06-896 Sigma-AldrichAnti-GFP (Green Fluorescent Protein) 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.

A transcriptional programme initiated by the proneural factors Neurog2 and Ascl1 controls successive steps of neurogenesis in the embryonic cerebral cortex. Previous work has shown that proneural factors also confer a migratory behaviour to cortical neurons by inducing the expression of the small GTP-binding proteins such as Rnd2 and Rnd3. However, the directionality of radial migration suggests that migrating neurons also respond to extracellular signal-regulated pathways. Here we show that the Plexin B2 receptor interacts physically and functionally with Rnd3 and stimulates RhoA activity in migrating cortical neurons. Plexin B2 competes with p190RhoGAP for binding to Rnd3, thus blocking the Rnd3-mediated inhibition of RhoA and also recruits RhoGEFs to directly stimulate RhoA activity. Thus, an interaction between the cell-extrinsic Plexin signalling pathway and the cell-intrinsic Ascl1-Rnd3 pathway determines the level of RhoA activity appropriate for cortical neuron migration.

When nanoparticles/proteins are infused into the brain, they are often transported to distal sites in a manner that is dependent both on the characteristics of the infusate and the region targeted. We have previously shown that adeno-associated virus (AAV) is disseminated within the brain by perivascular flow and also by axonal transport. Perivascular distribution usually does not depend strongly on the nature of the infusate. Many proteins, neutral liposomes and AAV particles distribute equally well by this route when infused under pressure into various parenchymal locations. In contrast, axonal transport requires receptor-mediated uptake of AAV by neurons and engagement with specific transport mechanisms previously demonstrated for other neurotropic viruses. Cerebrospinal fluid (CSF) represents yet another way in which brain anatomy may be exploited to distribute nanoparticles broadly in the central nervous system. In this study, we assessed the distribution and perivascular transport of nanoparticles of different sizes delivered into the parenchyma of rodents and CSF in non-human primates.

In rodents and most other mammals studied, neuronal precursors generated in the subventricular zone (SVZ) migrate to the adult olfactory bulb (OB) to differentiate into interneurons called granule and periglomerular cells. How the newborn cells navigate in the postnatal forebrain to reach precisely their target area is largely unknown. However, it is often thought that postnatal neurogenesis recapitulates the neuronal development occurring during embryogenesis. During brain development, intracellular kinases are key elements for controlling cell polarization as well as the coupling between polarization and cellular movement. We show here that the polarity kinase MARK2 maintains its expression in the postnatal SVZ-OB system. We therefore investigated the potential role of this kinase in adjusting postnatal neuroblast migration. We employed mouse brain slices maintained in culture, in combination with lentiviral vector injections designed to label neuronal precursors with GFP and to diminish the expression of MARK2. Time-lapse video microscopy was used to monitor neuroblast migration in the postnatal forebrain from SVZ precursors to cells populating the OB. We found that reduced MARK2 expression resulted in altered migratory patterns and stalled neuroblasts in the rostral migratory stream (RMS). In agreement with the observed migratory defects, we report a diminution of the proportion of cells reaching the OB layers. Our study reveals the involvement of MARK2 in the maintenance of the migratory direction in postnatally-generated neuroblasts and consequently on the control of the number of newly-generated neurons reaching and integrating the appropriate target circuits.

Electrical and pharmacological stimulation methods are commonly used to study neuronal brain circuits in vivo, but are problematic, because electrical stimulation has limited specificity, while pharmacological activation has low temporal resolution. A recently developed alternative to these methods is the use of optogenetic techniques, based on the expression of light sensitive channel proteins in neurons. While optogenetics have been applied in in vitro preparations and in in vivo studies in rodents, their use to study brain function in nonhuman primates has been limited to the cerebral cortex. Here, we characterize the effects of channelrhodopsin-2 (ChR2) transfection in subcortical areas, i.e., the putamen, the external globus pallidus (GPe) and the ventrolateral thalamus (VL) of rhesus monkeys. Lentiviral vectors containing the ChR2 sequence under control of the elongation factor 1α promoter (pLenti-EF1α -hChR2(H134R)-eYFP-WPRE, titer 10⁹ particles/ml) were deposited in GPe, putamen and VL. Four weeks later, a probe combining a conventional electrode and an optic fiber was introduced in the previously injected brain areas. We found light-evoked responses in 31.5% and 32.7% of all recorded neurons in the striatum and thalamus, respectively, but only in 2.5% of recorded GPe neurons. As expected, most responses were time-locked increases in firing, but decreases or mixed responses were also seen, presumably via ChR2-mediated activation of local inhibitory connections. Light and electron microscopic analyses revealed robust expression of ChR2 on the plasma membrane of cell somas, dendrites, spines and terminals in the striatum and VL. This study demonstrates that optogenetic experiments targeting the striatum and basal ganglia-related thalamic nuclei can be successfully achieved in monkeys. Our results indicate important differences of the type and magnitude of responses in each structure. Experimental conditions such as the vector used, the number and rate of injections, or the light stimulation conditions have to be optimized for each structure studied.

