Detailed examination of the midbrain Edinger–Westphal (EW) nucleus revealed the existence of two distinct nuclei. One population of EW preganglionic (EWpg) neurons was found to control oculomotor functions, and a separate population of EW centrally projecting (EWcp) neurons was found to contain stress- and feeding-related neuropeptides. Although it has been shown that EWcp neurons are highly responsive to drugs of abuse and behavioral stress, a genetic characterization of the EWcp was needed. To identify genetic differences in the EWcp of inbred mouse strains that differ in behaviors relevant to EWcp function, we used publicly available tools from the Allen Brain Atlas to identify 68 transcripts that were selectively expressed in the EWcp, and examined their expression within tissue punch microdissection samples containing the EWcp of adult male C57BL/6J (B6) and DBA/2J (D2) mice. Using 96-well quantitative real-time PCR (qPCR) arrays that included the EWcp-specific genes, several other genes of interest, and five housekeeping genes, we identified strain differences in expression of 11 EWcp-specific genes (BC023892, Btg3, Bves, Cart, Cck, Ghsr, Neto1, Postn, Ptprn, Rcn1, and Ucn), two immediate early genes (Egr1 and Fos), and one dopamine-related gene (Drd5). All significant expression differences were greater in B6 vs. D2 mice, and several of these were verified either at the protein level using immunohistochemistry (IHC) or in silico using microarray data sets from whole brain and other brain areas. These results demonstrate a significant advance in our understanding of the EWcp on three levels. First, we generated a list of EWcp-specific genes (most of which had not yet been reported within the EWcp in the literature) that will be informative for future studies of EWcp function. Second, due to similarity in results from qPCR and IHC, we revealed that strain differences in basal EWcp neuropeptide content are accounted for by differential transcription and number of peptidergic neurons, rather than by differential rates of peptide release. And third, our identification of differentially expressed EWcp-specific genes between B6 and D2 mice may hold powerful insight into the neurogenetic contributions of the EWcp to stress- and addiction-related behaviors.

The neural circuitry that processes natural rewards converges with that engaged by addictive drugs. Because of this common neurocircuitry, drugs of abuse have been able to engage the hedonic mechanisms normally associated with the processing of natural rewards. Ghrelin is an orexigenic peptide that stimulates food intake by activating GHS-R1A receptors in the hypothalamus. However, ghrelin also activates GHS-R1A receptors on extrahypothalamic targets that mediate alcohol reward. The central nucleus of the amygdala (CeA) has a critical role in regulating ethanol consumption and the response to ethanol withdrawal. We previously demonstrated that rat CeA GABAergic transmission is enhanced by acute and chronic ethanol treatment. Here, we used quantitative RT-PCR (qRT-PCR) to detect Ghsr mRNA in the CeA and performed electrophysiological recordings to measure ghrelin effects on GABA transmission in this brain region. Furthermore, we examined whether acute or chronic ethanol treatment would alter these electrophysiological effects. Our qRT-PCR studies show the presence of Ghsr mRNA in the CeA. In naive animals, superfusion of ghrelin increased the amplitude of evoked inhibitory postsynaptic potentials (IPSPs) and the frequency of miniature inhibitory postsynaptic currents (mIPSCs). Coapplication of ethanol further increased the ghrelin-induced enhancement of IPSP amplitude, but to a lesser extent than ethanol alone. When applied alone, ethanol significantly increased IPSP amplitude, but this effect was attenuated by the application of ghrelin. In neurons from chronic ethanol-treated (CET) animals, the magnitude of ghrelin-induced increases in IPSP amplitude was not significantly different from that in naive animals, but the ethanol-induced increase in amplitude was abolished. Superfusion of the GHS-R1A antagonists D-Lys3-GHRP-6 and JMV 3002 decreased evoked IPSP and mIPSC frequency, revealing tonic ghrelin activity in the CeA. D-Lys3-GHRP-6 and JMV 3002 also blocked ghrelin-induced increases in GABAergic responses. Furthermore, D-Lys3-GHRP-6 did not affect ethanol-induced increases in IPSP amplitude. These studies implicate a potential role for the ghrelin system in regulating GABAergic transmission and a complex interaction with ethanol at CeA GABAergic synapses.

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The perioculomotor urocortin-containing population of neurons (pIIIu: otherwise known as the nonpreganglionic Edinger-Westphal nucleus) is sensitive to alcohol and is involved in the regulation of alcohol intake. A recent study indicated that this brain region is also sensitive to psychostimulants. Since pIIIu has been shown to respond to stress, we investigated how psychostimulant-induced pIIIu activation compares to stress-and ethanol-induced activation, and whether it is independent from a generalized stress response. Several experiments were performed to test how the pIIIu responds to psychostimulants by quantifying the number of Fos immunoreactive nuclei after acute intraperitoneal injections of saline, 10-30 mg/kg cocaine, 5 mg/kg methamphetamine, 5 mg/kg amphetamine, 2.5 g/kg ethanol, 2 hours of restraint stress, 10 minutes of swim stress, or six applications of mild footshock in male C57BL/6J mice. We also compared Fos immunoreactivity in pIIIu after acute (20 mg/kg cocaine) and repeated cocaine exposure (7 days of 20 mg/kg cocaine) injections in male C57BL/6J mice in order to investigate the potential habituation of this response. Finally, we quantified the number of Fos immunoreactive nuclei in pIIIu after administration of saline, 2.5 g/kg ethanol, 20 mg/kg cocaine, or 2 hours of restraint stress in male Sprague-Dawley rats. We found that exposure to psychostimulants and ethanol induced significantly higher Fos levels in pIIIu compared to stress in mice. Furthermore, repeated cocaine injections did not decrease Fos immunoreactivity as would be expected if this response was due to stress. In rats, exposure to ethanol, psychostimulant and restraint stress all induced pIIIu Fos immunoreactivity compared to saline-injected controls. In both mice and rats, ethanol-and cocaine-induced Fos immunoreactivity occured exclusively in urocortin 1-, but not in tyrosine hydroxylase-, positive cells. These results provide evidence that the pIIIu Fos-response to psychostimulants is independent of a generalized stress in mice, but not rats. They additionally show that the pIIIu response to stress differs significantly between species.

