Andrey E. Ryabinin

Alcohol drinking produces brain region-selective changes in expression of inducible transcription factors.

Mapping the effects of alcohol consumption on neural activity could provide valuable information on mechanisms of alcohols effects on behavior. The present study sought to identify effects of alcohol consumption on expression of inducible transcription factors (ITFs) in mouse brain. C57BL/6J mice were trained to consume 10% ethanol/10% sucrose solution during a 30-min limited access period. Control animals were given access to 10% sucrose solution or water. Following the final day of the procedure, animals were sacrificed and immunohistochemical analyses were performed for three ITFs (c-Fos, FosB, and Zif268). Alcohol-consuming animals had increased ITF expression in several brain areas. Specifically, c-Fos was significantly induced in the nucleus accumbens core (AcbC), the medial posteroventral portion of the central nucleus of the amygdala (CeMPV), and the Edinger-Westphal nucleus (EW). Expression of c-Fos was significantly lower in the dentate gyrus of alcohol-consuming animals vs. sucrose-consuming animals. However, it was not significantly different from the water controls. Induction of c-Fos in AcbC, CeMPV and EW was significantly related to blood alcohol concentrations (BAC). Furthermore, FosB expression in the CeMPV and the EW was also significantly higher in the alcohol-consuming animals vs. water controls. FosB expression in the EW was significantly related to BAC. The significance of these results is two-fold. First, our experiments demonstrate that ITF mapping is an effective strategy in identifying alcohol-induced changes following voluntary consumption. Second, they suggest a relationship between ITF expression in AcbC, CeMPV and EW and the level of alcohol intoxication.

Different levels of Fos immunoreactivity after repeated handling and injection stress in two inbred strains of mice

Expression of Fos and Fos-related antigens was immunohistochemically analyzed in DBA/2J and C57BL/6J inbred mice in response to acute or repeated handling and injection stress. Both strains showed a strong induction of Fos and Fos-related antigens in discrete areas of hypothalamus, amygdala, neocortex, septum, and thalamus 2 h after an acute intraperitoneal injection of normal saline. To habituate animals to this procedure, mice were subjected to repeated handling and injections during 2 weeks preceding the experiment. This procedure led to complete habituation of the immediate early gene response to injection stress in stress-responsive brain areas of C57BL/6J mice, such that no significant difference was found between expression of these proteins in brains of saline-injected animals after repeated stress vs. control animals. In contrast, many brain areas of saline-injected DBA/2J mice still showed elevated Fos and Fos-related antigen expression after repeated injections. These results indicate that identical habituation procedures do not necessarily lead to identical levels of gene expression in brains of inbred strains of mice. In turn, they suggest that genetic components for some behavioral and pharmacological traits identified using inbred strains could be related to different rates of habituation to experimental procedures.

Role of hippocampus in alcohol-induced memory impairment: Implications from behavioral and immediate early gene studies

Abstract Acute alcohol intoxication disrupts memory acquisition in humans and laboratory animals. This review summarizes recent behavioral and immediate early gene expression studies addressing the mechanisms of this phenomenon. Most behavioral investigations agree that the amnestic effect of alcohol is due to its preferential detrimental effect on hippocampus-dependent than on hippocampus-independent forms of learning. However, some hippocampal lesion studies contradict these results. Learning in behavioral paradigms is accompanied by induction of c-fos and other immediate early genes in many brain regions of the animal. In contrast, studies on alcohol-mediated changes in expression of this gene confirm selective hippocampal suppression of basal and experience-induced expression of c-fos after acute and repeated administration of alcohol. This hippocampal suppression is in marked contrast with alcohol-mediated induction of c-fos expression in other brain areas. However, the selective suppression of hippocampal gene expression and memory by alcohol is most likely mediated by a number of interacting neurotransmitter systems. Thus, effects of lower doses of alcohol (0.5 g/kg or lower in rats) seem to be preferentially mediated through GABAergic systems. At intermediate doses (0.75–2 g/kg), several other neurotransmitter systems are affected besides GABA. Higher doses lead to none-specific effects, probably involving even more neurotransmitter systems. Elucidation of these neurotransmitter systems will be highly important for developing rational approaches for correction of alcohol-related cognitive disorders.

