Cellular Neuroscience

Recent evidence in vitro suggests that the tuft dendrites of pyramidal neurons are capable of evoking local NMDA receptordependent electrogenesis, so-called NMDA spikes. However, it has so far proved difficult to demonstrate their existence in vivo. Moreover, it is not clear whether NMDA spikes are relevant to the output of pyramidal neurons. We found that local NMDA spikes occurred in tuft dendrites of layer 2/3 pyramidal neurons both spontaneously and following sensory input, and had a large influence on the number of output action potentials. Using two-photon activation of an intracellular caged NMDA receptor antagonist (tc-MK801), we found that isolated NMDA spikes typically occurred in multiple branches simultaneously and that sensory stimulation substantially increased their probability. Our results demonstrate that NMDA receptors have a vital role in coupling the tuft region of the layer 2/3 pyramidal neuron to the cell body, enhancing the effectiveness of layer 1 input.

SynCAM1 is an adhesion molecule involved in synaptic differentiation and organization. SynCAM1 is also expressed in astroglial cells where it mediates astrocyte-to astrocyte and glial-neuronal adhesive communication. In astrocytes, SynCAM1 is functionally linked to erbB4 receptors, which are involved in the control of both neuronal/glial development and mature neuronal and glial function. Here we report that mice carrying a dominant-negative form of SynCAM1 specifically targeted to astrocytes (termed GFAP-DNSynCAM1 mice) exhibit disrupted diurnal locomotor activity with enhanced and more frequent episodes of activity than control littermates during the day (when the animals are normally sleeping) accompanied by shorter periods of rest. GFAP-DNSynCAM1 mice also display high levels of basal activity in the dark period (the rodent's awake/active time) that are attenuated by the psychostimulant D,L-amphetamine, and reduced anxiety levels in response to both avoidable and unavoidable provoking stimuli. These results indicate that disruption of SynCAM1-dependent astroglial function results in behavioral abnormalities similar to those described in animals model of attention-deficit hyperactive disorder (ADHD), and suggest a hitherto unappreciated contribution of glial cells to the pathophysiology of this disorder.

Mutations in SHANK3 and large duplications of the region spanning SHANK3 both cause a spectrum of neuropsychiatric disorders, indicating that proper SHANK3 dosage is critical for normal brain function. However, SHANK3 overexpression per se has not been established as a cause of human disorders because 22q13 duplications involve several genes. Here we report that Shank3 transgenic mice modelling a human SHANK3 duplication exhibit manic-like behaviour and seizures consistent with synaptic excitatory/inhibitory imbalance. We also identified two patients with hyperkinetic disorders carrying the smallest SHANK3-spanning duplications reported so far. These findings indicate that SHANK3 overexpression causes a hyperkinetic neuropsychiatric disorder. To probe the mechanism underlying the phenotype, we generated a Shank3 in vivo interactome and found that Shank3 directly interacts with the Arp2/3 complex to increase F-actin levels in Shank3 transgenic mice. The mood-stabilizing drug valproate, but not lithium, rescues the manic-like behaviour of Shank3 transgenic mice raising the possibility that this hyperkinetic disorder has a unique pharmacogenetic profile.

Balance, symmetry and normality dominate our thinking and culture, perhaps because of their simplicity. In neuroscience, we seek for and tend to present 'typical' or 'representative' neurons, dendritic arbors, spines, axon calibres and connectivity in our communications. For example, population activity is typically summarized by the mean firing rate of the contributing neurons or by the average number of neurons participating in various network patterns. Similarly, changes in synaptic strength, spike transfer function and other evoked and induced features are condensed to a single mean to characterize drug-or plasticity-induced effects or changes brought about by learning. Likewise, the power of the electro encephalogram (EEG) or the strength of the blood-oxygen-level-dependent (BOLD) signal is typically reported by a single mean value for comparison across experimental groups. The differences between mean values across groups or experimental conditions are then quantified by statistics based on symmetrical, bell-shaped Gaussian (normal) probability distributions. However, recent advances, which are summarized in this Review, suggest that such simplification is no longer tenable because the majority of interactions in highly interconnected systems, especially in biological systems, are multiplicative and synergistic rather than additive.

