Periadolescent ethanol exposure reduces adult forebrain ChAT+IR neurons: correlation with behavioral pathology.

Substance abuse typically begins in adolescence; therefore, the impact of alcohol during this critical time in brain development is of particular importance. Epidemiological data indicate that excessive alcohol consumption is prevalent among adolescents and may have lasting neurobehavioral consequences. Loss of cholinergic input to the forebrain has been demonstrated following fetal alcohol exposure and in adults with Wernicke-Korsakoff syndrome. In the present study, immunohistochemistry for choline acetyltransferase (ChAT) was determined to assess forebrain cholinergic neurons (Ch1-4), and behavioral changes following periadolescent alcohol exposure. Wistar rats were exposed to intermittent ethanol vapor (14 h on/10 h off/day) for 35 days from postnatal day (PD) 22 to PD 57 (average blood alcohol concentration (BAC): 163 mg%). Rats were withdrawn from vapor and assessed for locomotor activity, startle response, conflict behavior in the open field, and immobility in the forced swim test, as adults. Rats were then sacrificed at day 71/72 and perfused for histochemical analyses. Ethanol vapor-exposed rats displayed: increased locomotor activity 8 h after the termination of vapor delivery for that 24 h period at day 10 and day 20 of alcohol vapor exposure, significant reductions in the amplitude of their responses to prepulse stimuli during the startle paradigm at 24 h withdrawal, and at 2 weeks following withdrawal, less anxiety-like and/or more "disinhibitory" behavior in the open field conflict, and more immobility in the forced swim test. Quantitative analyses of ChAT immunoreactivity revealed a significant reduction in cell counts in the Ch1-2 and Ch3-4 regions of the basal forebrain in ethanol vapor-exposed rats. This reduction in cell counts was significantly correlated with less anxiety-like and/or more "disinhibitory" behavior in the open field conflict test. These studies demonstrate that behavioral measures of arousal, affective state, disinhibitory behavior, and ChAT+IR, are all significantly impacted by periadolescent ethanol exposure and withdrawal in Wistar rats.

N-methyl-D-aspartate receptor subunit expression in adult and adolescent brain following chronic ethanol exposure.

Substantial evidence suggests that glutamatergic neurotransmission is a critical mediator of the experience-dependent synaptic plasticity that may underlie alcohol dependence. Substance abuse typically begins in adolescence; therefore, the impact of alcohol on glutamatergic systems during this critical time in brain development is of particular importance. The N-methyl-d-aspartate receptor (NMDAR) is involved in developmental mechanisms underlying neuronal differentiation and synaptogenesis and as such may be a target system for alcohol effects during adolescence. In the present study quantitative biochemical determinations were made of the relative abundance of different protein expressions of NMDAR subunits in adolescents and adults after 2 weeks of ethanol vapor exposure, and 24 h and 2 weeks following withdrawal. After 2 weeks of ethanol vapor exposure N-methyl-d-aspartate receptor NR1 subunit (NR1), N-methyl-d-aspartate receptor NR2A subunit (NR2A), and N-methyl-d-aspartate receptor NR2B subunit (NR2B) subunit expression was found to be increased in hippocampus of the adults. In contrast, 2 weeks of ethanol exposure resulted in no significant changes in NR1 and NR2B subunits and a reduction NR2A subunit expression in hippocampus in adolescents. Twenty-four h and 2 weeks following withdrawal from ethanol vapor NR1 and NR2A subunit expression in hippocampus was decreased in adolescents, whereas in adults it had returned to control levels. In frontal cortex, 2 weeks of chronic ethanol exposure produced decreases in NR1 subunit expression in both adults and adolescents but also produced decreases in NR2A and NR2B subunit expression in adults that returned or exceeded control levels by 2 weeks following withdrawal from ethanol vapor. These results demonstrate that NMDAR subunit composition can be modulated differentially between adolescents and adults by chronic ethanol exposure and withdrawal. These developmental differences in NMDAR subunits composition may also be associated with the enhanced vulnerability of the adolescent brain to ethanol dependence.

