Astrocytes mediate the effect of oxytocin in the central amygdala on neuronal activity and affective states in rodents.

Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.

Injured Axons Instruct Schwann Cells to Build Constricting Actin Spheres to Accelerate Axonal Disintegration.

After a peripheral nerve lesion, distal ends of injured axons disintegrate into small fragments that are subsequently cleared by Schwann cells and later by macrophages. Axonal debris clearing is an early step of the repair process that facilitates regeneration. We show here that Schwann cells promote distal cut axon disintegration for timely clearing. By combining cell-based and in vivo models of nerve lesion with mouse genetics, we show that this mechanism is induced by distal cut axons, which signal to Schwann cells through PlGF mediating the activation and upregulation of VEGFR1 in Schwann cells. In turn, VEGFR1 activates Pak1, leading to the formation of constricting actomyosin spheres along unfragmented distal cut axons to mediate their disintegration. Interestingly, oligodendrocytes can acquire a similar behavior as Schwann cells by enforced expression of VEGFR1. These results thus identify controllable molecular cues of a neuron-glia crosstalk essential for timely clearing of damaged axons.

GLUT2-Expressing Neurons as Glucose Sensors in the Brain: Electrophysiological Analysis.

Brain glucose sensing plays an essential role in the regulation of energy homeostasis. Recent publications report that neurons expressing glucose transporter GLUT2 act as glucose sensors in different regions of the brain and contribute to the control of glucose homeostasis and feeding behavior. In this chapter we describe the methods used to explore glucose sensing in genetically tagged GLUT2-expressing neurons with slice electrophysiology.

Glucagon-Like Peptide 1 and Its Analogs Act in the Dorsal Raphe and Modulate Central Serotonin to Reduce Appetite and Body Weight.

Glucagon-like peptide 1 (GLP-1) and serotonin play critical roles in energy balance regulation. Both systems are exploited clinically as antiobesity strategies. Surprisingly, whether they interact in order to regulate energy balance is poorly understood. Here we investigated mechanisms by which GLP-1 and serotonin interact at the level of the central nervous system. Serotonin depletion impaired the ability of exendin-4, a clinically used GLP-1 analog, to reduce body weight in rats, suggesting that serotonin is a critical mediator of the energy balance impact of GLP-1 receptor (GLP-1R) activation. Serotonin turnover and expression of 5-hydroxytryptamine (5-HT) 2A (5-HT2A) and 5-HT2C serotonin receptors in the hypothalamus were altered by GLP-1R activation. We demonstrate that the 5-HT2A, but surprisingly not the 5-HT2C, receptor is critical for weight loss, anorexia, and fat mass reduction induced by central GLP-1R activation. Importantly, central 5-HT2A receptors are also required for peripherally injected liraglutide to reduce feeding and weight. Dorsal raphe (DR) harbors cell bodies of serotonin-producing neurons that supply serotonin to the hypothalamic nuclei. We show that GLP-1R stimulation in DR is sufficient to induce hypophagia and increase the electrical activity of the DR serotonin neurons. Finally, our results disassociate brain metabolic and emotionality pathways impacted by GLP-1R activation. This study identifies serotonin as a new critical neural substrate for GLP-1 impact on energy homeostasis and expands the current map of brain areas impacted by GLP-1R activation.

Notch1 Regulates Hippocampal Plasticity Through Interaction with the Reelin Pathway, Glutamatergic Transmission and CREB Signaling.

UNLABELLED: Notch signaling plays a crucial role in adult brain function such as synaptic plasticity, memory and olfaction. Several reports suggest an involvement of this pathway in neurodegenerative dementia. Yet, to date, the mechanism underlying Notch activity in mature neurons remains unresolved. In this work, we investigate how Notch regulates synaptic potentiation and contributes to the establishment of memory in mice. We observe that Notch1 is a postsynaptic receptor with functional interactions with the Reelin receptor, apolipoprotein E receptor 2 (ApoER2) and the ionotropic receptor, N-methyl-D-aspartate receptor (NMDAR). Targeted loss of Notch1 in the hippocampal CA fields affects Reelin signaling by influencing Dab1 expression and impairs the synaptic potentiation achieved through Reelin stimulation. Further analysis indicates that loss of Notch1 affects the expression and composition of the NMDAR but not AMPAR. Glutamatergic signaling is further compromised through downregulation of CamKII and its secondary and tertiary messengers resulting in reduced cAMP response element-binding (CREB) signaling. Our results identify Notch1 as an important regulator of mechanisms involved in synaptic plasticity and memory formation. These findings emphasize the possible involvement of this signaling receptor in dementia. HIGHLIGHTS: In this paper, we propose a mechanism for Notch1-dependent plasticity that likely underlies the function of Notch1 in memory formation: Notch1 interacts with another important developmental pathway, the Reelin cascade.Notch1 regulates both NMDAR expression and composition.Notch1 influences a cascade of cellular events culminating in CREB activation.

