Jessica Santollo

Trace Conditioning and the Hippocampus: The Importance of Contiguity

Trace conditioning, a form of classical conditioning in which the presentation of the conditioned stimulus (CS) and the unconditioned stimulus (US) is separated in time by an interstimulus interval, requires an intact hippocampus. In contrast, classical conditioning procedures in which the CS and US are not separated by an interstimulus interval (i.e., delay conditioning procedures) typically do not (Solomon et al., 1986). However, why trace conditioning is dependent on the hippocampus is unknown. Several theories suggest that it is specifically the discontiguity between the CS and US in trace conditioning that critically engages the hippocampus. However, there are other explanations that do not depend on discontiguity. To determine whether the lack of contiguity renders trace conditioning hippocampal dependent, we designed a “contiguous trace conditioning” (CTC) paradigm in which CS–US contiguity is restored by re-presenting the CS simultaneously with the US. Although rats with excitotoxic lesions of the hippocampus could not learn a standard trace fear-conditioning paradigm, lesioned rats trained on CTC showed significant conditioning, at levels similar to those with sham surgeries. Importantly, lesioned rats trained solely with simultaneous CS–US presentations did not demonstrate conditioning. Together, these data suggest that rats with hippocampal lesions can form a memory of a trace CS–US association when contiguity is restored. Therefore, the dependence of traditional trace paradigms on the hippocampus can be attributed to the absence of temporal contiguity.

Amelioration of binge eating by nucleus accumbens shell deep brain stimulation in mice involves D2 receptor modulation.

Hedonic overconsumption contributing to obesity involves altered activation within the mesolimbic dopamine system. Dysregulation of dopamine signaling in the nucleus accumbens shell (NAS) has been implicated in reward-seeking behaviors, such as binge eating, which contributes to treatment resistance in obesity (Wise, 2012). Direct modulation of the NAS with deep brain stimulation (DBS), a surgical procedure currently under investigation in humans for the treatment of major depression, obsessive–compulsive disorder, and addiction, may also be effective in ameliorating binge eating. Therefore, we examined the ability of DBS of the NAS to block this behavior in mice. c-Fos immunoreactivity was assessed as a marker of DBS-mediated neuronal activation. NAS DBS was found to reduce binge eating and increased c-Fos levels in this region. DBS of the dorsal striatum had no influence on this behavior, demonstrating anatomical specificity for this effect. The dopamine D2 receptor antagonist, raclopride, attenuated the action of DBS, whereas the D1 receptor antagonist, SCH-23390, was ineffective, suggesting that dopamine signaling involving D2 receptors underlies the effect of NAS DBS. To determine the potential translational relevance to the obese state, chronic NAS DBS was also examined in diet-induced obese mice and was found to acutely reduce caloric intake and induce weight loss. Together, these findings support the involvement of the mesolimbic dopamine pathways in the hedonic mechanisms contributing to obesity, and the efficacy of NAS DBS to modulate this system.

Estradiol decreases the orexigenic effect of neuropeptide Y, but not agouti-related protein, in ovariectomized rats.

Available data suggest that estradiol exerts an inhibitory effect on food intake by modulating the actions of multiple gut- and brain-derived peptides implicated in the control of food intake. For example, recent studies have shown that estradiol decreases the orexigenic effects of ghrelin and melanin-concentrating hormone. In the present study, we examined estradiols ability to decrease the actions of two additional orexigenic peptides, neuropeptide Y (NPY) and agouti-related protein (AgRP). Food intake was monitored following lateral ventricular infusions of 5 microg NPY, 10 microg AgRP, or saline vehicle in ovariectomized rats treated with either 1 microg estradiol or sesame oil vehicle. NPY increased food intake for 2h in both oil- and estradiol-treated ovariectomized rats. During this interval, the orexigenic effect of NPY was significantly greater in oil-treated rats, relative to estradiol-treated rats. In contrast to the short-term action of NPY, a single injection of AgRP increased food intake for 3 days in oil- and estradiol-treated rats. Meal pattern analysis revealed that the orexigenic effect of AgRP is mediated by an increase in meal size, not meal number. Unlike that observed following NPY treatment, estradiol failed to modulate the magnitude by which AgRP increased food intake and meal size. We conclude that a physiological regimen of estradiol treatment decreases the orexigenic effect of NPY, but not AgRP, in ovariectomized rats.

