MAB5504 Sigma-AldrichAnti-Vesicular Glutamate Transporter 2 Antibody

Regulation of GABAergic inhibitory inputs and alterations in POMC neuron activity by nutrients and adiposity signals regulate energy and glucose homeostasis. Thus, understanding how POMC neurons integrate these two signal molecules at the synaptic level is important. Here we show that leptin's action on GABA release to POMC neurons is influenced by glucose levels. Leptin stimulates the JAK2-PI3K pathway in both presynaptic GABAergic terminals and postsynaptic POMC neurons. Inhibition of AMPK activity in presynaptic terminals decreases GABA release at 10 mM glucose. However, postsynaptic TRPC channel opening by the PI3K-PLC signalling pathway in POMC neurons enhances spontaneous GABA release via activation of presynaptic MC3/4 and mGlu receptors at 2.5 mM glucose. High-fat feeding blunts AMPK-dependent presynaptic inhibition, whereas PLC-mediated GABAergic feedback inhibition remains responsive to leptin. Our data indicate that the interplay between glucose and leptin signalling in glutamatergic POMC neurons is critical for determining the strength of inhibitory tone towards POMC neurons.

Mesoaccumbens fibers are thought to co-release dopamine and glutamate. However, the mechanism is unclear, and co-release by mesoaccumbens fibers has not been documented. Using electron microcopy, we found that some mesoaccumbens fibers have vesicular transporters for dopamine (VMAT2) in axon segments that are continuous with axon terminals that lack VMAT2, but contain vesicular glutamate transporters type 2 (VGluT2). In vivo overexpression of VMAT2 did not change the segregation of the two vesicular types, suggesting the existence of highly regulated mechanisms for maintaining this segregation. The mesoaccumbens axon terminals containing VGluT2 vesicles make asymmetric synapses, commonly associated with excitatory signaling. Using optogenetics, we found that dopamine and glutamate were released from the same mesoaccumbens fibers. These findings reveal a complex type of signaling by mesoaccumbens fibers in which dopamine and glutamate can be released from the same axons, but are not normally released at the same site or from the same synaptic vesicles.

The ability of animals to respond to life-threatening stimuli is essential for survival. Although vision provides one of the major sensory inputs for detecting threats across animal species, the circuitry underlying defensive responses to visual stimuli remains poorly defined. Here, we investigate the circuitry underlying innate defensive behaviours elicited by predator-like visual stimuli in mice. Our results demonstrate that neurons in the superior colliculus (SC) are essential for a variety of acute and persistent defensive responses to overhead looming stimuli. Optogenetic mapping revealed that SC projections to the lateral posterior nucleus (LP) of the thalamus, a non-canonical polymodal sensory relay, are sufficient to mimic visually evoked fear responses. In vivo electrophysiology experiments identified a di-synaptic circuit from SC through LP to the lateral amygdale (Amg), and lesions of the Amg blocked the full range of visually evoked defensive responses. Our results reveal a novel collicular-thalamic-Amg circuit important for innate defensive responses to visual threats.

Cerebellar circuits are patterned into an array of topographic parasagittal domains called zones. The proper connectivity of zones is critical for motor coordination and motor learning, and in several neurological diseases cerebellar circuits degenerate in zonal patterns. Despite recent advances in understanding zone function, we still have a limited understanding of how zones are formed. Here, we focused our attention on Purkinje cells to gain a better understanding of their specific role in establishing zonal circuits. We used conditional mouse genetics to test the hypothesis that Purkinje cell neurotransmission is essential for refining prefunctional developmental zones into sharp functional zones. Our results show that inhibitory synaptic transmission in Purkinje cells is necessary for the precise patterning of Purkinje cell zones and the topographic targeting of mossy fiber afferents. As expected, blocking Purkinje cell neurotransmission caused ataxia. Using in vivo electrophysiology, we demonstrate that loss of Purkinje cell communication altered the firing rate and pattern of their target cerebellar nuclear neurons. Analysis of Purkinje cell complex spike firing revealed that feedback in the cerebellar nuclei to inferior olive to Purkinje cell loop is obstructed. Loss of Purkinje neurotransmission also caused ectopic zonal expression of tyrosine hydroxylase, which is only expressed in adult Purkinje cells when calcium is dysregulated and if excitability is altered. Our results suggest that Purkinje cell inhibitory neurotransmission establishes the functional circuitry of the cerebellum by patterning the molecular zones, fine-tuning afferent circuitry, and shaping neuronal activity.