Hypoglycemia-activated GLUT2 neurons of the nucleus tractus solitarius stimulate vagal activity and glucagon secretion.

Glucose-sensing neurons in the brainstem participate in the regulation of energy homeostasis but have been poorly characterized because of the lack of specific markers to identify them. Here we show that GLUT2-expressing neurons of the nucleus of the tractus solitarius form a distinct population of hypoglycemia-activated neurons. Their response to low glucose is mediated by reduced intracellular glucose metabolism, increased AMP-activated protein kinase activity, and closure of leak K(+) channels. These are GABAergic neurons that send projections to the vagal motor nucleus. Light-induced stimulation of channelrhodospin-expressing GLUT2 neurons in vivo led to increased parasympathetic nerve firing and glucagon secretion. Thus GLUT2 neurons of the nucleus tractus solitarius link hypoglycemia detection to counterregulatory response. These results may help identify the cause of hypoglycemia-associated autonomic failure, a major threat in the insulin treatment of diabetes.

Sodium sensing in neurons with a dendrimer-based nanoprobe.

Ion imaging is a powerful methodology to assess fundamental biological processes in live cells. The limited efficiency of some ion-sensing probes and their fast leakage from cells are important restrictions to this approach. In this study, we present a novel strategy based on the use of dendrimer nanoparticles to obtain better intracellular retention of fluorescent probes and perform prolonged fluorescence imaging of intracellular ion dynamics. A new sodium-sensitive nanoprobe was generated by encapsulating a sodium dye in a PAMAM dendrimer nanocontainer. This nanoprobe is very stable and has high sodium sensitivity and selectivity. When loaded in neurons in live brain tissue, it homogenously fills the entire cell volume, including small processes, and stays for long durations, with no detectable alterations of cell functional properties. We demonstrate the suitability of this new sodium nanosensor for monitoring physiological sodium responses such as those occurring during neuronal activity.

Optical probing of sodium dynamics in neurons and astrocytes.

Changes in intracellular Na(+) concentration underlie essential neurobiological processes, but few reliable tools exist for their measurement. Here we characterize a new synthetic Na(+)-sensitive fluorescent dye, Asante Natrium Green (ANG), with unique properties. This indicator was excitable in the visible spectrum and by two-photon illumination, suffered little photobleaching and located to the cytosol were it remained for long durations without noticeable unwanted effects on basic cell properties. When used in brain tissue, ANG yielded a bright fluorescent signal during physiological Na(+) responses both in neurons and astrocytes. Synchronous electrophysiological and fluorometric recordings showed that ANG produced accurate Na(+) measurement in situ. This new Na(+) indicator opens innovative ways of probing neuronal circuits.

Swim stress differentially blocks CRF receptor mediated responses in dorsal raphe nucleus.

Modulation of the serotonergic (5-HT) neurotransmitter system arising from the dorsal raphe nucleus (DR) is thought to support the behavioral effects of swim stress, i.e., immobility. In vivo pharmacological and anatomical studies suggest that corticotropin-releasing factor (CRF) and γ-aminobutyric acid (GABA) synaptic transmission closely interact to set the response of the DR to swim stress. To investigate the cellular basis of these physiological mechanisms the effects of ovine CRF (oCRF) on GABA(A)-dependent miniature inhibitory postsynaptic currents (mIPSCs) in 5-HT and non-5-HT DR neurons in acute mesencephalic slices obtained from rats either naïve or 24h after a 15 min swim stress session were tested. In this study, the effect of swim stress alone was to decrease the holding current, i.e., hyperpolarize the neuron, and to increase the amplitude and charge of mIPSCs recorded from non-5-HT neurons. Ovine CRF (10 nM) induced an increase in mIPSC frequency in 5-HT neurons recorded from naïve rats, an effect that was suppressed by swim stress. The inward current elicited by oCRF in both 5-HT and non-5-HT neurons was also blocked by swim stress. Ovine CRF increased mIPSCs amplitude and charge in both 5-HT and non-5-HT neurons, but this effect was not modified by swim stress. In concert with our previous findings that swim stress decreased input resistance, action potential threshold and action potential duration and increased glutamatergic synaptic activity the overall primary effect of swim stress is to increase the excitability of 5-HT neurons. These data provide a mechanism at the cellular level for the immobility induced by swim stress and identifies critical components of the raphe circuitry responsible for the altered output of 5-HT neurons induced by swim stress.