Sensitivity to the euphoric and locomotor-activating effects of drugs of abuse may contribute to risk for excessive use and addiction. Repeated administration of psychostimulants such as methamphetamine (MA) can result in neuroadaptive consequences that manifest behaviorally as a progressive escalation of locomotor activation, termed psychomotor sensitization. The present studies addressed the involvement of specific components of the corticotropin-releasing factor (CRF) system in locomotor activation and psychomotor sensitization induced by MA (1, 2 mg/kg) by utilizing pharmacological approaches, as well as a series of genetic knockout (KO) mice, each deficient for a single component of the CRF system: CRF-R1, CRF-R2, CRF, or the CRF-related peptide Urocortin 1 (Ucn1). CRF-R1 KO mice did not differ from wild-type mice in sensitization to MA, and pharmacological blockade of CRF-R1 with CP-154,526 (15, 30 mg/kg) in DBA/2J mice did not selectively attenuate either the acquisition or expression of MA-induced sensitization. Deletion of either of the endogenous ligands of CRF-R1 (CRF, Ucn1) either enhanced or had no effect on MA-induced sensitization, providing further evidence against a role for CRF-R1 signaling. Interestingly, deletion of CRF-R2 attenuated MA-induced locomotor activation, elucidating a novel contribution of the CRF system to MA sensitivity, and suggesting the participation of the endogenous urocortin peptides Ucn2 and Ucn3. Immunohistochemistry for Fos was used to visualize neural activation underlying CRF-R2-dependent sensitivity to MA, identifying the basolateral and central nuclei of the amygdala as neural substrates involved in this response. Our results support further examination of CRF-R2 involvement in neural processes associated with MA addiction.

Urocortin 1 (Ucn 1) is an endogenous corticotropin releasing factor (CRF)-related peptide. Ucn 1 is most highly expressed in the perioculomotor urocortin containing neurons (pIIIu), previously known as the non-preganglionic Edinger-Westphal nucleus (npEW). Various studies indicate that these cells are involved in stress adaptation and the regulation of ethanol (EtOH) intake. However, the developmental trajectory of these neurons remained unexamined. Expression of the cocaine-and amphetamine-regulated transcript (CART), which co-localizes with Ucn 1 in the perioculomotor area (pIII) has been examined prenatally, but not postnatally. The goal of the current study was to characterize the ontogenetic profile of Ucn 1 and CART during postnatal development in C57BL/6J (B6) mice. B6 mice were bred, and brains were collected at postnatal days (PND) 1, 4, 8, 12, 16, 24 and 45. Brightfield immunohistochemical staining for Ucn 1 and CART showed that Ucn 1 -immunoreactivity (ir) was absent at PND 1, while CART -ir was already apparent in pIIIu at birth, a finding indicating that although the pIIIu neurons have already migrated to their adult position, Ucn 1 expression is triggered in them at later postnatal stages. Ucn1 -ir gradually increased with age, approaching adult levels at PND 16. This developmental profile was confirmed by doubleimmunofluorescence, which showed that Ucn 1 was absent in CART -positive cells of pIII at PND 4 and that Ucn 1 and CART are strongly but not completely co-localized in pIII at PND 24. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis confirmed that Ucn 1 mRNA levels are significantly lower at PND 4 and PND 12 than in adult animals. The lack of brain Ucn 1 immunoreactivity at birth and the gradual postnatal increase in Ucn 1 in pIIIu suggests that this peptide plays a greater behavioral role in adulthood than during the early postnatal development of an organism.

Noninvasive functional imaging holds great promise for the future of translational research, due to the ability to directly compare between preclinical and clinical models of psychiatric disorders. Despite this potential, concerns have been raised regarding the necessity to anesthetize rodent and monkey subjects during these procedures, because anesthetics may alter neuronal activity. For example, in studies of drugs of abuse and alcohol, it is not clear to what extent anesthesia can interfere with drug-induced neural activity. Therefore, the current study investigated whole brain c-Fos activation following isoflurane anesthesia as well as ethanol-induced activation of c-Fos in anesthetized mice. In the first experiment, we examined effects of 1 or 3 sessions of gaseous isoflurane on c-Fos activation across the brain in male C57BL/6J mice. Isoflurane administration led to c-Fos activation in several areas, including the piriform cortex and lateral septum. Lower or similar levels of activation in these areas were detected after three sessions of isoflurane, suggesting that multiple exposures may eliminate some of the enhanced neuronal activation caused by acute isoflurane. In the second experiment, we investigated the ability of ethanol injection (1.5 or 2.5 g/kg i.p.) to induce c-Fos activation under anesthesia. Following three sessions of isoflurane, 1.5 g/kg of ethanol induced c-Fos in the central nucleus of amygdala, piriform cortex, and centrally-projecting Edinger-Westphal nucleus. This induction was lower after 2.5 g/kg of ethanol. These results demonstrate that ethanol-induced neural activation can be detected in the presence of isoflurane anesthesia. They also suggest, that while habituation to isoflurane helps reduce neuronal activation, interaction between effects of anesthesia and alcohol can occur. Studies using fMRI imaging could benefit from using habituated animals and dose response analyses.

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