The layers and sublayers of primary visual cortex, or V1, in primates are easily distinguishable compared to those in other cortical areas, and are especially distinct in anthropoid primates - monkeys, apes, and humans - where they also vary in histological appearance. This variation in primate-specific specialization has led to a longstanding confusion over the identity of layer 4 and its proposed sublayers in V1. As the application of different histological markers relate to the issue of defining and identifying layers and sublayers, we applied four traditional and four more recent histological markers to brain sections of V1 and adjoining secondary visual cortex (V2) in macaque monkeys, chimpanzees, and humans in order to compare identifiable layers and sublayers in both cortical areas across these species. The use of Nissl, neuronal nuclear antigen (NeuN), Gallyas myelin, cytochrome oxidase (CO), acetylcholinesterase (AChE), nonphosphorylated neurofilament H (SMI-32), parvalbumin (PV), and vesicular glutamate transporter 2 (VGLUT2) preparations support the conclusion that the most popular scheme of V1 lamination, that of Brodmann, misidentifies sublayers of layer 3 (3Bβ and 3C) as sublayers of layer 4 (4A and 4B), and that the specialized sublayer of layer 3 in monkeys, 3Bβ, is not present in humans. These differences in interpretation are important as they relate to the proposed functions of layer 4 in primate species, where layer 4 of V1 is a layer that receives and processes information from the visual thalamus, and layer 3 is a layer that transforms and distributes information to other cortical areas.

Torsin A (TA) is a ubiquitous protein belonging to the superfamily of proteins called "ATPases associated with a variety of cellular activities" (AAA(+) ATPase). To date, a great deal of attention has been focused on neuronal TA since its mutant form causes early-onset (DYT1) torsion dystonia, an inherited movement disorder characterized by sustained muscle contractions and abnormal postures. Interestingly, it has been proposed that TA, by interacting with the cytoskeletal network, may contribute to the control of neurite outgrowth and/or by acting as a chaperone at synapses could affect synaptic vesicle turnover and neurotransmitter release. Accordingly, both its peculiar developmental expression in striatum and cerebellum and evidence from DYT1 knock-in mice suggest that TA may influence dendritic arborization and synaptogenesis in the brain. Therefore, to better understand TA function a detailed description of its localization at synaptic level is required. Here, we characterized by means of rigorous quantitative confocal analysis TA distribution in the mouse cerebellum at postnatal day 14 (P14), when both cerebellar synaptogenesis and TA expression peak. We observed that the protein is broadly distributed both in cerebellar cortex and in the deep cerebellar nuclei (DCN). Of note, Purkinje cells (PC) express high levels of TA also in the spines and axonal terminals. In addition, abundant expression of the protein was found in the main GABA-ergic and glutamatergic inputs of the cerebellar cortex. Finally, TA was observed also in glial cells, a cellular population little explored so far. These results extend our knowledge on TA synaptic localization providing a clue to its potential role in synaptic development.