Corticotropin-releasing factor increases GABA synaptic activity and induces inward current in 5-hydroxytryptamine dorsal raphe neurons.

Stress-related psychiatric disorders such as anxiety and depression involve dysfunction of the serotonin [5-hydroxytryptamine (5-HT)] system. Previous studies have found that the stress neurohormone corticotropin-releasing factor (CRF) inhibits 5-HT neurons in the dorsal raphe nucleus (DRN) in vivo. The goals of the present study were to characterize the CRF receptor subtypes (CRF-R1 and -R2) and cellular mechanisms underlying CRF-5-HT interactions. Visualized whole-cell patch-clamp recording techniques in brain slices were used to measure spontaneous or evoked GABA synaptic activity in DRN neurons of rats and CRF effects on these measures. CRF-R1 and -R2-selective agonists were bath applied alone or in combination with receptor-selective antagonists. CRF increased presynaptic GABA release selectively onto 5-HT neurons, an effect mediated by the CRF-R1 receptor. CRF increased postsynaptic GABA receptor sensitivity selectively in 5-HT neurons, an effect to which both receptor subtypes contributed. CRF also had direct effects on DRN neurons, eliciting an inward current in 5-HT neurons mediated by the CRF-R2 receptor and in non-5-HT neurons mediated by the CRF-R1 receptor. These results indicate that CRF has direct membrane effects on 5-HT DRN neurons as well as indirect effects on GABAergic synaptic transmission that are mediated by distinct receptor subtypes. The inhibition of 5-HT DRN neurons by CRF in vivo may therefore be primarily an indirect effect via stimulation of inhibitory GABA synaptic transmission. These results regarding the cellular mechanisms underlying the complex interaction between CRF, 5-HT, and GABA systems could contribute to the development of novel treatments for stress-related psychiatric disorders.

Functional maps of neocortical local circuitry.

This review aims to summarize data obtained with different techniques to provide a functional map of the local circuit connections made by neocortical neurones, a reference for those interested in cortical circuitry and the numerical information required by those wishing to model the circuit. A brief description of the main techniques used to study circuitry is followed by outline descriptions of the major classes of neocortical excitatory and inhibitory neurones and the connections that each layer makes with other cortical and subcortical regions. Maps summarizing the projection patterns of each class of neurone within the local circuit and tables of the properties of these local circuit connections are provided.This review relies primarily on anatomical studies that have identified the classes of neurones and their local and long distance connections and on paired intracellular and whole-cell recordings which have documented the properties of the connections between them. A large number of different types of synaptic connections have been described, but for some there are only a few published examples and for others the details that can only be obtained with paired recordings and dye-filling are lacking. A further complication is provided by the range of species, technical approaches and age groups used in these studies. Wherever possible the range of available data are summarised and compared. To fill some of the more obvious gaps for the less well-documented cases, data obtained with other methods are also summarized.

Selective 5-HT receptor inhibition of glutamatergic and GABAergic synaptic activity in the rat dorsal and median raphe.

The dorsal (DR) and median (MR) raphe nuclei contain 5-hydroxytryptamine (5-HT) cell bodies that give rise to the majority of the ascending 5-HT projections to the forebrain. The DR and MR have differential roles in mediating stress, anxiety and depression. Glutamate and GABA activity sculpt putative 5-HT neuronal firing and 5-HT release in a seemingly differential manner in the MR and DR, yet isolated glutamate and GABA activity within the DR and MR has not been systematically characterized. Visualized whole-cell voltage-clamp techniques were used to record excitatory and inhibitory postsynaptic currents (EPSC and IPSC) in 5-HT-containing neurons. There was a regional variation in action potential-dependent (spontaneous) and basal [miniature (m)] glutamate and GABAergic activity. mEPSC activity was greater than mIPSC activity in the DR, whereas in the MR the mIPSC activity was greater. These differences in EPSC and IPSC frequency indicate that glutamatergic and GABAergic input have distinct cytoarchitectures in the DR and MR. 5-HT(1B) receptor activation decreased mEPSC frequency in the DR and the MR, but selectively inhibited mIPSC activity only in the MR. This finding, in concert with its previously described function as an autoreceptor, suggests that 5-HT(1B) receptors influence the ascending 5-HT system through multiple mechanisms. The disparity in organization and integration of glutamatergic and GABAergic input to DR and MR neurons and their regulation by 5-HT(1B) receptors may contribute to the distinction in MR and DR regulation of forebrain regions and their differential function in the aetiology and pharmacological treatment of psychiatric disease states.