<p><b>A:</b> Rhythmic changes in AGR inst. ff. disappear after desheathing. During a gastric mill rhythm, AGR inst. ff. (top trace) changed rhythmically. Bottom traces: extracellular recordings of <i>mvn</i>, <i>dgn</i> and <i>lgn</i> showing the timing of the gastric mill rhythm. Left: before desheathing; right: after desheathing the STG. Raw data are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103459#pone.0103459.s003" target="_blank">Raw Data.xlsx S1</a>. <b>B:</b> Phase diagram of normalized averaged AGR inst. ff. and the gastric mill motor neurons LG, GM and DG before (left) and after (right) desheathing, from the same animal. AGR inst. ff. was binned (bin width 0.1). The grey surface shows the standard deviation. ***significant difference with p<0.001. <b>C:</b> Rhythmic changes in AGR inst. ff. were not affected by RH795. Left: non-desheathed STG, before staining, right: non-desheathed STG after 1 h staining and 20 minutes washout. <b>D:</b> Phase diagram of the same animal before (left) and after (right) staining with RH795. For detail see (B). Phases 0.3 and 0.7 were significantly different. ***p<0.001 before staining and **p<0.01 after staining. There was no significant difference between the mean values of all phases before and after staining (see text for details).</p

<p>Lucifer yellow (0.25 mg/ml) uptake in response to P2X7 stimulation by ATP (3 mM) was measured using a fixed time incubation at 37°C. Coverslips were briefly washed in standard extracellular solution before being imaged. (A) Phase contrast and fluorescent images for each stimulation condition; Control, ATP (3 mM) and ATP+PRO (2.5 mM) and (B) mean data from 100–300 cells (regions of interest) from multiple images.</p

Microglia are myeloid cells of the central nervous system (CNS) that participate both in normal CNS function and disease. We investigated the molecular signature of microglia and identified 239 genes and 8 microRNAs that were uniquely or highly expressed in microglia vs. myeloid and other immune cells. Out of 239 genes, 106 were enriched in microglia as compared to astrocytes, oligodendrocytes and neurons. This microglia signature was not observed in microglial lines or in monocytes recruited to the CNS and was also observed in human microglia. Based on this signature, we found a crucial role for TGF-β in microglial biology that included: 1) the requirement of TGF-β for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia; and 2) the absence of microglia in CNS TGF-β1 deficient mice. Our results identify a unique microglial signature that is dependent on

SynCAM1 is an adhesion molecule involved in synaptic differentiation and organization. SynCAM1 is also expressed in astroglial cells where it mediates astrocyte-to astrocyte and glial-neuronal adhesive communication. In astrocytes, SynCAM1 is functionally linked to erbB4 receptors, which are involved in the control of both neuronal/glial development and mature neuronal and glial function. Here we report that mice carrying a dominant-negative form of SynCAM1 specifically targeted to astrocytes (termed GFAP-DNSynCAM1 mice) exhibit disrupted diurnal locomotor activity with enhanced and more frequent episodes of activity than control littermates during the day (when the animals are normally sleeping) accompanied by shorter periods of rest. GFAP-DNSynCAM1 mice also display high levels of basal activity in the dark period (the rodent's awake/active time) that are attenuated by the psychostimulant D,L-amphetamine, and reduced anxiety levels in response to both avoidable and unavoidable provoking stimuli. These results indicate that disruption of SynCAM1-dependent astroglial function results in behavioral abnormalities similar to those described in animals model of attention-deficit hyperactive disorder (ADHD), and suggest a hitherto unappreciated contribution of glial cells to the pathophysiology of this disorder.

The blood vessel is no longer viewed as passive plumbing for the brain. Increasingly, experimental and clinical findings suggest that cerebral endothelium may possess endocrine and paracrine properties -actively releasing signals into and receiving signals from the neuronal parenchyma. Hence, metabolically perturbed microvessels may contribute to central nervous system (CNS) injury and disease. Furthermore, cerebral endothelium can serve as sensors and integrators of CNS dysfunction, releasing measurable biomarkers into the circulating bloodstream. Here, we define and analyze the concept of a brain vasculome, i.e. a database of gene expression patterns in cerebral endothelium that can be linked to other databases and systems of CNS mediators and markers. Endothelial cells were purified from mouse brain, heart and kidney glomeruli. Total RNA were extracted and profiled on Affymetrix mouse 430 2.0 micro-arrays. Gene expression analysis confirmed that these brain, heart and glomerular preparations were not contaminated by brain cells (astrocytes, oligodendrocytes, or neurons), cardiomyocytes or kidney tubular cells respectively. Comparison of the vasculome between brain, heart and kidney glomeruli showed that endothelial gene expression patterns were highly organ-dependent. Analysis of the brain vasculome demonstrated that many functionally active networks were present, including cell adhesion, transporter activity, plasma membrane, leukocyte transmigration, Wnt signaling pathways and angiogenesis. Analysis of representative genome-wide-association-studies showed that genes linked with Alzheimer's disease, Parkinson's disease and stroke were detected in the brain vasculome. Finally, comparison of our mouse brain vasculome with representative plasma protein databases demonstrated significant overlap, suggesting that the vasculome may be an important source of circulating signals in blood. Perturbations in cerebral endothelial function may profoundly affect CNS homeostasis. Mapping and dissecting the vasculome of the brain in health and disease may provide a novel database for investigating disease mechanisms, assessing therapeutic targets and exploring new biomarkers for the CNS.

