James W. Sharp

The Neuron-specific K-Cl Cotransporter, KCC2 ANTIBODY DEVELOPMENT AND INITIAL CHARACTERIZATION OF THE PROTEIN

The neuron-specific K-Cl cotransporter (KCC2) is hypothesized to function as an active Cl− extrusion pathway important in postsynaptic inhibition mediated by ligand-gated anion channels, like γ-aminobutyric acid type A (GABAA) and glycine receptors. To understand better the functional role of KCC2 in the nervous system, we developed polyclonal antibodies to a KCC2 fusion protein and used these antibodies to characterize and localize KCC2 in the rat cerebellum. The antibodies specifically recognized the KCC2 protein which is an ∼140-kDa glycoprotein detectable only within the central nervous system. The KCC2 protein displayed a robust and punctate distribution in primary cultured retinal amacrine cells known to form exclusively GABAAergic synapses in culture. In immunolocalization studies, KCC2 was absent from axons and glia but was highly expressed at neuronal somata and dendrites, indicating a specific postsynaptic distribution of the protein. In the granule cell layer, KCC2 exhibited a distinct colocalization with the β2/β3-subunits of the GABAAreceptor at the plasma membrane of granule cell somata and at cerebellar glomeruli. KCC2 lightly labeled the plasma membrane of Purkinje cell somata. Within the molecular layer, KCC2 exhibited a distinctly punctate distribution along dendrites, indicating it may be highly localized at inhibitory synapses along these processes. The distinct postsynaptic localization of KCC2 and its colocalization with GABAA receptor in the cerebellum are consistent with the putative role of KCC2 in neuronal Cl− extrusion and postsynaptic inhibition.

Glucose sensing by gut endocrine cells and activation of the vagal afferent pathway is impaired in a rodent model of type 2 diabetes mellitus.

Glucose in the gut lumen activates gut endocrine cells to release 5-HT, glucagon-like peptide 1/2 (GLP-1/2), and glucose-dependent insulinotropic polypeptide (GIP), which act to change gastrointestinal function and regulate postprandial plasma glucose. There is evidence that both release and action of incretin hormones is reduced in type 2 diabetes (T2D). We measured cellular activation of enteroendocrine and enterochromaffin cells, enteric neurons, and vagal afferent neurons in response to intestinal glucose in a model of type 2 diabetes mellitus, the UCD-T2DM rat. Prediabetic (PD), recent-diabetic (RD, 2 wk postonset), and 3-mo diabetic (3MD) fasted UCD-T2DM rats were given an orogastric gavage of vehicle (water, 0.5 ml /100 g body wt) or glucose (330 μmol/100 g body wt); after 6 min tissue was removed and cellular activation was determined by immunohistochemistry for phosphorylated calcium calmodulin-dependent kinase II (pCaMKII). In PD rats, pCaMKII immunoreactivity was increased in duodenal 5-HT (P < 0.001), K (P < 0.01) and L (P < 0.01) cells in response to glucose; glucose-induced activation of all three cell types was significantly reduced in RD and 3MD compared with PD rats. Immunoreactivity for GLP-1, but not GIP, was significantly reduced in RD and 3MD compared with PD rats (P < 0.01). Administration of glucose significantly increased pCaMKII in enteric and vagal afferent neurons in PD rats; glucose-induced pCaMKII immunoreactivity was attenuated in enteric and vagal afferent neurons (P < 0.01, P < 0.001, respectively) in RD and 3MD. These data suggest that glucose sensing in enteroendocrine and enterochromaffin cells and activation of neural pathways is markedly impaired in UCD-T2DM rats.

Intestinal glucose‐induced calcium‐calmodulin kinase signaling in the gut–brain axis in awake rats

Background  Lumenal glucose initiates changes in gastrointestinal (GI) function, including inhibition of gastric emptying, stimulation of pancreatic exocrine and endocrine secretion, and intestinal fluid secretion. Glucose stimulates the release of GI hormones and 5‐hydroxytryptamine (5‐HT), and activates intrinsic and extrinsic neuronal pathways to initiate changes in GI function. The precise mechanisms involved in luminal glucose‐sensing are not clear; studying gut endocrine cells is difficult due to their sparse and irregular localization within the epithelium.

The mGlu2/3 receptor agonist LY379268 injected into cortex or thalamus decreases neuronal injury in retrosplenial cortex produced by NMDA receptor antagonist MK-801: possible implications for psychosis

The non-competitive NMDA receptor antagonists, including PCP (phencyclidine), ketamine, and MK-801 (dizocilpine) produce psychosis in humans and injure neurons in retrosplenial cortex in adult rodent brain. This study examined the effects of the metabotropic mGlu2/3 agonist LY379268 and antagonist LY341495 on cortical injury produced by systemic MK-801 (1 mg/kg i.p.) in adult female rats. Systemic injections of mGlu2/3 agonist LY379268, but not mGlu2/3 antagonist LY341495, decreased the injury in the retrosplenial cortex produced by systemic MK-801 as assessed by Hsp70 induction. Bilateral injections of LY379268, but not vehicle, into retrosplenial cortex or bilateral injections of LY379268 into anterior thalamus also decreased the injury in retrosplenial cortex produced by systemic MK-801. The data show that bilateral activation of mGlu2/3 glutamate receptors in cortex or anterior thalamus decreases the neuronal injury in retrosplenial cortex produced by systemic MK-801. Because antipsychotic medications decrease cortical injury produced by NMDA antagonists in rodents and decrease psychosis in humans, mGlu2/3 agonists that decrease cortical injury produced by NMDA antagonists in rodents might be evaluated for decreasing psychosis in people.