Role of corticotropin-releasing factor pathways in stress-related alterations of colonic motor function and viscerosensibility in female rodents.

BACKGROUND: Clinical reports have shown that irritable bowel syndrome (IBS) is comorbid with anxiety/depression and stress-related events, and that the disorder is more prevalent among women than among men. In rodents, colorectal distention (CRD) induces abdominal contractions, and this visceromotor response is used to assess visceral pain. The activation of brain corticotropin-releasing factor (CRF) pathways has a key role in the behavioral and visceral responses to stress. OBJECTIVE: In this review of experimental studies that delineate the underlying mechanisms of the stress response, we focused on CRF signaling pathways and sex hormones in modulating visceral hypersensitivity induced by CRD in rodents. METHODS: The findings of our recent research on the development of an experimental model of visceral pain in female rats and the modulation of the hyperalgesic response to CRD by CRF antagonists were integrated with those of the published literature. A MEDLINE search of the years 1981 to 2005 was conducted using the key words stress, CRF, CRH, CRF1 receptor, IBS, CRD, female rat, visceral pain, estrogen, and anxiety. RESULTS: CRF and other related mammalian peptides (urocortins) interact with the distinct CRF subtype 1 and 2 receptors. Well-documented preclinical studies have established the role of brain CRF1 receptors in mediating stress-related anxiogenic and visceral (stimulation of colonic motor function and sensitization to repeated CRD) responses in male rodents, whereas more limited studies have been performed in female rats. Our recent study indicated that the CRF1 antagonist antalarmin prevents visceral hypersensitivity induced by 2 sets of CRD in female rats. In several models of visceral pain induced by CRD, sex differences and a sensitization action of estrogen were reported. Our preliminary evidence indicated a potentiating interaction between CRF-CRF1 pathways and estrogen in the stimulation of colonic motor responses that may take place within the enteric neurons of the colon, where both CRF1 and estrogen receptors are present. CONCLUSIONS: The results of this review suggest that overactivity of CRF1 signaling in the brain and the gut may have relevance in understanding the comorbidity of anxiety/depression and IBS in diarrhea-predominant female patients. Targeting these mechanisms with CRF1 antagonists may provide a novel therapeutic strategy.

Repeated exposure to water avoidance stress in rats: a new model for sustained visceral hyperalgesia.

Chronic stress plays an important role in the development and exacerbation of symptoms in functional gastrointestinal disorders. To better understand the mechanisms underlying this relationship, we aimed to characterize changes in visceral and somatic nociception, colonic motility, anxiety-related behavior, and mucosal immune activation in rats exposed to 10 days of chronic psychological stress. Male Wistar rats were submitted daily to either 1-h water avoidance (WA) stress or sham WA for 10 consecutive days. The visceromotor response to colorectal distension, thermal somatic nociception, and behavioral responses to an open field test were measured at baseline and after chronic WA. Fecal pellets were counted after each WA stress or sham WA session as a measure of stress-induced colonic motility. Colonic samples were collected from both groups and evaluated for structural changes and neutrophil infiltration, mast cell number by immunohistochemistry, and cytokine expression by quantitative RT-PCR. Rats exposed to chronic WA (but not sham stress) developed persistent visceral hyperalgesia, whereas only transient changes in somatic nociception were observed. Chronically stressed rats also exhibited anxiety-like behaviors, enhanced fecal pellet excretion, and small but significant increases in the mast cell numbers and the expression of IL-1beta and IFN-gamma. Visceral hyperalgesia following chronic stress persisted for at least a month. Chronic psychological stress in rats results in a robust and long-lasting alteration of visceral, but not somatic nociception. Visceral hyperalgesia is associated with other behavioral manifestations of stress sensitization but was only associated with minor colonic immune activation arguing against a primary role of mucosal immune activation in the maintenance of this phenomenon.