB354;RRID:AB_2255365;goat polyclonalantibody; 1:1000
Systematic genetic access to GABAergic cell types will facilitate studying the function and development of inhibitory circuitry. However, single gene-driven recombinase lines mark relatively broad and heterogeneous cell populations. Although intersectional approaches improve precision, it remains unclear whether they can capture cell types defined by multiple features. Here we demonstrate that combinatorial genetic and viral approaches target restricted GABAergic subpopulations and cell types characterized by distinct laminar location, morphology, axonal projection, and electrophysiological properties. Intersectional embryonic transcription factor drivers allow finer fate mapping of progenitor pools that give rise to distinct GABAergic populations, including laminar cohorts. Conversion of progenitor fate restriction signals to constitutive recombinase expression enables viral targeting of cell types based on their lineage and birth time. Properly designed intersection, subtraction, conversion, and multi-color reporters enhance the precision and versatility of drivers and viral vectors. These strategies and tools will facilitate studying GABAergic neurons throughout the mouse brain.
Literature context: onoclonal RRID:AB_2255365 1:200
Many neurological diseases including major depression and schizophrenia manifest as dysfunction of the GABAergic system within the cingulate cortex. However, relatively little is known about the properties of GABAergic interneurons in the cingulate cortex. Therefore, we investigated the neurochemical properties of GABAergic interneurons in the cingulate cortex of FVB-Tg(GadGFP)45704Swn/J mice expressing green fluorescent protein (GFP) in a subset of GABAergic interneurons (GFP-expressing inhibitory interneurons [GINs]) by means of immunocytochemical and design-based stereologic techniques. We found that GINs represent around 12% of all GABAergic interneurons in the cingulate cortex. In contrast to other neocortical areas, GINs were only found in cortical layers II and III. More than 98% of GINs coexpressed the neuropeptide somatostatin (SOM), but only 50% of all SOM + neurons were GINs. By analyzing the expression of calretinin (CR), calbindin (CB), parvalbumin, and various neuropeptides, we identified several distinct GIN subgroups. In particular, we observed coexpression of SOM with CR and CB. In addition, we found neuropeptide Y expression almost exclusively in those GINs that coexpressed SOM and CR. Thus, with respect to the expression of calcium-binding proteins and neuropeptides, GINs are surprisingly heterogeneous in the mouse cingulate cortex, and the minority of GINs express only one marker protein or peptide. Furthermore, our observation of overlap between the SOM + and CR + interneuron population was in contrast to earlier findings of non-overlapping SOM + and CR + interneuron populations in the human cortex. This might indicate that findings in mouse models of neuropsychiatric diseases may not be directly transferred to human patients. J. Comp. Neurol. 524:2281-2299, 2016. © 2015 Wiley Periodicals, Inc.
Literature context: . MAB354; RRID:AB_2255365) or a mous
Within the basolateral amygdaloid complex (BLA), neuropeptide Y (NPY) buffers against protracted anxiety and fear. Although the importance of NPY's actions in the BLA is well documented, little is known about the source(s) of NPY fibers to this region. The current studies identified sources of NPY projections to the BLA by using a combination of anatomical and neurochemical approaches. NPY innervation of the BLA was assessed in rats by examining the degree of NPY coexpression within interneurons or catecholaminergic fibers with somatostatin and tyrosine hydroxylase (TH) or dopamine β-hydroxylase (DβH), respectively. Numerous NPY(+) /somatostatin(+) and NPY(+) /somatostatin(-) fibers were observed, suggesting at least two populations of NPY fibers within the BLA. No colocalization was noted between NPY and TH or DβH immunoreactivities. Additionally, Fluorogold (FG) retrograde tracing with immunohistochemistry was used to identify the precise origin of NPY projections to the BLA. FG(+) /NPY(+) cells were identified within the amygdalostriatal transition area (AStr) and stria terminalis and scattered throughout the bed nucleus of the stria terminalis. The subpopulation of NPY neurons in the AStr also coexpressed somatostatin. Subjecting animals to a conditioned fear paradigm increased NPY gene expression within the AStr, whereas no changes were observed within the BLA or stria terminalis. Overall, these studies identified limbic regions associated with stress circuits providing NPY input to the BLA and demonstrated that a unique NPY projection from the AStr may participate in the regulation of conditioned fear. J. Comp. Neurol. 524:2418-2439, 2016. © 2016 Wiley Periodicals, Inc.
