Effect of recurrent yohimbine on immediate and post-hoc behaviors, stress hormones, and energy homeostatic parameters.

Evidence from experimental models has suggested that acute activation of brain stress and anxiety pathways impacts subsequent behaviors that are mediated or modulated by limbic circuitry. There have been limited investigations of prior or chronic activation of these pathways on subsequent limbic-mediated behaviors. In this study, we tested whether recurrent administration of the anxiogenic compound yohimbine (YOH) could have post-injection effects on brain activation, stress hormones, and performance in sucrose self-administration and startle response paradigms. Rats received six injections across two weeks of either 2mg/kg YOH or saline. Behavioral evaluation confirmed the continued efficacy of the YOH regimen, and increased adrenal corticosterone (CORT) was observed. Several days following YOH or SAL administration, cFos, CORT and adrenocorticotropin hormone (ACTH), and behavioral performance were measured. cFos was elevated post-YOH in the hippocampus; ventral tegmental area/zona inserta; and central and medial nuclei of the amygdala. This activation is consistent with a sustained effect of YOH to activate fear and anxiety circuitries in the CNS. CORT but not ACTH was elevated in the YOH-rats following startle testing. Self-administration and startle tests suggested an increase of non-specific activity in the post-YOH rats; there was no increase in sucrose self-administration or startle response per se. Our findings suggest that recurrent YOH administration may prove a useful and reliable model for simulating recurrent stress/anxiety, and that enhancements to the paradigm such as higher or more frequent dosing of YOH could yield stronger or more extensive behavioral effects.

Loss of islet sympathetic nerves and impairment of glucagon secretion in the NOD mouse: relationship to invasive insulitis.

AIMS/HYPOTHESIS: We hypothesised that non-obese diabetic mice (NOD) mice have an autoimmune-mediated loss of islet sympathetic nerves and an impairment of sympathetically mediated glucagon responses. We aimed: (1) to determine whether diabetic NOD mice have an early impairment of the glucagon response to insulin-induced hypoglycaemia (IIH) and a coincident loss of islet sympathetic nerves; (2) to determine whether invasive insulitis is required for this nerve loss; and (3) to determine whether sympathetically mediated glucagon responses are also impaired. METHODS: We measured glucagon responses to both IIH and tyramine in anaesthetised mice. We used immunohistochemistry to quantify islet sympathetic nerves and invasive insulitis. RESULTS: The glucagon response to IIH was markedly impaired in NOD mice after only 3 weeks of diabetes (change, -70%). Sympathetic nerve area within the islet was also markedly reduced at this time (change, -66%). This islet nerve loss was proportional to the degree of invasive insulitis. More importantly, blocking the infiltration prevented the nerve loss. Mice with autoimmune diabetes had an impaired glucagon response to sympathetic nerve activation, whereas those with non-autoimmune diabetes did not. CONCLUSIONS/INTERPRETATION: The invasive insulitis seen in diabetic NOD mice causes early sympathetic islet neuropathy. Further studies are needed to confirm that early sympathetic islet neuropathy is responsible for the impaired glucagon response to tyramine.

Effect of moderate intake of sweeteners on metabolic health in the rat.

The rise in prevalence of obesity, diabetes, metabolic syndrome, and fatty liver disease has been linked to increased consumption of fructose-containing foods or beverages. Our aim was to compare the effects of moderate consumption of fructose-containing and non-caloric sweetened beverages on feeding behavior, metabolic and serum lipid profiles, and hepatic histology and serum liver enzymes, in rats. Behavioral tests determined preferred (12.5-15%) concentrations of solutions of agave, fructose, high fructose corn syrup (HFCS), a combination of HFCS and Hoodia (a putative appetite suppressant), or the non-caloric sweetener Stevia (n=5/gp). HFCS intake was highest, in preference and self-administration tests. Groups (n=10/gp) were then assigned to one of the sweetened beverages or water as the sole source of liquid at night (3 nights/wk, 10wks). Although within the normal range, serum cholesterol was higher in the fructose and HFCS groups, and serum triglycerides were higher in the Agave, HFCS, and HFCS/Hoodia groups (vs. water-controls, p<0.05). Liver histology was normal in all groups with no evidence of steatosis, inflammation, or fibrosis; however serum alanine aminotransferase was higher in the fructose and HFCS groups (vs. water-controls, p<0.05). Serum inflammatory marker levels were comparable among Stevia, agave, fructose, HFCS, and water-consuming groups, however levels of IL-6 were significantly lower in association with the ingestion of Hoodia. There were no differences in terminal body weights, or glucose tolerance assessed by 120-min IVGTTs performed at the end of the 10-week regimen. We conclude that even moderate consumption of fructose-containing liquids may lead to the onset of unfavorable changes in the plasma lipid profile and one marker of liver health, independent of significant effects of sweetener consumption on body weight.