Event-related oscillations as risk markers in genetic mouse models of high alcohol preference.

Mouse models have been developed to simulate several relevant human traits associated with alcohol use and dependence. However, the neurophysiological substrates regulating these traits remain to be completely elucidated. We have previously demonstrated that differences in the event-related potential (ERP) responses can be found that distinguish high-alcohol preferring from low alcohol preferring mice that resemble differences seen in human studies of individuals with high and low risk for alcohol dependence. Recently, evidence of genes that affect event-related oscillations (EROs) and the risk for alcohol dependence has emerged, however, to date EROs have not been evaluated in genetic mouse models of high and low alcohol preference. Therefore, the objective of the present study was to characterize EROs in mouse models of high (C57BL/6 [B6] and high alcohol preference 1 [HAP-1] mice) and low (DBA/2J [D2] and low alcohol preference-1 [LAP-1] mice) alcohol preference. A time-frequency representation method was used to determine delta, theta and alpha/beta ERO energy and the degree of phase variation in these mouse models. The present results suggest that the decrease in P3 amplitudes previously shown in B6 mice, compared to D2 mice, is related to reductions in evoked delta ERO energy and delta and theta phase locking. In contrast, the increase in P1 amplitudes reported in HAP-1 mice, compared to LAP-1 mice, is associated with increases in evoked theta ERO energy. These studies suggest that differences in delta and theta ERO measures in mice mirror changes observed between groups at high- and low-risk for alcoholism where changes in EROs were found to be more significant than group differences in P3 amplitudes, further suggesting that ERO measures are more stable endophenotypes in the study of alcohol dependence. Further studies are needed to determine the relationship between expression of these neurophysiological endophenotypes and the genetic profile of these mouse models.

Apolipoprotein D mRNA expression is elevated in PDAPP transgenic mice.

Apolipoprotein D (apoD) expression is known to be elevated in select regions of rodent and human brain in association with different types of CNS pathology. To investigate a potential role for apoD in the neuropathology of Alzheimer's disease, we have measured apoD mRNA expression in transgenic mice expressing mutated human amyloid precursor protein under control of platelet-derived growth factor promoter (PDAPP mice). In situ hybridization analysis revealed increased apoD mRNA expression in brains of aged (26 months) PDAPP transgenic mice compared to aged littermate controls. These increases were most prominent in the hippocampal fimbria, corpus callosum and other white matter tracts. No substantial increases in expression were observed in white matter regions in young (6 months) PDAPP transgenic mice compared to young controls. Comparison between aged and young control mice revealed increased apoD expression in similar white matter regions of the aged animals. These findings suggest that, although increases in apoD expression are a normal feature of brain aging, super-increases may represent a glial cell compensatory response to beta-amyloid deposition in Alzheimer's disease.

Hypocretin-1 modulates rapid eye movement sleep through activation of locus coeruleus neurons.

The hypocretins (hcrts), also known as orexins, are two recently identified excitatory neuropeptides that in rat are produced by approximately 1200 neurons whose cell bodies are located in the lateral hypothalamus. The hypocretins/orexins have been implicated in the regulation of rapid eye movement (REM) sleep and the pathophysiology of narcolepsy. In the present study, we investigated whether the locus coeruleus (LC), a structure receiving dense hcrtergic innervation, which is quiescent during REM sleep, might be a target for hcrt to regulate REM sleep. Local administration of hcrt1 but not hcrt2 in the LC suppressed REM sleep in a dose-dependent manner and increased wakefulness at the expense of deep, slow-wave sleep. These effects were blocked with an antibody that neutralizes hcrt binding to hcrt receptor 1. In situ hybridization and immunocytochemistry showed the presence of hcrt receptor 1 but not the presence of hcrt receptor 2 in the LC. Iontophoretic application of hcrt1 enhanced the firing rate of LC neurons in vivo, and local injection of hcrt1 into the LC induced the expression of c-fos in the LC area. We propose that hcrt receptor 1 in the LC is a key target for REM sleep regulation and might be involved in the pathophysiological mechanisms of narcolepsy.

