Dervla O'Malley

Leptin inhibits epileptiform-like activity in rat hippocampal neurones via PI 3-kinase-driven activation of BK channels

The obese gene product, leptin is an important circulating satiety factor that regulates energy balance via its actions in the hypothalamus. However, leptin receptors are also expressed in brain regions not directly associated with energy homeostasis, such as the hippocampus. Here, leptin inhibits hippocampal neurones via activation of large conductance Ca2+‐activated K+ (BK) channels, a process that may be important in regulating neuronal excitability. We now show that leptin receptor labelling is expressed on somata, dendrites and axons, and is also concentrated at synapses in hippocampal cultures. In functional studies, leptin potently and reversibly reduces epileptiform‐like activity evoked in lean, but not leptin‐resistant Zucker fa/fa rats. Furthermore, leptin also depresses enhanced Ca2+ levels evoked following Mg2+ removal in hippocampal cultures. The ability of leptin to modulate this activity requires activation of BK, but not KATP, channels as the effects of leptin were mimicked by the BK channel activator NS‐1619, and inhibited by the BK channel inhibitors, iberiotoxin and charybdotoxin. The signalling mechanisms underlying this process involve stimulation of phosphoinositide 3‐kinase (PI 3‐kinase), but not mitogen‐activated protein kinase (MAPK), as two structurally unrelated inhibitors of PI 3‐kinase, LY294002 and wortmannin, blocked the actions of leptin. These data indicate that leptin, via PI 3‐kinase‐driven activation of BK channels, elicits a novel mechanism for controlling neuronal excitability. As uncontrolled excitability in the hippocampus is one underlying cause of temporal lobe epilepsy, this novel action of leptin could provide an alternative therapeutic target in the management of epilepsy.

Region specific decrease in glial fibrillary acidic protein immunoreactivity in the brain of a rat model of depression

A growing body of evidence from human postmortem and animal studies suggests that deficits in glial cell (particularly astrocytes) density and function, in limbic regions of the brain contribute to the etiology of depressive disorders. Despite the widespread use of Wistar-Kyoto (WKY) rat strain as a model of depression and stress susceptibility, there is a paucity of data examining whether alterations in brain astrocytic population are present in the model. In the present study, we investigated the expression of the astrocytic markers glial fibrillary acidic protein (GFAP) in various brain regions in WKY rats in comparison to Sprague-Dawley rats. A significant deficit in GFAP-immunoreactive cells was found in the prefrontal cortex region (infralimbic, prelimbic and anterior cingulate cortex), in the basolateral amygdala as well as in the hippocampus (CA3 and dentate gyrus) in WKY rat brain. No statistical difference was found in the other brain regions analyzed (insular cortex, somatosensory cortex, CA1 and callosal white matter). No difference was found in the total density of astrocytes (assessed by s-100beta immunoreactivity), neurons (determined by NeuN expression) or in the total number of cells in the regions of interest. A slight increase in the intensity of s-100beta immunoreactivity was observed. The lower expression of GFAP in WKY rats was further confirmed by Western-blot analysis. These results suggest that specific astrocytic deficits in GFAP expression in corticolimbic circuits may be a general correlate of depressive-like behavior in animal models in addition to human major depression. Moreover, they suggest that glial physiology may become a therapeutic target in depression and other stress-related conditions.

Sodium-Coupled Glucose Cotransporters Contribute to Hypothalamic Glucose Sensing

Specialized neurons within the hypothalamus have the ability to sense and respond to changes in ambient glucose concentrations. We investigated the mechanisms underlying glucose-triggered activity in glucose-excited neurons, using primary cultures of rat hypothalamic neurons monitored by fluorescence calcium imaging. We found that 35% (738 of 2,139) of the neurons were excited by increasing glucose from 3 to 15 mmol/l, but only 9% (6 of 64) of these glucose-excited neurons were activated by tolbutamide, suggesting the involvement of a ATP-sensitive K+ channel–independent mechanism. α-Methylglucopyranoside (αMDG; 12 mmol/l), a nonmetabolizable substrate of sodium glucose cotransporters (SGLTs), mimicked the effect of high glucose in 67% of glucose-excited neurons, and both glucose- and αMDG-triggered excitation were blocked by Na+ removal or by the SGLT inhibitor phloridzin (100 nmol/l). In the presence of 0.5 mmol/l glucose and tolbutamide, responses could also be triggered by 3.5 mmol/l αMDG, supporting a role for an SGLT-associated mechanism at low as well as high substrate concentrations. Using RT-PCR, we detected SGLT1, SGLT3a, and SGLT3b in both cultured neurons and adult rat hypothalamus. Our findings suggest a novel role for SGLTs in glucose sensing by hypothalamic glucose-excited neurons.

