Amanda J. Roberts

Interaction between the Corticotropin-Releasing Factor System and Hypocretins (Orexins): A Novel Circuit Mediating Stress Response

The hypothalamic neuropeptides hypocretins (orexins) play a crucial role in the stability of arousal and alertness. We tested whether the hypocretinergic system is a critical component of the stress response activated by the corticotropin-releasing factor (CRF). Our results show that CRF-immunoreactive terminals make direct contact with hypocretin-expressing neurons in the lateral hypothalamus and that numerous hypocretinergic neurons express the CRF-R1/2 receptors. We also demonstrate that application of CRF to hypothalamic slices containing identified hypocretin neurons depolarizes membrane potential and increases firing rate in a subpopulation of hypocretinergic cells. CRF-induced depolarization was tetrodotoxin insensitive and was blocked by the peptidergic CRF-R1 antagonist astressin. Moreover, activation of hypocretinergic neurons in response to acute stress was severely impaired in CRF-R1 knock-out mice. Together, our data provide evidence of a direct neuroanatomical and physiological input from CRF peptidergic system onto hypocretin neurons. We propose that, after stressor stimuli, CRF stimulates the release of hypocretins and that this circuit contributes to activation and maintenance of arousal associated with the stress response.

Excessive Ethanol Drinking Following a History of Dependence: Animal Model of Allostasis

Alcohol withdrawal symptoms, particularly negative emotional states, can persist for months following the removal of alcohol. These protracted withdrawal symptoms have been implicated as an important trigger of relapse to excessive drinking in alcoholics and may represent a long lasting shift in affective tone as a result of chronic alcohol exposure. It was shown previously that ethanol-dependent rats increased their operant responding for ethanol when tested during the first 12 hr after withdrawal. The purpose of the present experiments was to determine the persistence of this finding by examining operant oral ethanol self-administration in rats with a history of physical dependence upon ethanol, detoxified and then allowed a two week period of protracted abstinence. The results of these experiments indicate that operant responding for ethanol was enhanced during protracted abstinence by 30–100% and remained elevated for 4–8 weeks post acute withdrawal. These results have important implications for understanding the characteristics and mechanisms underlying vulnerability to relapse.

Enhanced alcohol self-administration after intermittent versus continuous alcohol vapor exposure

BACKGROUND Ethanol self-administering rats exhibit enhanced responding during withdrawal from continuous exposure to ethanol vapor. This study compared self-administration of ethanol during withdrawal from continuous versus intermittent ethanol vapor. METHODS Experiment 1 examined self-administration of ethanol in rats trained to self-administer ethanol after continuous, intermittent (14 hr on and 10 hr off), or no (i.e., controls) ethanol vapor exposure. Exposure time was equalized such that the intermittent group received 4 weeks of exposure and the continuous group received 2 weeks of exposure. Four self-administration tests were conducted 2 hr after removal from vapor, and each test was separated by 3 to 4 days of ethanol vapor. Experiment 2 examined self-administration of ethanol after 2 weeks of intermittent vapor either 2 or 8 hr after removal from vapor. Experiment 3 addressed the specificity of the increased responding for ethanol by examining saccharin self-administration after 2 weeks of intermittent vapor. RESULTS Four weeks of intermittent exposure produced an increase in ethanol self-administration during the first withdrawal relative to controls and relative to animals receiving 2 weeks of continuous exposure. The continuous group was indistinguishable from controls on the first test and gradually increased their responding across tests. Two weeks of intermittent exposure also increased ethanol self-administration, and there was no difference in this effect 2 or 8 hr after removal from vapor. There was no difference in saccharin self-administration in control rats and those given 2 weeks of intermittent exposure. CONCLUSIONS The finding that intermittent exposure produces more rapid increases in self-administration of ethanol relative to continuous exposure suggests that intermittent exposure may be associated with a more rapid escalation of the allostatic processes responsible for excessive ethanol self-administration. The mechanisms that drive the increases in drinking during withdrawal are similar after 2 and 8 hr of withdrawal and seem to be specific to ethanol.