Hippocampal-dependent learning and experience-dependent activation of the hippocampus are preferentially disrupted by ethanol

A classical fear conditioning paradigm was used to examine the effect of acute ethanol on the acquisition of context conditioning, a hippocampal-dependent associative task, and tone conditioning, a hippocampal-independent task. Administration of ethanol before the presentation of seven tone-shock pairings severely disrupted the acquisition of context conditioning, but had only a slight effect on tone conditioning, when conditioned fear was measured 48 h later. This effect was dose dependent: a dose of 0.5 g/kg had no effect on either context or tone conditioning, while doses of 1.0 and 1.5 g/kg disrupted context conditioning by 78-86%, and tone conditioning by 9-17%. Subsequent experiments indicated that ethanols preferential effect on context conditioning could not be attributed to the fact that context conditioning is weaker than tone conditioning, ethanol-induced changes in motivational state or state-dependent learning. The effect of ethanol on stimulus-induced increases in hippocampal and neocortical expression of c-fos mRNA, a marker for changes in metabolic neuronal activity, was also examined. Ethanol completely blocked the induction of hippocampal c-fos mRNA by exposure to the conditioning context alone or seven tone-shock pairings, but only attenuated neocortical responses to these stimuli. Together, these results suggest that ethanol disrupts hippocampal-dependent learning by preferentially impairing stimulus processing at the level of the hippocampus.

Increasing Histone Acetylation in the Hippocampus-Infralimbic Network Enhances Fear Extinction

BACKGROUND A key finding from recent studies of epigenetic mechanisms of memory is that increasing histone acetylation after a learning experience enhances memory consolidation. This has been demonstrated in several preparations, but little is known about whether excitatory and inhibitory memories are equally sensitive to drugs that promote histone acetylation and how transcriptional changes in the hippocampal-medial prefrontal cortex network contribute to these drug effects. METHODS We compare the long-term behavioral consequences of systemic, intrahippocampal and intra-medial prefrontal cortex administration of the histone deacetylase inhibitor sodium butyrate (NaB) after contextual fear conditioning and extinction 1 and/or 14 days later in male c57BL/6J mice (n = 302). Levels of histone acetylation and expression of the product of the immediate-early gene c-Fos were assessed by immunohistochemistry following infusion of NaB into the hippocampus (n = 26). RESULTS Across a variety of conditions, the effects of NaB on extinction were larger and more persistent compared to the effects on initial memory formation. NaB administered following weak extinction induced behavioral extinction, infralimbic histone acetylation and c-Fos expression consistent with strong extinction. No similar effect was seen in the prelimbic cortex. The involvement of the infralimbic cortex was confirmed as infusions of NaB into the infralimbic, but not prelimbic cortex, induced extinction enhancements. CONCLUSIONS These studies show that the memory modulating ability of drugs that enhance acetylation is sensitive to a variety of behavioral and molecular conditions. We further identify transcriptional changes in the hippocampal-infralimbic circuit associated with extinction enhancements induced by the histone deacetylase inhibitor NaB.

THE EDINGER-WESTPHAL NUCLEUS: A HISTORICAL, STRUCTURAL AND FUNCTIONAL PERSPECTIVE ON A DICHOTOMOUS TERMINOLOGY

The eponymous term nucleus of Edinger‐Westphal (EW) has come to be used to describe two juxtaposed and somewhat intermingled cell groups of the midbrain that differ dramatically in their connectivity and neurochemistry. On one hand, the classically defined EW is the part of the oculomotor complex that is the source of the parasympathetic preganglionic motoneuron input to the ciliary ganglion (CG), through which it controls pupil constriction and lens accommodation. On the other hand, EW is applied to a population of centrally projecting neurons involved in sympathetic, consumptive, and stress‐related functions. This terminology problem arose because the name EW has historically been applied to the most prominent cell collection above or between the somatic oculomotor nuclei (III), an assumption based on the known location of the preganglionic motoneurons in monkeys. However, in many mammals, the nucleus designated as EW is not made up of cholinergic, preganglionic motoneurons supplying the CG and instead contains neurons using peptides, such as urocortin 1, with diverse central projections. As a result, the literature has become increasingly confusing. To resolve this problem, we suggest that the term EW be supplemented with terminology based on connectivity. Specifically, we recommend that 1) the cholinergic, preganglionic neurons supplying the CG be termed the Edinger‐Westphal preganglionic (EWpg) population and 2) the centrally projecting, peptidergic neurons be termed the Edinger‐Westphal centrally projecting (EWcp) population. The history of this nomenclature problem and the rationale for our solutions are discussed in this review. J. Comp. Neurol. 519:1413–1434, 2011.