Expression of receptors for insulin and leptin in the ventral tegmental area/substantia nigra (VTA/SN) of the rat.

Recent studies have demonstrated that the metabolic hormones insulin and leptin can modulate behavioral performance in reward-related paradigms. However, specific anatomical substrate(s) within the CNS for these effects remain to be identified. We hypothesize that midbrain dopamine neurons, which have been implicated to be critical in the mediation of motivational and reward aspects of stimuli, contribute to these behavioral effects of insulin and leptin. As one approach to evaluate this hypothesis, we used double-labeling fluorescence immunohistochemistry to determine whether the midbrain dopamine neurons express insulin receptors or leptin receptors. Extensive co-expression of tyrosine hydroxylase (a marker for dopamine neurons) with both the insulin receptor and the leptin receptor was observed in the ventral tegmentum and substantia nigra. These findings suggest that midbrain dopamine neurons are direct targets of insulin and leptin, and that they participate in mediating the effects of these hormones on reward-seeking behavior.

Effects of repetitive hypoglycemia on neuroendocrine response and brain tyrosine hydroxylase activity in the rat.

Hypoglycemia-associated autonomic failure (HAAF) is a syndrome of acute adaptation to a metabolic stressor, in which neuroendocrine responses to repetitive hypoglycemic bouts are blunted. The CNS mechanisms that contribute to HAAF are unknown. In the present study, we modeled HAAF in the rat and measured the activity of tyrosine hydroxylase (TH) as an index of acute noradrenergic activation, to test the hypothesis that noradrenergic activation of the hypothalamus might be impaired. In association with a significant counter-regulatory response to a single bout of hypoglycemia (elevated corticosterone, catecholamines, and glucagon), TH activity was elevated overall in brainstem NE cell body areas and hypothalamus. With multiple hypoglycemic episodes in a 24 h period, the counter-regulatory response was blunted, and hypothalamic TH activity was comparable to that of saline-infused controls. In a similar paradigm, multiple bouts of CNS neuroglucopenia did not blunt the hyperglycemic or corticosterone responses, and were required for elevation of TH activity. This alternate response pattern suggests that insulin-induced hypoglycemia and cerebral neuroglucopenia represent somewhat different metabolic stressors at the CNS.

Intraventricular insulin decreases kappa opioid-mediated sucrose intake in rats.

The hormone insulin acts in the central nervous system (CNS) as a regulator of body adiposity and food intake. Recent work from our laboratory has provided evidence that one way by which insulin may decrease food intake is by decreasing the rewarding properties of food. Evidence from others suggests that endogenous opioids may mediate the palatable properties of foods, and insulin may decrease nonfood-related reward via interaction with some CNS kappa opioid systems. In the present study we examined the ability of insulin to interact with exogenous or endogenous kappa opioids to modulate feeding of palatable sucrose pellets by nondeprived rats. Insulin (5 mU intracerebroventricular (i.c.v.), t=-3h) completely reversed the ability of the exogenous kappa agonist U50,488 (26 microg, i.c.v., t=-15 min) to stimulate 90-min sucrose feeding (211+/-32% reduced to 125+/-23% of 90-min baseline intake). Further, i.c.v. insulin (5 mU, t=-3h) interacted with a subthreshold dose of the kappa receptor antagonist norbinaltorphimine (5 microg, i.c.v., t=-15 min) to decrease the 90-min sucrose intake baseline (77+/-11% versus 109+/-10% of 90 min baseline intake, insulin/norbinaltorphimine versus norbinaltorphimine). Together these studies provide new evidence that insulin in the CNS may decrease the action of CNS kappa opioid system(s) that mediate palatable feeding.