In mammalian neurons, targeting and translation of specific mRNAs in dendrites contribute to synaptic plasticity. After nuclear export, mRNAs designated for dendritic transport are generally assumed to be translationally dormant and activity of individual synapses may locally trigger their extrasomatic translation. We show that the long, GC-rich 59-untranslated region of dendritic SAPAP3 mRNA restricts translation initiation via a mechanism that involves an upstream open reading frame (uORF). In addition, the uORF enables the use of an alternative translation start site, permitting synthesis of two SAPAP3 isoforms from a single mRNA. While both isoforms progressively accumulate at postsynaptic densities during early rat brain development, their levels relative to each other vary in different adult rat brain areas. Thus, alternative translation initiation events appear to regulate relative expression of distinct SAPAP3 isoforms in different brain regions, which may function to influence synaptic plasticity.

Forebrain circuits rely upon a relatively small but remarkably diverse population of GABAergic interneurons to bind and entrain large principal cell assemblies for network synchronization and rhythmogenesis. Despite the high degree of heterogeneity across cortical interneurons, members of a given subtype typically exhibit homogeneous developmental origins, neuromodulatory response profiles, morphological characteristics, neurochemical signatures and electrical features. Here we report a surprising divergence among hippocampal oriens-lacunosum moleculare (O-LM) projecting interneurons that have hitherto been considered a homogeneous cell population. Combined immunocytochemical, anatomical and electrophysiological interrogation of Htr3a-GFP and Nkx2-1-cre:RCE mice revealed that O-LM cells parse into a caudal ganglionic eminence-derived subpopulation expressing 5-HT 3A receptors (5-HT 3A Rs) and a medial ganglionic eminence-derived subpopulation lacking 5-HT 3A Rs. These two cohorts differentially participate in network oscillations, with 5-HT 3A R-containing O-LM cell recruitment dictated by serotonergic tone. Thus, members of a seemingly uniform interneuron population can exhibit unique circuit functions and neuromodulatory properties dictated by disparate developmental origins. npg

In the developing mammalian brain, gamma-aminobutyric acid (GABA) is thought to play an excitatory rather than an inhibitory role due to high levels of intracellular Cl 2 in immature neurons. This idea, however, has been questioned by recent studies which suggest that glucose-based artificial cerebrospinal fluid (ACSF) may be inadequate for experiments on immature and developing brains. These studies suggest that immature neurons may require alternative energy sources, such as lactate or pyruvate. Lack of these other energy sources is thought to result in artificially high intracellular Cl 2 concentrations, and therefore a more depolarized GABA receptor (GABAR) reversal potential. Since glucose metabolism can vary widely among different species, it is important to test the effects of these alternative energy sources on different experimental preparations. We tested whether pyruvate affects GABAergic transmission in isolated brains of developing wild type Xenopus tadpoles in vitro by recording the responsiveness of tectal neurons to optic nerve stimulation, and by measuring currents evoked by local GABA application in a gramicidin perforated patch configuration. We found that, in contrast with previously reported results, the reversal potential for GABAR-mediated currents does not change significantly between developmental stages 45 and 49. Partial substitution of glucose by pyruvate had only minor effects on both the GABA reversal potential, and the responsiveness of tectal neurons at stages 45 and 49. Total depletion of energy sources from the ACSF did not affect neural responsiveness. We also report a strong spatial gradient in GABA reversal potential, with immature cells adjacent to the lateral and caudal proliferative zones having more positive reversal potentials. We conclude that in this experimental preparation standard glucose-based ACSF is an appropriate extracellular media for in vitro experiments.

The problem of gain control in a neuron that faces thousands of synaptic stimuli has received much attention. Studies of relatively simple models established that synaptic gain can be modulated by multiple factors, such as presynaptic plasticity and/or intrinsic neuronal nonlinearities . However, most central synapses are characterized by small active zones and high stochasticity of vesicular release . In addition, dendritic trees abound with active conductances . All of the above calls for a revised look at the simplified mean-field models of gain modulation.