Estradiol acts in the medial preoptic area, arcuate nucleus, and dorsal raphe nucleus to reduce food intake in ovariectomized rats.

Estradiol (E2) exerts an inhibitory effect on food intake in a variety of species. While compelling evidence indicates that central, rather than peripheral, estrogen receptors (ERs) mediate this effect, the exact brain regions involved have yet to be conclusively identified. In order to identify brain regions that are sufficient for E2s anorectic effect, food intake was monitored for 48 h following acute, unilateral, microinfusions of vehicle and two doses (0.25 and 2.5 μg) of a water-soluble form of E2 in multiple brain regions within the hypothalamus and midbrain of ovariectomized rats. Dose-related decreases in 24-h food intake were observed following E2 administration in the medial preoptic area (MPOA), arcuate nucleus (ARC), and dorsal raphe nucleus (DRN). Within the former two brain areas, the larger dose of E2 also decreased 4-h food intake. Food intake was not influenced, however, by similar E2 administration in the paraventricular nucleus, lateral hypothalamus, or ventromedial nucleus. These data suggest that E2-responsive neurons within the MPOA, ARC, and DRN participate in the estrogenic control of food intake and provide specific brain areas for future investigations of the cellular mechanism underlying estradiols anorexigenic effect.

The bed nucleus of the stria terminalis is critically involved in enhancing associative learning after stressful experience.

Exposure to an acute stressful event enhances trace eyeblink conditioning in male rats, even when rats begin training days after the stressor (Shors, 2001). The authors examined whether the bed nucleus of the stria terminalis (BNST), an area involved in stress and anxiety, is critically involved in this effect and, if so, when. The authors found that excitotoxic lesions to the BNST prevented the enhanced conditioning after stressor exposure. In addition, temporary inactivation of the BNST during the stressor did not alter enhanced responding, whereas inactivation during training prevented the enhancement. These data indicate that stressful experience induces persistent changes in the BNST that are necessary for enhancing learning well after the stressful event has ceased.

Activation of ERα is necessary for estradiol's anorexigenic effect in female rats.

While there is considerable evidence that the ovarian hormone estradiol reduces food intake in female rats, it is unclear which estrogen receptor (ER) subtype, ERα or ERβ, mediates this effect. While several studies have demonstrated that activation of ERα, but not ERβ, is sufficient to reduce food intake in ovariectomized (OVX) rats, there are limited data regarding which receptor subtype is necessary. Here we used the selective ERα and ERß antagonists, MPrP and PHTPP, respectively, to investigate this question. We found that antagonism of ERα, but not ERβ, prevented the decrease in food intake following acute administration of estradiol in OVX rats. In addition, antagonism of ERα prevented the estrous-related, phasic reduction in food intake that occurs in response to the rise in circulating levels of estradiol in cycling rats. We conclude that activation of ERα is necessary for the anorexigenic effects of exogenous and endogenous estradiol in female rats.

Effect of a putative ERα antagonist, MPP, on food intake in cycling and ovariectomized rats

Estrogens exert many of their behavioral effects by binding to nuclear estrogen receptor (ER) proteins, ERalpha and ERbeta. Recent studies involving ER knockout mice and selective ER agonists suggest that estradiols anorexigenic effect is mediated via activation of ERalpha. To investigate this hypothesis, we examined whether the presumptive ERalpha antagonist, MPP, could block estradiols anorexigenic effect. In the first series of experiments, the effects of MPP on food intake and uterine weight were monitored in ovariectomized (OVX) rats treated with either a physiological dose of estradiol benzoate (EB) or a selective ERalpha agonist (PPT). In the final experiment, food intake was monitored following acute administration of MPP in ovarian-intact (cycling) female rats. Contrary to our hypothesis, MPP failed to attenuate either EBs or PPTs ability to decrease food intake and increase uterine weight in OVX rats. However, in ovarian-intact rats, a similar regimen of MPP treatment attenuated the phasic decrease in food intake that is associated with estrus. We conclude that MPP may be a useful tool to investigate the behavioral actions of endogenous estradiol, but may have limited utility in studying the behavioral effects of exogenous estradiol in OVX rats.

The orexigenic effect of melanin-concentrating hormone (MCH) is influenced by sex and stage of the estrous cycle.