PVN activation is suppressed by repeated hypoglycemia but not antecedent corticosterone in the rat.

The mechanism(s) underlying hypoglycemia-associated autonomic failure (HAAF) are unknown. To test the hypothesis that the activation of brain regions involved in the counterregulatory response to hypoglycemia is blunted with HAAF, rats were studied in a 2-day protocol. Neuroendocrine responses and brain activation (c-Fos immunoreactivity) were measured during day 2 insulin-induced hypoglycemia (0.5 U insulin x 100 g body x wt(-1) x h(-1) iv for 2 h) after day 1 hypoglycemia (Hypo-Hypo) or vehicle. Hypo-Hypo animals demonstrated HAAF with blunted epinephrine, glucagon, and corticosterone (Cort) responses and decreased activation of the medial hypothalamus [the paraventricular (PVN), dorsomedial (DMH), and arcuate (Arc) nuclei]. To evaluate whether increases in day 1 Cort were responsible for the decreased hypothalamic activation, Cort was infused intracerebroventricularly (72 microg) on day 1 and the response to day 2 hypoglycemia was measured. Intracerebroventricular Cort infusion failed to alter the neuroendocrine response to day 2 hypoglycemia, despite elevating both central nervous system and peripheral Cort levels. However, day 1 Cort blunted responses in two of the same hypothalamic regions as Hypo-Hypo (the DMH and Arc) but not in the PVN. These results suggest that decreased activation of the PVN may be important in the development of HAAF and that antecedent exposure to elevated levels of Cort is not always sufficient to produce HAAF.

Leptin reverses sucrose-conditioned place preference in food-restricted rats.

Previous studies have suggested that food restriction can modify performance in the conditioned place preference (CPP) paradigm. In the present study, we tested the hypotheses that food restriction would enhance the development of a CPP to low-calorie sucrose pellets and that peripheral leptin replacement in food-restricted animals would reverse this effect. Using a range of 45-mg sucrose pellets (0-15 pellets) as a reward, we observed that a significant place preference was conditioned in food-restricted, but not ad libitum-fed rats. This CPP was reversed either by treatment of food-restricted rats with the dopamine receptor antagonist alpha-flupenthixol (200 microg/kg ip) during the training protocol or by chronic subcutaneous replacement of leptin (125 microg/kg/day) that attenuated the food restriction-induced decrease of circulating leptin. We conclude that dopaminergic signaling and the fall of plasma leptin concentrations contribute to the CPP of food-restricted rats. This finding suggests that in addition to metabolic adaptations, hypoleptinemia results in behavioral adaptations during states of energy deprivation.

Insulin and raclopride combine to decrease short-term intake of sucrose solutions.

We have previously reported that the hormone insulin can modulate synaptic function of dopamine neurons. To evaluate whether insulin can alter performance of a task which is dependent on intact dopaminergic signaling, we tested rats in a five minute lick rate task, with a range of concentrations of sucrose or oil solutions. Rats received either ip (t -15 min) saline or the D2 receptor antagonist raclopride (50 microg/kg), and intraventricular (t -4 h) saline or insulin (5 mU). Although ineffective on its own, insulin combined with raclopride treatment resulted in significant suppression of sucrose lick rates compared to the saline/saline group. The overall results are consistent with our hypothesis that insulin may modify performance in tasks that are dependent on dopaminergic signaling.

Intraventricular insulin suppresses the acoustic startle response in rats.

We and others have previously reported that the hormone insulin alters brain noradrenergic function at the synaptic and molecular levels. In the present study, we examined the in vivo effect of insulin (administered chronically via osmotic minipumps at a dose of 5 mU/day into the third cerebral ventricle) on the acoustic startle response. Rats receiving chronic intraventricular insulin had a significantly reduced startle response relative to vehicle-treated controls (i.e., 47 +/- 21% of baseline control startle response). Because our previous findings suggest that on an acute basis, insulin may enhance endogenous noradrenergic activity by inhibiting norepinephrine reuptake, we speculate here that the chronic effect of insulin is similar to that of the noradrenergic reuptake blocker, desipramine, which has been reported to decrease baseline startle performance.