Literature context: RRID:AB_2255365 1:200
The bed nucleus of the stria terminalis (BNST) plays an important role in fear, stress, and anxiety. It contains a collection of subnuclei delineated by gross cytoarchitecture features; however, there has yet to be a systematic examination of specific BNST neuronal types and their associated neurochemical makeup. The present study focuses on improved characterization of the anterior BNST based on differing molecular and chemical expression aided by mouse genetics. Specific Cre driver lines crossed with a fluorescent reporter line were used for genetic cell targeting and immunochemical staining. Using this new approach, we were able to robustly identify specific excitatory and inhibitory cell types in the BNST. The presence and distribution of excitatory neurons were firmly established; glutamatergic neurons in the anterior BNST accounted for about 14% and 31% of dorsal and ventral BNST cells, respectively. GABAergic neurons expressing different isoforms of glutamic acid decarboxylase were found to have differential subregional distributions. Almost no parvalbumin-expressing cells were found in the BNST, while somatostatin-expressing cells and calretinin-expressing cells account for modest proportions of BNST cells. In addition, vasoactive intestinal peptide-expressing axonal plexuses were prominent in the oval and juxtacapsular subregions. In addition, we discovered that corticotropin-releasing hormone-expressing cells contain GABAergic and glutamatergic subpopulations. Together, this study reveals new information on excitatory and inhibitory neurons in the BNST, which will facilitate genetic dissection and functional studies of BNST subregions. J. Comp. Neurol. 524:2379-2399, 2016. © 2016 Wiley Periodicals, Inc.
Literature context: illipore, RRID:AB_2255365), GAD67 (m
Hippocampal neurons activated during encoding drive the recall of contextual fear memory. Little is known about how such ensembles emerge during acquisition and eventually form the cellular engram. Manipulating the activity of granule cells (GCs) of the dentate gyrus (DG), we reveal a mechanism of lateral inhibition that modulates the size of the cellular engram. GCs engage somatostatin-positive interneurons that inhibit the dendrites of surrounding GCs. Our findings reveal a microcircuit within the DG that controls the size of the cellular engram and the stability of contextual fear memory.
Literature context: e1:1000MilliporeMab354 2060939 AB_2255365 Anti- Somatostatin 1, 2 Rabb
The activity of excitatory neurons is controlled by a highly diverse population of inhibitory interneurons. These cells show a high level of physiological, morphological and neurochemical heterogeneity, and play highly specific roles in neuronal circuits. In the mammalian hippocampus, these are divided into 21 different subtypes of GABAergic interneurons based on their expression of different markers, morphology and their electrophysiological properties. Ideally, all can be marked using an antibody directed against the inhibitory neurotransmitter GABA, but parvalbumin, calbindin, somatostatin, and calretinin are also commonly used as markers to narrow down the specific interneuron subtype. Here, we describe a journey to find the necessary immunological reagents for studying GABAergic interneurons of the mouse hippocampus. Based on web searches there are several hundreds of different antibodies on the market directed against these four markers. Searches in the literature databases allowed us to narrow it down to a subset of antibodies most commonly used in publications. However, in our hands the most cited ones did not work for immunofluorescence stainings of formaldehyde fixed tissue sections and cultured hippocampal neurons, and we had to immunostain our way through thirteen different commercial antibodies before finally finding a suitable antibody for each of the four markers. The antibodies were evaluated based on signal-to-noise ratios as well as if positive cells were found in layers of the hippocampus where they have previously been described. Additionally, the antibodies were also tested on sections from mouse spinal cord with similar criteria for specificity of the antibodies. Using the antibodies with a high rating on pAbmAbs, an antibody review database, stainings with high signal-to-noise ratios and location of the immunostained cells in accordance with the literature could be obtained, making these antibodies suitable choices for studying the GABAergic system.
GABAergic interneurons are key elements regulating the activity of local circuits, and abnormal inhibitory circuits are implicated in certain psychiatric and neurodevelopmental diseases. The glutamatergic input that interneurons receive is a key determinant of their activity, yet its molecular structure and development, which are often distinct from those of glutamatergic input to pyramidal cells, are poorly defined. The membrane-associated guanylate kinase (MAGUK) homologs PSD-95/SAP90, PSD-93/chapsyn110, SAP97, and SAP102 are central organizers of the postsynaptic density at excitatory synapses on pyramidal neurons. We therefore studied the cell-type-specific and developmental expression of MAGUKs in the nonoverlapping parvalbumin (PV)- and somatostatin (SOM)-positive interneurons in the visual cortex. These interneuron subtypes account for the vast majority of interneurons in the cortex and have different functional properties and postsynaptic structures, being either axodendritic (PV(+)) or axospinous (SOM(+)). To study cell-type-specific MAGUK expression, we used DIG-labeled riboprobes against each MAGUK along with antibodies against either PV or SOM and examined tissue from juvenile (P15) and adult mice. Both PV(+) and SOM(+) interneurons express mRNA for PSD-95, PSD-93, and SAP102 in P15 and adult tissue. In contrast, these interneuron subtypes express SAP97 at P15, but for adult visual cortex we found that most PV(+) and SOM(+) interneurons show low or no expression of SAP97. Given the importance of SAP97 in regulating AMPA receptor GluA1 subunit and NMDA receptor subunits at glutamatergic synapses, these results suggest a developmental shift in glutamate receptor subunit composition and regulation of glutamatergic synapses on PV(+) and SOM(+) interneurons.