Recently, it was shown that the orexigenic effect of melanin-concentrating hormone (MCH) is attenuated by estradiol treatment in ovariectomized (OVX) rats. This suggests that female rats may be less responsive than male rats to the behavioral effects of MCH. To investigate this hypothesis, the effects of lateral ventricular infusions of MCH on food intake, water intake, meal patterns, and running wheel activity were examined in male and female rats. To further characterize the impact of estradiol on MCH-induced food intake, female rats were OVX and tested with and without 17-beta-estradiol benzoate (EB) replacement. In support of our hypothesis, food and water intakes following MCH treatment were greater in male rats, relative to female rats. Specifically, the orexigenic effect of MCH was maximal in male rats and minimal in EB-treated OVX rats. In both sexes, the orexigenic effect of MCH was mediated by a selective increase in meal size, which was attenuated in EB-treated OVX rats. MCH-induced a short-term (2 h) decrease in wheel running that, unlike its effects on ingestive behavior, was similar in males and females. Thus, estradiol decreases some, but not all, of the behavioral effects of MCH. To examine the influence of endogenous estradiol, food intake was monitored following MCH treatment in ovarian-intact, cycling rats. As predicted by our findings in OVX rats, the orexigenic effect of MCH was attenuated in estrous rats, relative to diestrous rats. We conclude that the female rats reduced sensitivity to the orexigenic effect of MCH may contribute to sex- and estrous cycle-related differences in food intake.

Activation of Membrane-Associated Estrogen Receptors Decreases Food and Water Intake in Ovariectomized Rats

Estradiol (E2) decreases food and water intake in a variety of species, including rats. Available evidence suggests that this is mediated by genomic mechanisms that are most often attributed to nuclear estrogen receptors. More recent studies indicate that membrane-associated estrogen receptors (mERs) also can influence gene expression through the activation of transcription factors, yet it is unclear whether mERs are involved in mediating the hypophagic and antidipsetic effects of E2. In the present experiments, we injected E2 or a membrane-impermeable form of E2 (E2-BSA) into the lateral cerebral ventricle of ovariectomized female rats and evaluated the effect on 23 h food and water intake. First, we found that higher doses of E2 were necessary to reduce water intake than were sufficient to reduce food intake. Analysis of drinking microstructure revealed that the decrease in water intake after E2 treatment was mediated by both a decrease in burst number and burst size. Next, the activation of mERs with E2-BSA decreased both overnight food and water intake and analysis of drinking microstructure indicated that the decreased water intake resulted from a decrease in burst number. Finally, E2-BSA did not condition a taste aversion, suggesting that the inhibitory effects on food and water intake were not secondary to malaise. Together these findings suggest that activation of mERs is sufficient to decrease food and water intake in female rats.

Oestradiol Decreases Melanin-Concentrating Hormone (MCH) and MCH Receptor Expression in the Hypothalamus of Female Rats

Previous studies have shown that oestradiol (E2) decreases the orexigenic effect of melanin‐concentrating hormone (MCH). In the present study, we examined whether this action of E2 is mediated by its ability to decrease the expression of MCH or its receptor (MCHR1). Using immunocytochemistry and western blotting, we examined whether E2 decreases MCH‐immunoreactive neurones or MCHR1 protein content in the hypothalamus of female rats. We found that both MCH and MCHR1 protein expression was decreased by acute E2 treatment in ovariectomised rats, and by the peri‐ovulatory increase in circulating E2 in pro‐oestrous rats, relative to rats at other cycle stages. To determine whether these changes in MCH/MCHR1 protein expression may be mediated by E2s ability to directly regulate the transcription of MCH and MCHR1 genes, the effect of E2 treatment on MCH and MCHR1 mRNA expression in a neuronal hypothalamic cell line was examined using real‐time reverse transcriptase‐polymerase chain reaction. We also determined whether MCH and oestrogen receptor (ER)α are co‐expressed in the hypothalamus of female rats. E2 treatment did not decrease MCH or MCHR1 mRNA expression in vitro, and no hypothalamic neurones were identified that co‐expressed MCH and ERα. We conclude that E2‐dependent decreases in hypothalamic MCH/MCHR1 protein expression mediate the ability of E2 to decrease MCH‐induced feeding. The current findings suggest, however, that E2 exerts these actions indirectly, most likely though interactions with other neuronal systems that provide afferent input to MCH and MCHR1 neurones.