Endocrine regulation of neurotransmitter transporters.

Aminergic signalling in the CNS is terminated by clearance of neurotransmitters from the synapse via high affinity transporter molecules in the presynaptic membrane. Relatively recent sequence identification of these molecules has now permitted the initiation of studies of regulation of transporter function at the cellular and systems levels. In vitro studies provide evidence that the transporters for dopamine, serotonin, and gamma-aminobutyric acid (GABA) may be substrates for regulation by protein kinase C and protein kinase A signalling. Changes in energy balance and metabolic status, such as starvation, result in major shifts in hormonal output. It is now recognized that metabolic hormones such as insulin or the adrenal steroids can have significant acute and chronic effects on several aspects of CNS function. Data from this laboratory and others now provide evidence that insulin and adrenal and gonadal steroid hormones may regulate the synthesis and activity of the transporters. Future studies should permit elucidation of the cellular basis for endocrine regulation of neurotransmitter clearance, and thus, the role of endocrines in the maintenance of normal CNS aminergic signalling. The potential relevance of transporter regulation for the ketogenic diet is discussed.

Neurotransmitter transporters: target for endocrine regulation.

Aminergic signaling in the CNS is terminated by clearance from the synapse via high-affinity transporter molecules in the presynaptic membrane. Relatively recent sequence identification of these molecules has now permitted the initiation of studies of regulation of transporter function at the cellular and systems levels. In vitro studies provide evidence that the transporters for dopamine, serotonin, and gamma-aminobutyric acid are substrates for regulation by protein kinase C signaling. In vivo studies provide evidence that insulin and adrenal and gonadal steroid hormones may regulate the synthesis and activity of the transporters. Future directions should permit evaluation of the role of endocrine regulation in neurotransmitter clearance, and thus in the maintenance of normal CNS aminergic signaling.

Desipramine treatment decreases 3H-nisoxetine binding and norepinephrine transporter mRNA in SK-N-SHSY5Y cells.

The antidepressant desipramine has been shown to decrease synaptic membrane concentrations of the norepinephrine re-uptake transporter (NET) in vivo and in vitro, on both an acute and a chronic basis. The possible contribution of decreased NET synthesis to the chronic downregulation of the NETs has not been definitively established. In this study, we treated SK-N-SHSY5Y cells with 100 nM desipramine for 24 or 72 h, and measured 3H-nisoxetine binding (as an estimate of NETs) and NET mRNA by quantitative reverse transcription polymerase chain reaction. Similar to what has been reported previously, membrane 3H-nisoxetine binding was significantly decreased at both 24 and 72 h (approximately 50% at both time points). However, a significant decrease (64 +/- 8% of paired control) of NET mRNA was observed only at the 72-h time point. We conclude that decreased NET synthesis may contribute to the chronic, but not acute, effect of desipramine to downregulate the NET.

Sodium depletion and aldosterone decrease dopamine transporter activity in nucleus accumbens but not striatum.

Motivated behaviors, including sodium (Na) appetite, are correlated with increased dopamine (DA) transmission in the nucleus accumbens (NAc). DA transporter (DAT) modulation affects DA transmission and may play a role in motivated behaviors. In vivo Na depletion, which reliably induces Na appetite, was correlated with robust decreases in DA uptake via the DAT in the rat NAc with rotating disk electrode voltammetry [1,277 +/- 162 vs. 575 +/- 89 pmol. s-1. g-1; Vmax of transport for control vs. Na-depleted tissue]. Plasma aldosterone (Aldo) levels increase after in vivo Na depletion and contribute to Na appetite. Decreased DAT activity in the NAc was observed after in vitro Aldo treatment (428 +/- 28 vs. 300 +/- 25 pmol. s-1. g-1). Neither treatment affected DAT activity in the striatum. These results suggest that a direct action of Aldo is one possible mechanism by which Na depletion induces a reduction in DAT activity in the NAc. Reduced DAT activity may play a role in generating increased NAc DA transmission during Na appetite, which may underlie the motivating properties of Na for the Na-depleted rat.

NPY and food intake: discrepancies in the model.