Distinct alterations in colonic morphology and physiology in two rat models of enhanced stress-induced anxiety and depression-like behaviour

Stress and anxiety are important causal and exacerbating factors in functional gastro-intestinal (GI) disorders such as irritable bowel syndrome. Stress affects GI motility, faecal transit and visceral pain sensitivity. Additionally, permeability and function of the gut epithelium, which acts as a barrier between the external environment and the bodys internal milieu is altered by stress. However, the effects of an enhanced stress response on colonic morphology require further investigation. We have used two animal models of stress and anxiety, the maternally separated (MS) and Wistar Kyoto (WKY) rats to examine colonic morphology. These rats exhibit increased anxiety behaviours, visceral hypersensitivity and increased stress-induced defecation in the open field arena. At a morphological level, increased mucus secretion and an associated elevation in the number of mucosal goblet cells was observed in the high anxiety rats. Additionally, the mucosal layer was flattened in MS and WKY rats, a finding indicative of mild mucosal damage. Furthermore, the muscular layer of the distal colon in these animals was thickened, an observation that may have implications for faecal transit and visceral pain perception. This study provides evidence of altered colonic function and morphology in two animal models with a heightened response to stress.

The neurotransmitters glycine and GABA stimulate glucagon‐like peptide‐1 release from the GLUTag cell line

The incretin hormone, glucagon‐like peptide‐1 (GLP‐1) is released from intestinal L‐cells following food ingestion. Its secretion is triggered by a range of nutrients, including fats, carbohydrates and proteins. We reported previously that Na+‐dependent glutamine uptake triggered electrical activity and GLP‐1 release from the L‐cell model line GLUTag. However, whereas alanine also triggered membrane depolarization and GLP‐1 secretion, the response was Na+ independent. A range of alanine analogues, including d‐alanine, β‐alanine, glycine and l‐serine, but not d‐serine, triggered similar depolarizing currents and elevation of intracellular [Ca2+], a sensitivity profile suggesting the involvement of glycine receptors. In support of this idea, glycine‐induced currents and GLP‐1 release were blocked by strychnine, and currents showed a 58.5 mV shift in reversal potential per 10‐fold change in [Cl−], consistent with the activation of a Cl−‐selective current. GABA, an agonist of related Cl− channels, also triggered Cl− currents and secretion, which were sensitive to picrotoxin. GABA‐triggered [Ca2+]i increments were abolished by bicuculline and partially impaired by (1,2,5,6‐tetrahydropyridine‐4‐yl)methylphosphinic acid (TPMPA), suggesting the involvement of both GABAA and GABAC receptors. Expression of GABAA, GABAC and glycine receptor subunits was confirmed by RT‐PCR. Glycine‐triggered GLP‐1 secretion was impaired by bumetanide but not bendrofluazide, suggesting that a high intracellular [Cl−] maintained by Na+–K+–2Cl− cotransporters is necessary for the depolarizing response to glycine receptor ligands. Our results suggest that GABA and glycine stimulate electrical activity and GLP‐1 release from GLUTag cells by ligand‐gated ion channel activation, a mechanism that might be important in responses to endogenous ligands from the enteric nervous system or dietary sources.

Colonic soluble mediators from the maternal separation model of irritable bowel syndrome activate submucosal neurons via an interleukin-6-dependent mechanism