Urocortin 1 distribution in mouse brain is strain-dependent

Urocortin 1 has been implicated in a number of specific behaviors, which include energy balance, stress reactivity and ethanol consumption. To elucidate genetically influenced differences in the mouse urocortin 1 system, we performed immunohistochemical characterization of urocortin 1 distribution in C57BL/6J and DBA/2J mouse brain. Urocortin 1 analysis reveals strain-dependent differences in distribution of urocortin 1 immunoreactive neurons and neuronal fibers. In both strains, the highest number of urocortin 1-positive neurons was observed in the Edinger-Westphal nucleus and lateral superior olive. Urocortin 1-positive neurons were detected in the dorsal nucleus of the lateral lemniscus of DBA/2J mice, but were absent in the C57BL/6J strain. Differences in urocortin 1 fibers were detected in many areas throughout the brain, and were most apparent in the septal areas, thalamic areas, several midbrain regions, and medulla. Strain-dependent distribution of urocortin 1-containing cells and fibers suggests that differences in this neuropeptide system may underlie differences in behavior and physiological responses between these strains. Further, we found that in both mouse strains, urocortin 1 in the Edinger-Westphal nucleus and choline acetyltransferase are not coexpressed. We show that the urocortin 1-positive neurons of this brain area form a separate population of cells that we propose to be called the non-preganglionic Edinger-Westphal nucleus.

CRF receptors in the nucleus accumbens modulate partner preference in prairie voles

Recent evidence suggests a role for corticotropin-releasing factor (CRF) in the regulation of pair bonding in prairie voles. We have previously shown that monogamous and non-monogamous vole species have dramatically different distributions of CRF receptor type 1 (CRF(1)) and CRF receptor type 2 (CRF(2)) in the brain and that CRF(1) and CRF(2) receptor densities in the nucleus accumbens (NAcc) are correlated with social organization. Monogamous prairie and pine voles have significantly lower levels of CRF receptor type 1 (CRF(1)), and significantly higher levels of type 2 (CRF(2)) binding, in NAcc than non-monogamous meadow and montane voles. Here, we report that microinjections of CRF directly into the NAcc accelerate partner preference formation in male prairie voles. Control injections of CSF into NAcc, and CRF into caudate-putamen, did not facilitate partner preference. Likewise, CRF injections into NAcc of non-monogamous meadow voles also did not facilitate partner preference. In prairie voles, this CRF facilitation effect was blocked by co-injection of either CRF(1) or CRF(2) receptor antagonists into NAcc. Immunocytochemical staining for CRF and Urocortin-1 (Ucn-1), two endogenous ligands for CRF(1) and CRF(2) receptors in the brain, revealed that CRF, but not Ucn-1, immunoreactive fibers were present in NAcc. This supports the hypothesis that local CRF release into NAcc could activate CRF(1) or CRF(2) receptors in the region. Taken together, our results reveal a novel role for accumbal CRF systems in social behavior.

Stress-related neuropeptides and addictive behaviors: beyond the usual suspects.

Addictive disorders are chronic, relapsing conditions that cause extensive disease burden. Genetic factors partly account for susceptibility to addiction, but environmental factors such as stressful experiences and prolonged exposure of the brain to addictive drugs promote its development. Progression to addiction involves neuroadaptations within neurocircuitry that mediates stress responses and is influenced by several peptidergic neuromodulators. While corticotrophin releasing factor is the prototypic member of this class, recent work has identified several additional stress-related neuropeptides that play an important role in regulation of drug intake and relapse, including the urocortins, nociceptin, substance P, and neuropeptide S. Here, we review this emerging literature, discussing to what extent the properties of these neuromodulators are shared or distinct and considering their potential as drug targets.

Urocortin 1-containing neurons in the human Edinger-Westphal nucleus

The topographical location of neurons containing urocortin 1, a peptide related to corticotropin-releasing factor was investigated in human postmortem brain by immunohistochemistry, and compared with the location of neurons containing choline acetyltransferase, a marker for cholinergic cells. A three-dimensional computer reconstruction of the urocortin 1 and choline acetyltransferase-positive population of neurons within the oculomotor area was made. It was shown that the urocortin 1-positive neurons are located within the area identified as the Edinger-Westphal nucleus according to the human brain stem atlas, and that the neurons identified as Edinger-Westphal nucleus in the atlas are not choline acetyltransferase-positive. This finding agrees with recent animal studies showing that urocortin 1-positive neurons are not identical with the parasympathetic cholinergic neurons projecting to the ciliary ganglion. They indicate that the neurons identified as Edinger-Westphal nucleus in the human brain stem atlas belong to the non-preganglionic Edinger-Westphal nucleus, whereas the location of preganglionic Edinger-Westphal nucleus remains unidentified.