In vivo synaptic transmission in young and aged amyloid precursor protein transgenic mice.

Alzheimer's disease (AD) is characterized by progressive neurodegeneration and cognitive impairment. We examined in vivo alterations in hippocampal neurotransmission in both young and aged PDAPP transgenic mice and nontransgenic littermates. We now report that in vivo abnormal neurotransmission in hippocampal circuits of PDAPP mice precedes beta deposition and neurodegeneration. These in vivo data provide the first evidence that dysfunction in hippocampal neuronal circuits may not be correlated with age-related extracellular beta plaque deposition.

Ethanol inhibition of N-methyl-D-aspartate responses involves presynaptic gamma-aminobutyric acid(B) receptors.

Ethanol alters N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid subtype A (GABA(A)) receptor-mediated neurotransmission. We have previously demonstrated that GABA(B) receptor blockade uncovers ethanol enhancement of GABA(A) responses in the hippocampus. Therefore, we evaluated in vivo and in vitro the role of GABA(B) receptors in ethanol-induced inhibition of neuronal activity as well as NMDA responses in the hippocampus, ventral tegmental area (VTA), and nucleus accumbens (NAcc), three brain areas with known sensitivity to low doses of ethanol. In vivo, in situ microelectrophoretic application of ethanol enhanced inhibition of VTA GABA neuron firing rate by the GABA(B) agonist baclofen and reduced inhibition of VTA GABA firing rate by the GABA(A) agonist muscimol. The GABA(B) antagonist CGP35348 blocked baclofen- and ethanol-induced, but not muscimol-induced, reduction of NMDA-activated firing of hippocampal hilar mossy cells, hilar interneurons, and VTA GABA neurons, as well as ethanol inhibition of NMDA receptor-sensitive, amygdala-driven NAcc neurons. We performed in vitro studies in NAcc slices to evaluate the mechanism of GABA(B) receptor-mediated ethanol inhibition of NMDA neurotransmission. In the presence of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and the GABA(A) receptor antagonist bicuculline, superfusion of the GABA(B) antagonist CGP55845 blocked ethanol (66 mM) inhibition of evoked NMDA receptor-mediated excitatory postsynaptic potentials. However, CGP55845 did not significantly affect ethanol inhibition of NMDA currents produced by pressure application of NMDA or non-NMDA glutamatergic excitatory postsynaptic potentials evoked in the presence of the bicuculline and the NMDA antagonist DL-2-amino-5-phosphonovalerate. Taken together, these findings suggest that the sensitivity of NMDA receptor-mediated neurotransmission to ethanol is regulated by GABA(B) receptors, possibly at presynaptic sites.

Acute ethanol induction of c-Fos immunoreactivity in pre-pro-enkephalin expressing neurons of the central nucleus of the amygdala.

Previous studies have shown that acute ethanol administration induces expression of c-Fos immunoreactivity in the central nucleus of the amygdala (CNA) [S.L. Chang, N.A. Patel, A.A. Romero, Activation and desensitization of Fos immunoreactivity in the rat brain following ethanol administration, Brain Res., 679 (1995) 89-98; M. Morales, J.R. Criado, P.P. Sanna, S.J. Henriksen, F.E. Bloom, Acute ethanol induces c-fos immunoreactivity in GABAergic neurons of the central nucleus of the amygdala, Brain Res., 798 (1998) 333-336; A.E. Ryabinin, J.R. Criado, S.J. Henriksen, F.E. Bloom, M.C. Wilson, Differential sensitivity of c-Fos expression in hippocampus and other brain regions to moderate and low doses of alcohol, Mol. Psychiatry, 2 (1997) 32-43]. We recently showed that over 70% of these c-Fos immunoreactive neurons are GABAergic [M. Morales, J.R. Criado, P.P. Sanna, S.J. Henriksen, F.E. Bloom, Acute ethanol induces c-fos immunoreactivity in GABAergic neurons of the central nucleus of the amygdala, Brain Res, 798 (1998) 333-336]. In the present study, we report that ethanol-induced c-Fos immunoreactivity was mainly confined to neurons that express pro-enkephalin (ENK). In contrast, a small number of c-Fos immunoreactive neurons express corticotrophin releasing factor (CRF). Our results thus provide anatomical evidence indicating that within the amygdala, GABAergic neurons that contain ENK are responsive to acute ethanol exposure.