Irritable bowel syndrome (IBS) is characterized by episodic bouts of abdominal pain, bloating, and altered bowel habit. Accumulating evidence has linked immune activation with IBS, including reports of increases in circulating levels of the proinflammatory cytokine interleukin (IL)-6. However, it is unknown whether IL-6 contributes directly to disease manifestation. As enteric nervous activity mediates motility and secretory function, the aims of this study were to determine the effects of IL-6 on submucosal neurons and related gastrointestinal (GI) function. In these studies, we examined the colons of maternally separated (MS) rats, which exhibit elevated circulating levels of IL-6 in addition to GI dysfunction. To our knowledge, these studies are the first to provide evidence of the sensitivity of submucosal neurons to colonic secretions from MS rats (n = 50, P < 0.05), thus recapitulating clinical biopsy data. Moreover, we demonstrated that the excitatory action is IL-6 dependent. Thereafter, the impact of IL-6 on neuronal and glial activation and absorpto/secretory function was pharmacologically characterized. Other proinflammatory cytokines including IL-8 (n = 30, P > 0.05), IL-1β (n = 56, P > 0.05), and TNF-α (n = 56, P > 0.05) excited fewer neurons. Both muscarinic and nicotinic cholinergic receptors participate in the effect and cause downstream activation of ERK, JAK-STAT, and NF-κB signaling cascades. Functionally, IL-6 increases transepithelial resistance and enhances neurally and cholinergically mediated ion transport. These data provide a role for IL-6 in colonic secretory functions and relate these effects to GI dysfunction in an animal model of IBS, thereby elucidating a potential relationship between circulating levels of IL-6 and aberrant GI function.

Modulation of enteric neurons by interleukin-6 and corticotropin-releasing factor contributes to visceral hypersensitivity and altered colonic motility in a rat model of irritable bowel syndrome.

Hyperactivity of the stress system and low‐grade immune activation characterize the functional bowel disorder irritable bowel syndrome (IBS). These studies show that interleukin (IL)‐6 and IL‐8 and the stress hormone corticotropin‐releasing factor (CRF), present in IBS plasma, have functional effects on gastrointestinal activity by stimulating myenteric neurons and colonic contractions. Moreover, in the Wistar Kyoto rat model of IBS, which exhibits altered gastrointestinal motility and visceral pain sensitivity, blocking IL‐6 and/or CRF1 receptors alleviates these IBS‐like symptoms. Underlying these effects are altered colonic protein expression of tight junction proteins which regulate gut barrier function and the T‐type calcium channel CaV3.2, which has been linked to visceral pain. These findings demonstrate the importance of the enteric nervous system and intestinal physiology in bowel dysfunction.

MAPK-dependent actin cytoskeletal reorganization underlies BK channel activation by insulin

Numerous brain regions are enriched with insulin and insulin receptors, and several lines of evidence indicate that insulin is an important modulator of neuronal function. Indeed, recent studies have demonstrated that insulin inhibits hippocampal epileptiform‐like activity, in part by activating large‐conductance Ca2+‐activated potassium (BK) channels. Moreover, the mitogen‐activated protein kinase (MAPK) signalling cascade has been found to couple insulin to BK channel activation. However, the cellular events downstream of MAPK that underlie this action of insulin are unknown. Here we demonstrate that in hippocampal neurons, BK channel activation by insulin is blocked by actin filament stabilization, suggesting that this process is dependent on the actin cytoskeleton. Stabilizing actin filaments also markedly attenuated the ability of insulin to inhibit the aberrant hippocampal synaptic activity evoked following Mg2+ removal. Insulin also promoted rapid reorganization of fluorescently labelled polymerized actin filaments; an action that was prevented by inhibitors of MAPK activation. Moreover, in parallel studies, insulin increased the level of phospho‐MAPK immunostaining in hippocampal neurons. These data are consistent with BK channel activation by insulin involving MAPK‐dependent alterations in actin dynamics. This process may have important implications for the role of insulin in regulating hippocampal excitability.

Differential stress-induced alterations of colonic corticotropin-releasing factor receptors in the Wistar Kyoto rat

Background  A growing body of data implicates increased life stresses with the initiation, persistence and severity of symptoms associated with functional gut disorders such as irritable bowel syndrome (IBS). Activation of central and peripheral corticotropin‐releasing factor (CRF) receptors is key to stress‐induced changes in gastrointestinal (GI) function.

Early-life stress selectively affects gastrointestinal but not behavioral responses in a genetic model of brain-gut axis dysfunction.

Early‐life stress and a genetic predisposition to display an anxiety‐ and depressive‐like phenotype are associated with behavioral and gastrointestinal (GI) dysfunction. Animals exposed to early‐life stress, and those genetically predisposed to display anxiety or depressive behaviors, have proven useful tools in which to study stress‐related GI disorders, such as irritable bowel syndrome (IBS). IBS is a heterogeneous disorder, and likely a consequence of both genetic and environmental factors. However, the combined effects of early‐life stress and a genetic predisposition to display anxiety‐ and depression‐like behaviors on GI function have not been investigated.