The evidence that NPY is an endogenous neurotransmitter that modulates both sides of the energy equation is clear and compelling. While agreeing with this (and indeed contributing to the growing literature supporting the concept), we have found that the interpretation of the increased food intake stimulated by intraventricular (i.v.t.) NPY is more complex than first appears. We discuss evidence suggesting that NPY additionally (and presumably at other receptor populations in the brain) causes sensations that produce aversion or illness. Specifically, the i.v.t. administration of NPY at doses that stimulate eating also cause the formation of a conditioned taste aversion and the animal engages in a form of pica behavior (kaolin consumption). It also suppresses an otherwise robust increase of sodium consumption. We discuss evidence suggesting that whereas NPY activates feeding behavior by stimulating the complex sequence of behaviors beginning with the seeking and finding of food and ending with food ingestion, NPY does not stimulate increased eating in the absence of the anticipatory preliminary behaviors. Finally, we briefly review evidence suggesting that whatever sensation is aroused by i.v.t. NPY, it is not necessarily the same sensation that is aroused when animals are food-deprived. Hence, one must be cautious in interpreting NPY as solely an orexigen.

Effects of third intracerebroventricular injections of corticotropin-releasing factor (CRF) on ethanol drinking and food intake.

Corticotropin releasing factor (CRF), a neuropeptide secreted by hypothalamic and extrahypothalamic neurons, is thought to mediate stress-related behaviors. The tension reduction hypothesis suggests that ethanol drinking reduces stress; that drinking is reinforced by this reduced stress; and that the probability of drinking therefore subsequently increases. CRF also decrease food intake, and might decrease ethanol drinking similarly. We addressed these hypotheses directly by assessing the effects of intracerebroventricular (i.c.v.) CRF upon ethanol drinking (1 h/day). Rats were provided drinking tubes containing ethanol solutions that were gradually incremented in concentration (from 2% to 8% w/v, over 38 days). Ethanol intakes remained stable, ranging from 0.4 to 0.5 g/kg per hour on average, and a two-bottle choice test revealed that ethanol was preferred reliably to water. Third-i.c.v. cannulae were surgically implanted and CRF or vehicle was acutely injected immediately prior to the sessions. CRF dose-dependently reduced ethanol intake by 31% (0.5 microg) and 64% (5.0 microg), and reduced 24-h food by 9% and 21%, respectively, but did not alter body weights. I.c.v. CRF reduced ethanol drinking despite any acute stress-like effects that may have been present. Hence, these data are inconsistent with the tension reduction hypothesis. On the other hand, our results support the concept that food intake and ethanol drinking may be mediated by similar mechanisms.

Food deprivation decreases mRNA and activity of the rat dopamine transporter.

We have hypothesized that the midbrain dopamine (DA) neurons are a target for insulin action in the central nervous system (CNS). In support of this hypothesis, we have previously demonstrated that direct intracerebroventricular infusion of insulin results in an increase in mRNA levels for the DA reuptake transporter (DAT). In this study, 24- to 36-hour food deprivation was used as a model of decreased CNS insulin levels, to test whether DAT mRNA levels, DAT protein concentration or DAT functional activity would be decreased. DAT mRNA levels, assessed by in situ hybridization, were significantly decreased in the ventral tegmental area/substantia nigra pars compacta (VTA/SNc) (77 +/- 7% of controls, p < 0.05) of food-deprived (hypoinsulinemic) rats. Binding of a specific high-affinity DAT ligand (125I-RTI-121) to membranes from brain regions of fasted or free-feeding rats provided an estimate of DAT protein, which was unchanged in both of the major terminal projection fields, the striatum and nucleus accumbens (NAc). In addition, we utilized the rotating disk electrode voltametry technique to assess possible changes in the function of the DAT in fasting (hypoinsulinemic) rats. The Vmax of DA uptake was significantly decreased (87 +/- 7% of control, p < 0.05), without a change in the Km of uptake, in striatum from fasted rats. In vitro incubation with a physiological concentration (1 nM) of insulin resulted in an increase of striatal DA uptake to control levels. We conclude that striatal DAT function can be modulated by fasting and nutritional status, with a contribution by insulin.