Role of MAP kinase in signaling indispensable amino acid deficiency in the brain

Deficiencies of indispensable amino acids (IAAs) appear to be sensed in the anterior piriform cortex (APC) where neurons are activated and potentiated, however, the mediating intracellular signaling mechanisms are largely unexplored. It is postulated that signaling of amino acid deficiency may share many of the same pathways seen with long-term potentiation (LTP). Phosphorylation of mitogen-activated protein kinase (pMAP kinase) has been shown to be a necessary signaling event for the genesis and maintenance of LTP. Immunoperoxidase immunohistochemistry was used to determine the number of neurons showing activation of the MAP kinase signal transduction system. Relative to rats eating a corrected diet, rats consuming threonine-devoid diet showed significantly greater pMAP kinase labeling in the APC, dorsomedial hypothalamus, and the paraventricular hypothalamic nucleus. These are areas previously associated with control of food intake. However, since the dorsomedial hypothalamus and the paraventricular hypothalamic nucleus have not previously been implicated as chemosensory areas for IAAs, phosphorylated MAP kinase expression in these areas may reflect secondary activation.

Phosphorylation of Ca2+/calmodulin-dependent protein kinase type ii and the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (ampa) receptor in response to a threonine-devoid diet

The anterior piriform cortex (APC) functions as a chemosensor for indispensable amino acid deficiency and responds to this deficiency with increased activity, as indicated by observations including averaged evoked-potentials and c-fos expression in the APC. Little is known of the intracellular signaling mechanisms that mediate this deficiency-related increase in neuronal excitability, but previous studies have shown effects on intracellular Ca2+ in deficient APC slices in vitro. In the present study we hypothesized that indispensable amino acid deficiency increases intraneuronal Ca2+, resulting in autophosphorylation of calcium/calmodulin-dependent protein kinase type II (CaMKII) in vivo. Results demonstrated that phosphorylation levels of CaMKII (pCaMKII) in APC neurons increased at 20 and 40 min after a single meal of threonine-devoid diet. Phosphorylation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit (GluR1) at the serine 831 (S831) site was modestly increased in the APC in response to a threonine-devoid meal. The GluR1 subunit also showed increased phosphorylation at the 845 (S845) site, suggesting additional signaling mechanisms. Although phosphorylation of CaMKII was sustained, phosphorylation of the GluR1 subunit returned to control levels by 40 min. These effects of amino acid deficiency did not occur throughout the brain as neither CaMKII nor GluR1 showed increased phosphorylation in the neocortex. These findings support the notion that calcium and glutamate signaling in the APC, but not throughout the brain, are triggered during early responses to amino acid deficiency. They also suggest that longer-term changes in APC neurons in response to such a deficiency may be mediated at least in part by CaMKII.

Co‐localization of phosphorylated extracellular signal‐regulated protein kinases 1/2 (ERK1/2) and phosphorylated eukaryotic initiation factor 2α (eIF2α) in response to a threonine‐devoid diet

The anterior piriform cortex (APC) has been shown to be an essential brain structure for the detection of dietary indispensable amino acid (IAA) deficiency, but little has been known about possible molecular detection mechanisms. Increased phosphorylation of the α‐subunit of the eukaryotic initiation factor 2α (eIF2α) has been directly linked to amino acid deficiency in yeast. Recently, we have shown increased phosphorylation of eIF2α (p‐eIF2α) in the rat APC 20 minutes after ingestion of an IAA‐deficient meal. We suggest that if phosphorylation of eIF2α is an important mechanism in detection of IAA deficiency, then APC neurons that show p‐eIF2α should also show molecular evidence of potentiation. The present research demonstrates increased expression and co‐localization of p‐eIF2α and phosphorylated extracellular signal‐regulated protein kinase 1/2 (p‐ERK1/2) in APC neurons, but not in the primary motor or agranular insular cortices in response to an IAA‐deficient diet. ERK1/2 is an element of the mitogen‐activated protein kinase cascade, an intraneuronal signaling mechanism associated with neuronal activation. The region of the APC that responds to IAA deficiency with increased p‐eIF2α and p‐ERK1/2 labeling ranges from 3.1 to 2.5 mm rostral of bregma. Within this region, only a few neurons respond to IAA deficiency with co‐localization of abundant p‐eIF2α and p‐ERK1/2. These chemosensory neurons probably detect IAA deficiency and generate neuronal signaling to other portions of the brain, changing feeding behavior. J. Comp. Neurol. 494:485–494, 2006.