Adaptive responses of gamma-aminobutyric acid neurons in the ventral tegmental area to chronic ethanol.

We have recently identified a homogeneous population of gamma-aminobutyric acid (GABA)-containing neurons in the ventral tegmental area (VTA), an area implicated in the reinforcing properties of alcohol. We evaluated the effects of local and systemic ethanol on VTA GABA neuron spontaneous activity in ethanol naive and chronically treated freely behaving rats and in anesthetized rats. In freely behaving animals, acute i.p. administration of 0.2 to 2.0 g/kg ethanol reduced the firing rate of VTA GABA neurons. Chronic administration of 2.0 g/kg i.p. ethanol enhanced baseline activity of VTA GABA neurons and induced tolerance to ethanol inhibition of their firing rate. In a separate group of freely behaving animals, tolerance to 0.4 to 2.0 g/kg i.p. ethanol-induced inhibition of VTA GABA neuron firing rate was observed following 2 weeks of chronic exposure to ethanol vapors producing intermittent blood alcohol levels of 158 mg/100 ml. In acute studies in halothane-anesthetized animals, ethanol applied locally into the VTA decreased the spontaneous firing rate of VTA GABA neurons, whereas systemic ethanol produced an early inhibition followed by a late excitation at 30 to 60 min after the ethanol injection, suggesting that ethanol modulation of an extrinsic input may excite VTA GABA neurons. Tolerance to local ethanol inhibition of VTA GABA neuron firing rate was produced by 2 weeks of chronic exposure to intermittent ethanol vapors. These results demonstrate the marked sensitivity of these neurons to ethanol and suggest that chronic ethanol administration produces selective adaptive circuit responses within the VTA or in extrategmental structures that regulate VTA GABA neuron activity.

Structural and compositional determinants of cortistatin activity.

Cortistatin-14 (CST-14) is a putative novel neuropeptide that shares 11 of its 14 residues with somatostatin-14 (SRIF-14), yet its effects on sleep physiology, locomotor behavior and hippocampal function are different from those of somatostatin. We studied the structural basis for cortistatin's distinct biological activities. As with SRIF-14, CST-14 does not show any preferred conformation in solution, as determined by circular dichroism and nuclear magnetic resonance. Synthetic cortistatin analogs were designed and synthesized based on the cyclic structure of octreotide. Biological assays were carried out to determine their binding affinities to five somatostatin receptors (sstl-5) and their ability to produce changes in locomotor activity and to modulate hippocampal physiology and sleep. The results show that the compound with N-terminal proline and C-terminal lysine amide exhibits cortistatin-like biological activities, including reduction of population spike amplitudes in the hippocampal CA1 region, decrease in locomotor activity and enhancement of slow-wave sleep 2. These findings suggest that both proline and lysine are necessary for cortistatin binding to its specific receptor.

Cellular responses of nucleus accumbens neurons to opiate-seeking behavior: I. Sustained responding during heroin self-administration.