Photoreceptor terminals contain post-synaptic density (PSD) proteins e.g., PSD-95/PSD-93, but their role at photoreceptor synapses is not known. PSDs are generally restricted to post-synaptic boutons in central neurons and form scaffolding with multiple proteins that have structural and functional roles in neuronal signaling. The Shank family of proteins (Shank 1-3) functions as putative anchoring proteins for PSDs and is involved in the organization of cytoskeletal/signaling complexes in neurons. Specifically, Shank 1 is restricted to neurons and interacts with both receptors and signaling molecules at central neurons to regulate plasticity. However, it is not known whether Shank 1 is expressed at photoreceptor terminals. In this study we have investigated Shank 1A localization in the outer retina at photoreceptor terminals. We find that Shank 1A is expressed presynaptically in cone pedicles, but not rod spherules, and it is absent from mice in which the Shank 1 gene is deleted. Shank 1A co-localizes with PSD-95, peanut agglutinin, a marker of cone terminals, and glycogen phosphorylase, a cone specific marker. These findings provide convincing evidence for Shank 1A expression in both the inner and outer plexiform layers, and indicate a potential role for PSD-95/Shank 1 complexes at cone synapses in the outer retina.

Anti-apoptotic Bcl2 family proteins such as Bcl-x(L) protect cells from death by sequestering apoptotic molecules, but also contribute to normal neuronal function. We find in hippocampal neurons that Bcl-x(L) enhances the efficiency of energy metabolism. Our evidence indicates that Bcl-x(L)interacts directly with the β-subunit of the F(1)F(O) ATP synthase, decreasing an ion leak within the F(1)F(O) ATPase complex and thereby increasing net transport of H(+) by F(1)F(O) during F(1)F(O) ATPase activity. By patch clamping submitochondrial vesicles enriched in F(1)F(O) ATP synthase complexes, we find that, in the presence of ATP, pharmacological or genetic inhibition of Bcl-x(L) activity increases the membrane leak conductance. In addition, recombinant Bcl-x(L) protein directly increases the level of ATPase activity of purified synthase complexes, and inhibition of endogenous Bcl-x(L) decreases the level of F(1)F(O) enzymatic activity. Our findings indicate that increased mitochondrial efficiency contributes to the enhanced synaptic efficacy found in Bcl-x(L)-expressing neurons.

From an early postnatal period and throughout life there is a continuous production of olfactory bulb (OB) interneurons originating from neuronal precursors in the subventricular zone. To reach the OB circuits, immature neuroblasts migrate along the rostral migratory stream (RMS). In the present study, we employed cultured postnatal mouse forebrain slices and used lentiviral vectors to label neuronal precursors with GFP and to manipulate the expression levels of the Na-K-2Cl cotransporter NKCC1. We investigated the role of this Cl- transporter in different stages of postnatal neurogenesis, including neuroblast migration and integration in the OB networks once they have reached the granule cell layer (GCL). We report that NKCC1 activity is necessary for maintaining normal migratory speed. Both pharmacological and genetic manipulations revealed that NKCC1 maintains high [Cl-]i and regulates the resting membrane potential of migratory neuroblasts whilst its functional expression is strongly reduced at the time cells reach the GCL. As in other developing systems, NKCC1 shapes GABAA-dependent signaling in the RMS neuroblasts. Also, we show that NKCC1 controls the migration of neuroblasts in the RMS. The present study indeed indicates that the latter effect results from a novel action of NKCC1 on the resting membrane potential, which is independent of GABAA-dependent signaling. All in all, our findings show that early stages of the postnatal recruitment of OB interneurons rely on precise, orchestrated mechanisms that depend on multiple actions of NKCC1.

Repair of myelin injury in multiple sclerosis may fail, resulting in chronic demyelination, axonal loss, and disease progression. As cellular pathways regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN; eg, phosphatidylinositol-3-kinase [PI-3K]) have been reported to enhance axon regeneration and oligodendrocyte maturation, we investigated potentially beneficial effects of Pten loss of function in the oligodendrocyte lineage on remyelination.We characterized oligodendrocyte numbers and myelin sheath thickness in mice with conditional inactivation of Pten in oligodendrocytes, Olig2-cre, Pten(fl/fl) mice. Using a model of central nervous system demyelination, lysolecithin injection into the spinal cord white matter, we performed short- and long-term lesioning experiments and quantified oligodendrocyte maturation and myelin sheath thickness in remyelinating lesions.During development, we observed dramatic hypermyelination in the corpus callosum and spinal cord. Following white matter injury, however, there was no detectable improvement in remyelination. Moreover, we observed progressive myelin sheath abnormalities and massive axon degeneration in the fasciculus gracilis of mutant animals, as indicated by ultrastructure and expression of SMI-32, amyloid precursor protein, and caspase 6.These studies indicate adverse effects of chronic Pten inactivation (and by extension, activation PI-3K signaling) on myelinating oligodendrocytes and their axonal targets. We conclude that PTEN function in oligodendrocytes is required to regulate myelin thickness and preserve axon integrity. In contrast, PTEN is dispensable during myelin repair, and its inactivation confers no detectable benefit.

The use of transgenic mice in which subtypes of neurons are labeled with a fluorescent protein has greatly facilitated modern neuroscience research. GAD65-GFP mice, which have GABAergic interneurons labeled with GFP, are widely used in many research laboratories, although the properties of the labeled cells have not been studied in detail. Here we investigate these cells in the hippocampal area CA1 and show that they constitute ∼20% of interneurons in this area. The majority of them expresses either reelin (70±2%) or vasoactive intestinal peptide (VIP; 15±2%), while expression of parvalbumin and somatostatin is virtually absent. This strongly suggests they originate from the caudal, and not the medial, ganglionic eminence. GFP-labeled interneurons can be subdivided according to the (partially overlapping) expression of neuropeptide Y (42±3%), cholecystokinin (25±3%), calbindin (20±2%) or calretinin (20±2%). Most of these subtypes (with the exception of calretinin-expressing interneurons) target the dendrites of CA1 pyramidal cells. GFP-labeled interneurons mostly show delayed onset of firing around threshold, and regular firing with moderate frequency adaptation at more depolarized potentials.