Effects of essential amino acid deficiency: down‐regulation of KCC2 and the GABAA receptor; disinhibition in the anterior piriform cortex

The anterior piriform cortex (APC) is activated by, and is the brain area most sensitive to, essential (indispensable) amino acid (IAA) deficiency. The APC is required for the rapid (20 min) behavioral rejection of IAA deficient diets and increased foraging, both crucial adaptive functions supporting IAA homeostasis in omnivores. The biochemical mechanisms signaling IAA deficiency in the APC block initiation of translation in protein synthesis via uncharged tRNA and the general amino acid control kinase, general control nonderepressing kinase 2. Yet, how inhibition of protein synthesis activates the APC is unknown. The neuronal K+Cl− cotransporter, neural potassium chloride co‐transporter (KCC2), and GABAA receptors are essential inhibitory elements in the APC with short plasmalemmal half‐lives that maintain control in this highly excitable circuitry. After a single IAA deficient meal both proteins were reduced (vs. basal diet controls) in western blots of APC (but not neocortex or cerebellum) and in immunohistochemistry of APC. Furthermore, electrophysiological analyses support loss of inhibitory elements such as the GABAA receptor in this model. As the crucial inhibitory function of the GABAA receptor depends on KCC2 and the Cl− transmembrane gradient it establishes, these results suggest that loss of such inhibitory elements contributes to disinhibition of the APC in IAA deficiency.

Effects of kelthane on the estuarine shrimp Crangon tranciscorum

The chlorinated hydrocarbon pesticide Kelthane was assayed for effects on food consumption, molting rate, cannibalism, respiration, mortality and behavior of the estuarine shrimp Crangon franciscorum Stimpson. The test system was a single-pass pulse-flow apparatus employing a modified Mount-Brungs style diluter. Treatment levels were 0, 14, 33 and 81 μg l-1 Kelthane at 16.6°C and 19‰ S over an exposure period of 317 h. Exposure to Kelthane reduced food consumption, molting rate, cannibalism, and respiration. Behavior also was abnormal in exposed shrimp. They spent more time swimming, were less coordinated and had feeble escape reactions. Kelthane was very toxic to C. franciscorum with LT50 values of 163, 196 and 264 h for shrimp exposed to 81, 33 and 14 μg l-1 respectively. The incipient lethal level of Kelthane is probably below 14 μg l-1.

M1733 Glucose-Sensing in Enteroendocrine Cells and the Vagal Afferent Pathway is Attenuated in a Model of Type 2 Diabetes Mellitus in Rats

Introduction: Anorexia and hypermetabolism are a problem of paramount importance in children with cirrhosis. Plasma levels of both leptin and ghrelin are altered in patients with starvation and chronic diseases associated with anorexia. Ghrelin has an important role on feeding and weight homeostasis. Aim: We aimed to assess ghrelin and leptin levels in cirrhotic children and adolescents in comparison with controls. Methods: Forty children with cirrhosis (biliary atresia as etiology in 67.5%) aged between 3.7 and 188.7 months, 50% male, were evaluated, 50% were Child-Pugh A; 40%, B and 10%, C. Other 40 healthy children matched for sex and age, taken as controls, were also evaluated. Blood samples were collected at least after 3-hours fasting. Fasting leptin and acylated ghrelin levels were measured using a newly developed ELISA kit (Linco Research, St Charles-MI, USA). Body composition including body mass index (BMI), body fat mass and height for age were determined and classified according to WHO 2009 and Frisancho 2008 standard reference. All controls were eutrophic as well as 77.5% of cirrhotic patients. Other cirrhotics were either undernourished (21.3%) or overweighed (1.3%). Results: Ghrelin and leptin in both groups did not correlate with sex (P=0.023). Median values (25-75 centile) of acylated ghrelin were significantly lower in cirrhotics than in healthy controls [140.3 (93.9-269.7) pg/mL vs 277.8 (209.9-557.8) pg/mL, P=0.003]. Median values of total ghrelin and desacyl ghrelin of children with cirrhosis were 913.4 pg/mL (760.8-1443.7) and 1397.8 pg/mL (815.27-2047) respectively. Median values of leptin were not significantly different between cirrhotics and controls (P=0,26)]. From the cirrhotic group there was a significant difference between undernourished and well-nourished pactients [1.45ng/mL (0.66-1.88) 2.68 ng/mL (1.31-4.38)], P=0.023. Median values of Child-Pugh C were significantly lower than B and A [0.67 (0.17-1.11) ng/mL; 1.83 (1.24-4.04) ng/mL; 1.88 (0.67-3.67) ng/mL], P=0.033. Conclusions: Cirrhotic children showed lower plasma levels of acylated ghrelin compared with controls. This might be representative of the anorexia that consequently contributes to the malnourishment and illness severity of cirrhotic patients.