The nucleus accumbens (NAcc) has been hypothesized to be a critical component of the circuit mediating opiate-seeking behaviors. To further explore the electrophysiological correlates of opiate-seeking behavior, we recorded neurons in the NAcc and in the medial prefrontal cortex (mPFC) of rats trained to self-administer heroin for at least 2 weeks. Rats were trained to lever press (FR-1 schedule) for an intravenous (i.v.) infusion of heroin (0.06 mg/kg/injection) in an operant chamber. Spontaneous single unit activity in the NAcc and the mPFC was then recorded while animals were allowed to self-administer heroin. Our data suggest that about 20% (8/42) of the NAcc neurons studied exhibited an inhibitory response immediately after heroin self-administration. However, most of the NAcc neurons studied (76%; 32/42) were not affected during heroin self-administration. In contrast, noncontingent injection of a similar dose of heroin (0.06 mg/kg/injection) had no effect on NAcc spontaneous activity (0/6). On the other hand, passive administration of higher doses of heroin (0.2-0.6/mg/kg/injection) markedly suppressed the firing rate in 46% (6/13) of the neurons studied. These effects of heroin on NAcc activity were antagonized by systemic administration ofnaloxone (4-6 mg/kg, i.v.). Studies characterizing the responses of mPFC neurons during heroin self-administration showed that 40% (2/5) of the neurons tested exhibited an inhibitory effect immediately after heroin self-administration. These data suggest that in animals well-trained to self-administer heroin, only a small number (20%) of the NAcc neurons studied responded to heroin self-administration. Further research is necessary to determine whether these responses are a function of the opiate-seeking state of the animal and the mechanism(s) responsible for these effects of heroin.

Endogenous protein kinase A inhibitor (PKIalpha) modulates synaptic activity.

Protein kinase A (PKA) has long been known to be involved in major regulatory mechanisms underlying synaptic plasticity and complex behaviors such as learning and memory. The endogenous PKA inhibitor, PKIalpha, has been extensively studied for its effects on PKA and PKA-mediated signal transduction. Clear functions for PKIalpha in vivo, however, remain to be established. Here we describe that several forms of synaptic stimulation in the rat hippocampus cause a dramatic decrease in the concentration of PKIalpha in dentate granule cells. Furthermore, chronic infusion of antisense oligonucleotides against PKIalpha into the rat brain results in a dramatic reduction of the excitability of these neurons and elimination of their ability to exhibit long-term potentiation (LTP) and long-term depression (LTD), suggesting a stimulus-dependent regulatory role for PKIalpha in PKA signal transduction.

Acute ethanol induces c-fos immunoreactivity in GABAergic neurons of the central nucleus of the amygdala.

The central nucleus of the amygdala (CNA) is a component of the brain reward pathway which is believed to represent an anatomical substrate for drugs of abuse. Previous studies have shown that acute ethanol administration induces the expression of c-fos in the CNA of rat brains. We report here, that over 70% of these c-fos immunoreactive neurons are GABAergic. This observation provides the first anatomical evidence that GABAergic neurons of the CNA are responsive to acute ethanol exposure and suggest that the GABAergic system of the CNA is a key neuronal substrate for ethanol actions on the central nervous system.

Cortistatin is expressed in a distinct subset of cortical interneurons.

Cortistatin is a presumptive neuropeptide that shares 11 of its 14 amino acids with somatostatin. In contrast to somatostatin, administration of cortistatin into the rat brain ventricles specifically enhances slow wave sleep, apparently by antagonizing the effects of acetylcholine on cortical excitability. Here we show that preprocortistatin mRNA is expressed in a subset of GABAergic cells in the cortex and hippocampus that partially overlap with those containing somatostatin. A significant percentage of cortistatin-positive neurons is also positive for parvalbumin. In contrast, no colocalization was found between cortistatin and calretinin, cholecystokinin, or vasoactive intestinal peptide. During development there is a transient increase in cortistatin-expressing cells in the second postnatal week in all cortical areas and in the dentate gyrus. A transient expression of preprocortistatin mRNA in the hilar region at P16 is paralleled by electrophysiological changes in dentate granule cells. Together, these observations suggest mechanisms by which cortistatin may regulate cortical activity.