Aβ induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and synaptic loss

Significance Communication between nerve cells occurs at specialized cellular structures known as synapses. Loss of synaptic function is associated with cognitive decline in Alzheimer’s disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here we describe a pathway for synaptic damage whereby amyloid-β1–42 peptide (Aβ1–42) releases, via stimulation of α7 nicotinic receptors, excessive amounts of glutamate from astrocytes, in turn activating extrasynaptic NMDA-type glutamate receptors (eNMDARs) to mediate synaptic damage. The Food and Drug Administration-approved drug memantine offers some beneficial effect, but the improved eNMDAR antagonist NitroMemantine completely ameliorates Aβ-induced synaptic loss, providing hope for disease-modifying intervention in AD. Synaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer’s disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here, using FRET-based glutamate sensor imaging, we show that amyloid-β peptide (Aβ) engages α7 nicotinic acetylcholine receptors to induce release of astrocytic glutamate, which in turn activates extrasynaptic NMDA receptors (eNMDARs) on neurons. In hippocampal autapses, this eNMDAR activity is followed by reduction in evoked and miniature excitatory postsynaptic currents (mEPSCs). Decreased mEPSC frequency may reflect early synaptic injury because of concurrent eNMDAR-mediated NO production, tau phosphorylation, and caspase-3 activation, each of which is implicated in spine loss. In hippocampal slices, oligomeric Aβ induces eNMDAR-mediated synaptic depression. In AD-transgenic mice compared with wild type, whole-cell recordings revealed excessive tonic eNMDAR activity accompanied by eNMDAR-sensitive loss of mEPSCs. Importantly, the improved NMDAR antagonist NitroMemantine, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from Aβ-induced damage both in vitro and in vivo.

Central role of ceramide biosynthesis in body weight regulation, energy metabolism, and the metabolic syndrome

Although obesity is associated with multiple features of the metabolic syndrome (insulin resistance, leptin resistance, hepatic steatosis, chronic inflammation, etc.), the molecular changes that promote these conditions are not completely understood. Here, we tested the hypothesis that elevated ceramide biosynthesis contributes to the pathogenesis of obesity and the metabolic syndrome. Chronic treatment for 8 wk of genetically obese (ob/ob), and, high-fat diet-induced obese (DIO) mice with myriocin, an inhibitor of de novo ceramide synthesis, decreased circulating ceramides. Decreased ceramide was associated with reduced weight, enhanced metabolism and energy expenditure, decreased hepatic steatosis, and improved glucose hemostasis via enhancement of insulin signaling in the liver and muscle. Inhibition of de novo ceramide biosynthesis decreased adipose expression of suppressor of cytokine signaling-3 (SOCS-3) and induced adipose uncoupling protein-3 (UCP3). Moreover, ceramide directly induced SOCS-3 and inhibited UCP3 mRNA in cultured adipocytes suggesting a direct role for ceramide in regulation of metabolism and energy expenditure. Inhibition of de novo ceramide synthesis had no effect on adipose tumor necrosis factor-alpha (TNF-alpha) expression but dramatically reduced adipose plasminogen activator inhibitor-1 (PAI-1) and monocyte chemoattactant protein-1 (MCP-1). This study highlights a novel role for ceramide biosynthesis in body weight regulation, energy expenditure, and the metabolic syndrome.

Stress enhancement of craving during sobriety: a risk for relapse.

This report of the proceedings of a symposium presented at the 2004 Research Society on Alcoholism Meeting provides evidence linking stress during sobriety to craving that increases the risk for relapse. The initial presentation by Rajita Sinha summarized clinical evidence for the hypothesis that there is an increased sensitivity to stress-induced craving in alcoholics. During early abstinence, alcoholics who were confronted with stressful circumstances showed increased susceptibility for relapse. George Breese presented data demonstrating that stress could substitute for repeated withdrawals from chronic ethanol to induce anxiety-like behavior. This persistent adaptive change induced by multiple withdrawals allowed stress to induce an anxiety-like response that was absent in animals that were not previously exposed to chronic ethanol. Subsequently, Amanda Roberts reviewed evidence that increased drinking induced by stress was dependent on corticotropin-releasing factor (CRF). In addition, rats that were stressed during protracted abstinence exhibited anxiety-like behavior that was also dependent on CRF. Christopher Dayas indicated that stress increases the reinstatement of an alcohol-related cue. Moreover, this effect was enhanced by previous alcohol dependence. These interactive effects between stress and alcohol-related environmental stimuli depended on concurrent activation of endogenous opioid and CRF systems. A.D. Lê covered information that indicated that stress facilitated reinstatement to alcohol responding and summarized the influence of multiple deprivations on this interaction. David Overstreet provided evidence that restraint stress during repeated alcohol deprivations increases voluntary drinking in alcohol-preferring (P) rats that results in withdrawal-induced anxiety that is not observed in the absence of stress. Testing of drugs on the stress-induced voluntary drinking implicated serotonin and CRF involvement in the sensitized response. Collectively, the presentations provided convincing support for an involvement of stress in the cause of relapse and continuing alcohol abuse and suggested novel pharmacological approaches for treating relapse induced by stress.