Neurons in the subparafascicular area at the caudal border of the thalamus that contain the neuropeptide tuberoinfundibular peptide of 39 residues (TIP39) densely innervate several hypothalamic areas, including the paraventricular nucleus (PVN). These areas contain a matching distribution of TIP39's receptor, the parathyroid hormone receptor 2 (PTH2R). Frequent PTH2R coexpression with a vesicular glutamate transporter (VGlut2) suggests that TIP39 could presynaptically regulate glutamate release. By using immunohistochemistry we found CRH-ir neurons surrounded by PTH2R-ir fibers and TIP39-ir axonal projections in the PVN area of the mouse brain. Labeling hypothalamic neuroendocrine neurons by peripheral injection of fluorogold in PTH2R-lacZ knock-in mice showed that most PTH2Rs are on PVN and peri-PVN interneurons and not on neuroendocrine cells. Double fluorescent in situ hybridization revealed a high level of coexpression between PTH2R and VGlut2 mRNA by cells located in the PVN and nearby brain areas. Local TIP39 infusion (100 pmol) robustly increased pCREB-ir in the PVN and adjacent perinuclear zone. It also increased plasma corticosterone and decreased plasma prolactin. These effects of TIP39 on pCREB-ir, corticosterone, and prolactin were abolished by coinfusion of the ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonopentanoic acid (AP-5; 30 pmol each) and were absent in PTH2R knockout mice. Basal plasma corticosterone was slightly decreased in TIP39 knockout mice just before onset of their active phase. The present data indicate that the TIP39 ligand/PTH2 receptor system provides facilitatory regulation of the hypothalamic-pituitary-adrenal axis via hypothalamic glutamatergic neurons and that it may regulate other neuroendocrine systems by a similar mechanism.
The cerebral cortex has diverse types of inhibitory neurons. In rat cortex, past research has shown that parvalbumin (PV), somatostatin (SOM), calretinin (CR), and cholecystokinin (CCK) label four distinct chemical classes of GABAergic interneurons. However, in contrast to rat cortex, previous studies indicate that there is significant colocalization of SOM and CR in mouse cortical inhibitory neurons. In the present study we further characterized immunochemical distinctions among mouse inhibitory cortical neurons by double immunochemical labeling with chemical markers. We found that, PV, SOM, and vasointenstinal peptide (VIP) reliably identify three nonoverlapping distinct subpopulations, as there was no overlap of immunoreactivity between PV and all the other chemical markers tested, and SOM and VIP did not show any overlap in labeled neurons in all the cortical areas. In comparison, there was significant overlap in combinations of other chemical markers. With some laminar and regional variations, the average overlap of SOM/CR (percentage of SOM+ cells expressing CR) and SOM/neuropeptide tyrosine (NPY) across all examined layers and cortical regions was 21.6% and 7.1%, respectively. The average overlap of VIP/CR, VIP/NPY, and CR/NPY was 34.2%, 9.5%, and 10%, respectively. We quantified and assessed the percentages of marker-positive GABAergic cells, and showed that the nonoverlapping subpopulations (i.e., PV+, SOM+ and VIP+ cells) accounted for about 60% of the GABAergic cell population. Taken together, our data reveal important chemical distinctions between mouse inhibitory cortical neurons and indicate that PV, SOM, and VIP can differentially label a majority of mouse inhibitory cortical neurons.
Here we analyze the role of the Lhx6 lim-homeobox transcription factor in regulating the development of subsets of neocortical, hippocampal, and striatal interneurons. An Lhx6 loss-of-function allele, which expresses placental alkaline phosphatase (PLAP), allowed analysis of the development and fate of Lhx6-expressing interneurons in mice lacking this homeobox transcription factor. There are Lhx6+;Dlx+ and Lhx6-;Dlx+ subtypes of tangentially migrating interneurons. Most interneurons in Lhx6(PLAP/PLAP) mutants migrate to the cortex, although less efficiently, and exhibit defects in populating the marginal zone and superficial parts of the neocortical plate. By contrast, migration to superficial parts of the hippocampus is not seriously affected. Furthermore, whereas parvalbumin+ and somatostatin+ interneurons do not differentiate, NPY+ interneurons are present; we suggest that these NPY+ interneurons are derived from the Lhx6-;Dlx+ subtype. Striatal interneurons show deficits distinct from pallial interneurons, including a reduction in the NPY+ subtype. We provide evidence that Lhx6 mediates these effects through promoting expression of receptors that regulate interneuron migration (ErbB4, CXCR4, and CXCR7), and through promoting the expression of transcription factors either known (Arx) or implicated (bMaf, Cux2, and NPAS1) in controlling interneuron development.