Spinocerebellar ataxia type 7 (SCA7) is a dominantly inherited neurodegenerative disorder caused by a CAG - polyglutamine (polyQ) repeat expansion in the ataxin-7 gene. In polyQ disorders, synaptic dysfunction and neurodegeneration may develop prior to symptom onset. However, conditional expression studies of polyQ disease models demonstrate that suppression of gene expression can yield complete reversal of established behavioral abnormalities. To determine if SCA7 neurological and neurodegenerative phenotypes are reversible, we crossed PrP-floxed-SCA7-92Q BAC transgenic mice with a tamoxifen-inducible Cre recombinase transgenic line, CAGGS-Cre-ER™. PrP-floxed-SCA7-92Q BAC;CAGGS-Cre-ER™ bigenic mice were treated with a single dose of tamoxifen 1 month after the onset of detectable ataxia, which resulted in ~50% reduction of polyQ-ataxin-7 expression. Tamoxifen treatment halted or reversed SCA7 motor symptoms, reduced ataxin-7 aggregation in Purkinje cells (PCs), and prevented loss of climbing fiber (CF)-PC synapses in comparison to vehicle-treated bigenic animals and tamoxifen-treated PrP-floxed-SCA7-92Q BAC single transgenic mice. Despite this phenotype rescue, reduced ataxin-7 expression did not result in full recovery of cerebellar molecular layer thickness or prevent Bergmann glia degeneration. These results demonstrate that suppression of mutant gene expression by only 50% in a polyQ disease model can have a significant impact on disease phenotypes, even when initiated after the onset of detectable behavioral deficits. The findings reported here are consistent with the emerging view that therapies aimed at reducing neurotoxic gene expression hold the potential to halt or reverse disease progression in afflicted patients, even after the onset of neurological disability.

Previous studies indicate that while transgenic mice with ATXN1[30Q]-D776-induced disease share pathological features caused by ATXN1[82Q] having an expanded polyglutamine tract, they fail to manifest the age-related progressive neurodegeneration seen in spinocerebellar ataxia type 1. The shared features include morphological alterations in climbing fiber (CF) innervation of Purkinje cells (PCs). To further investigate the ability of ataxin-1 (ATXN1) to impact CF/PC innervation, this study used morphological and functional approaches to examine CF/PC innervation during postnatal development in ATXN1[30Q]-D776 and ATXN1[82Q] cerebella. Notably, ATXN1[30Q]-D776 induced morphological alterations consistent with the development of the innervation of PCs by CFs being compromised, including a reduction of CF translocation along the PC dendritic tree, and decreased pruning of CF terminals from the PC soma. As previously shown for ATXN1[82Q], ATXN1[30Q]-D776 must enter the nucleus of PCs to induce these alterations. Experiments using conditional ATXN1[30Q]-D776 mice demonstrate that both the levels and specific timing of mutant ATXN1 expression are critical for alteration of the CF-PC synapse. Together these observations suggest that ATXN1, expressed exclusively in PCs, alters expression of a gene(s) in the postsynaptic PC that are critical for its innervation by CFs. To investigate whether ATXN1[30Q]-D776 curbs the progressive disease in ATXN1[82Q]-S776 mice, we crossed ATXN1[30Q]-D776 and ATXN1[82Q]-S776 mice and found that double transgenic mice developed progressive PC atrophy. Thus, the results also show that to develop progressive cerebellar degeneration requires expressing ATXN1 with an expanded polyglutamine tract.

Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc-Brn1b and linc-Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr(-/-) neonates, whereas linc-Brn1b(-/-) mutants displayed distinct abnormalities in the generation of upper layer II-IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules. DOI: http://dx.doi.org/10.7554/eLife.01749.001.

The cerebellum is organized into zonal circuits that are thought to regulate ongoing motor behavior. Recent studies suggest that neuronal birthdates, gene expression patterning, and apoptosis control zone formation. Importantly, developing Purkinje cell zones are thought to provide the framework upon which afferent circuitry is organized. Yet, it is not clear whether altering the final placement of Purkinje cells affects the assembly of circuits into topographic zones. To gain insight into this problem, we examined zonal connectivity in scrambler mice; spontaneous mutants that have severe Purkinje cell ectopia due to the loss of reelin-disabled1 signaling. We used immunohistochemistry and neural tracing to determine whether displacement of Purkinje cell zones into ectopic positions triggers defects in zonal connectivity within sensory-motor circuits. Despite the abnormal placement of more than 95% of Purkinje cells in scrambler mice, the complementary relationship between molecularly distinct Purkinje cell zones is maintained, and consequently, afferents are targeted into topographic circuits. These data suggest that although loss of disabled1 distorts the Purkinje cell map, its absence does not obstruct the formation of zonal circuits. These findings support the hypothesis that Purkinje cell zones play an essential role in establishing afferent topography.