Impaired maturation of dendritic spines without disorganization of cortical cell layers in mice lacking NRG1/ErbB signaling in the central nervous system

Neuregulin-1 (NRG1) and its ErbB2/B4 receptors are encoded by candidate susceptibility genes for schizophrenia, yet the essential functions of NRG1 signaling in the CNS are still unclear. Using CRE/LOX technology, we have inactivated ErbB2/B4-mediated NRG1 signaling specifically in the CNS. In contrast to expectations, cell layers in the cerebral cortex, hippocampus, and cerebellum develop normally in the mutant mice. Instead, loss of ErbB2/B4 impairs dendritic spine maturation and perturbs interactions of postsynaptic scaffold proteins with glutamate receptors. Conversely, increased NRG1 levels promote spine maturation. ErbB2/B4-deficient mice show increased aggression and reduced prepulse inhibition. Treatment with the antipsychotic drug clozapine reverses the behavioral and spine defects. We conclude that ErbB2/B4-mediated NRG1 signaling modulates dendritic spine maturation, and that defects at glutamatergic synapses likely contribute to the behavioral abnormalities in ErbB2/B4-deficient mice.

High-priority communication I Antagonism of corticotropin-releasing factor attenuates the enhanced responsiveness to stress observed during protracted ethanol abstinence

One of the most critical attributes of chronic abstinence from alcohol is a state of anxiety, which can lead to mood disturbances and negative affect that can last for months or even years in alcoholics. Within hours after their final exposure to ethanol in experimental conditions, laboratory animals also exhibit an anxiety-like state. This state is accompanied by an enhanced stress response and can persist for weeks after withdrawal. One possible mechanism underlying these behavioral changes observed weeks after withdrawal is increased corticotropin-releasing factor (CRF) activity. In the present study, we sought to examine the role of CRF in the regulation of behavior in the elevated plus-maze during protracted abstinence by using intracerebroventricular administration of the CRF receptor antagonist [D-Phe(12),Nle(21,38),CalphaMeLeu(37)]rCRF((12-41)) (D-Phe-CRF((12-41))). Rats were surgically implanted with a guide cannula aimed at the lateral ventricles and subsequently fed a nutritionally complete ethanol [10% (vol./vol.)] or control liquid diet for 21 days. Rats were further divided into groups receiving microinjections of D-Phe-CRF((12-41)) or vehicle and 15 min of restraint stress, or D-Phe-CRF((12-41)) or vehicle and no restraint. Six weeks after removal of the liquid diet, rats were injected and then placed in a restraint tube or returned to their home cages for 15 min before testing in the elevated plus-maze. Rats with a history of ethanol dependence explored the open arms of the plus-maze significantly less when exposed to restraint stress compared with findings for all other groups, an effect attenuated by pretreatment with D-Phe-CRF((12-41)). Results of the current experiment demonstrated that continuous exposure to ethanol over a 3-week period leads to an increased behavioral responsiveness to stress, which seems to be regulated by CRF.

Glial ephrin-A3 regulates hippocampal dendritic spine morphology and glutamate transport

Increasing evidence indicates the importance of neuron-glia communication for synaptic function, but the mechanisms involved are not fully understood. We reported that the EphA4 receptor tyrosine kinase is in dendritic spines of pyramidal neurons of the adult hippocampus and regulates spine morphology. We now show that the ephrin-A3 ligand, which is located in the perisynaptic processes of astrocytes, is essential for maintaining EphA4 activation and normal spine morphology in vivo. Ephrin-A3-knockout mice have spine irregularities similar to those observed in EphA4-knockout mice. Remarkably, loss of ephrin-A3 or EphA4 increases the expression of glial glutamate transporters. Consistent with this, glutamate transport is elevated in ephrin-A3-null hippocampal slices whereas Eph-dependent stimulation of ephrin-A3 signaling inhibits glutamate transport. Furthermore, some forms of hippocampus-dependent learning are impaired in the ephrin-A3-knockout mice. Our results suggest that the interaction between neuronal EphA4 and glial ephrin-A3 bidirectionally controls synapse morphology and glial glutamate transport, ultimately regulating hippocampal function.

Transcription factor MEF2C influences neural stem/progenitor cell differentiation and maturation in vivo

Emerging evidence suggests that myocyte enhancer factor 2 (MEF2) transcription factors act as effectors of neurogenesis in the brain, with MEF2C the predominant isoform in developing cerebrocortex. Here, we show that conditional knockout of Mef2c in nestin-expressing neural stem/progenitor cells (NSCs) impaired neuronal differentiation in vivo, resulting in aberrant compaction and smaller somal size. NSC proliferation and survival were not affected. Conditional null mice surviving to adulthood manifested more immature electrophysiological network properties and severe behavioral deficits reminiscent of Rett syndrome, an autism-related disorder. Our data support a crucial role for MEF2C in programming early neuronal differentiation and proper distribution within the layers of the neocortex.