The homeodomain transcription factor Nkx2.1 is expressed in the pallidal (subcortical) telencephalon, including the medial ganglionic eminence (MGE) and preoptic area. Studies have shown that Nkx2.1 is required for normal patterning of the MGE and for the specification of the parvalbumin (PV)- and somatostatin (SST)-expressing cortical interneurons. To define the contribution of Nkx2.1 lineages to neurons in the mature telencephalon, we have generated transgenic mice carrying the genomic integration of a modified bacterial artificial chromosome (BAC) in which the second exon of Nkx2.1 is replaced by the Cre recombinase. Analysis of these mice has found that they express the Cre recombinase and Cre reporters within Nkx2.1-expressing domains of the brain, thyroid, pituitary, and lung. Telencephalic expression of reporters begins at about embryonic day 10.5. Expression both of Cre and of recombination-based Cre reporters is weaker within the dorsalmost region of the MGE than in other Nkx2.1-expressing regions. In this paper, we present fate-mapping data on Nkx2.1-lineage neurons throughout the telencephalon, including the cerebral cortex, amygdala, olfactory bulb, striatum, globus pallidus, septum, and nucleus basalis.
The amygdala controls emotional and social behavior and regulates instinctive reflexes such as defense and reproduction by way of descending projections to the hypothalamus and brainstem. The descending amygdalar projections are suggested to show a cortico-striato-pallidal organization similar to that of the basal ganglia (Swanson  Brain Res 886:113-164). To test this model we investigated the embryological origin and molecular properties of the mouse centromedial and extended amygdalar subdivisions, which constitute major sources of descending projections. We analyzed the distribution of key regulatory genes that show restricted expression patterns within the subpallium (Dlx5, Nkx2.1, Lhx6, Lhx7/8, Lhx9, Shh, and Gbx1), as well as genes considered markers for specific subpallial neuronal subpopulations. Our results indicate that most of the centromedial and extended amygdala is formed by cells derived from multiple subpallial subdivisions. Contrary to a previous suggestion, only the central--but not the medial--amygdala derives from the lateral ganglionic eminence and has striatal-like features. The medial amygdala and a large part of the extended amygdala (including the bed nucleus of the stria terminalis) consist of subdivisions or cell groups that derive from subpallial, pallial (ventral pallium), or extratelencephalic progenitor domains. The subpallial part includes derivatives from the medial ganglionic eminence, the anterior peduncular area, and possibly a novel subdivision, called here commissural preoptic area, located at the base of the septum and related to the anterior commissure. Our study provides a molecular and morphological foundation for understanding the complex embryonic origins and adult organization of the centromedial and extended amygdala.
Mammalian cortex contains a diversity of inhibitory neuron types, each with distinct morphological, immunochemical, and/or physiological properties. In rat cortex, chemical markers distinguish at least four distinct and nonoverlapping neuron classes based on expression of parvalbumin (PV), somatostatin (SST), calretinin (CR), and cholecystokinin (CCK). It has generally been assumed that these classifications should also apply to other rodent species. In mouse cortex, however, we found significant colocalization of SST and CR in inhibitory neurons; about 30% of SST-positive cells contained CR, and about 33% of CR-positive cells contained SST across frontal, somatosensory (S1), and visual cortex (V1). The SST and CR colocalized cells were concentrated in layer 2/3. We further characterized morphological and physiological properties of the mouse cortical inhibitory neuron types that express SST by using "GIN" transgenic mice, in which GFP is expressed in a subset of SST inhibitory neurons (see Oliva et al.  J Neurosci 20:3354-3368). Generally, both SST/CR+ cells and SST/CR- cells exhibited morphological features of Martinotti cells as described in rat cortex, and they also had similar accommodating spike-firing patterns. However, they differed significantly in quantitative comparisons of morphology and spike shapes. SST/CR+ cells had more horizontally extended dendritic fields and more primary process than did SST/CR- cells; and SST/CR- cells had narrower action potential widths and faster afterhyperpolarization than did SST/CR+ cells. Thus, our data show an important species difference in the chemical distinction of inhibitory neuron subtypes, and indicate that colocalization of CR in SST cells correlates with different morphological and physiological features.