Ethanol inhibits single-unit responses in the nucleus accumbens evoked by stimulation of the basolateral nucleus of the amygdala.

We studied the actions of intoxicating doses of ethanol on excitatory inputs from the basolateral nucleus of the amygdala, a major afferent system projecting to the nucleus accumbens (NAcc). In view of the hypothesized role of opioid receptors on the effects of ethanol on NAcc physiology, we also explored whether naloxone modulates ethanol-induced suppression of NAcc excitability in halothane anesthetized and freely moving unanesthetized rats. Intraperitoneal administration of ethanol (1.2-1.4 g/kg) markedly suppressed a subgroup of amygdala-activated NAcc neurons. The ethanol-induced reduction in amygdala-activated NAcc neurons was not reversed by naloxone (5.0 mg/kg, intraperitoneally). Moreover, naloxone had no effect on the suppressive effects of ethanol on NAcc spontaneous activity in either halothane-anesthetized or unanesthetized freely moving preparations. These findings suggest that opiate mechanisms either are not participating or are not solely responsible for the inhibitory effects of acute intoxicating doses of ethanol on NAcc physiology.

Differential sensitivity of c-Fos expression in hippocampus and other brain regions to moderate and low doses of alcohol.

Alcohol consumption in humans is characterized by a wide range of behavioral effects and pathological consequences that suggest several neuroanatomical targets for this drug. To identify these targets we have mapped alcohol-induced changes in the expression of the c-Fos protein in the rat brain. Administration of a moderate dose of alcohol (1.5 g kg-1) led to a suppression of basal and novel environment-induced c-Fos expression in the hippocampus and simultaneous induction of this protein in regions important for the reinforcing as well as aversive properties of drugs. These include the extended amygdala (including the central nucleus of amygdala, bed nucleus of stria terminals and nucleus accumbens), regions processing sensory information (including the Edinger-Westphal nucleus and the paraventricular nucleus of the thalamus) and in stress-related areas (including the paraventricular nucleus of the hypothalamus, nucleus of the solitary tract and several neocortical areas). Repeated administration of the same dose of alcohol did not decrease alcohol-mediated suppression of c-Fos in the hippocampus, but decreased alcohol-induced expression of c-Fos in other areas. A lower dose of acute alcohol (0.5 g kg-1) reduced basal c-Fos expression in several areas of the neocortex, hippocampus and hypothalamus. However, while this low dose of alcohol was unable to counteract the environmental novelty-induced c-Fos expression in these areas, it increased c-Fos expression in the central nucleus of amygdala (an effect similar to the one observed previously for diazepam). Our data suggest that the effects of low doses of alcohol may be due to selective GABA-like effects of ethanol, whereas higher doses of ethanol involve effects on multiple neurotransmitter systems.

A cortical neuropeptide with neuronal depressant and sleep-modulating properties.

Acetylcholine (ACh) plays a key role in the transitions between the different phases of sleep: Slow-wave sleep requires low ACh concentrations in the brain, whereas rapid-eye-movement (REM) sleep is associated with high levels of ACh. Also, these phases of sleep are differentially sensitive to a number of endogenous neuropeptides and cytokines, including somatostatin, which has been shown to increase REM sleep without significantly affecting other phases. Here we report the cloning and initial characterization of cortistatin, a neuropeptide that exhibits strong structural similarity to somatostatin, although it is the product of a different gene. Administration of cortistatin depresses neuronal electrical activity but, unlike somatostatin, induces low-frequency waves in the cerebral cortex and antagonizes the effects of acetylcholine on hippocampal and cortical measures of excitability. This suggests a mechanism for cortical synchronization related to sleep.

Microelectrophoretic application of SCH-23390 into the lateral septal nucleus blocks ethanol-induced suppression of LTP, in vivo, in the adult rodent hippocampus.

Ethanol intoxication produces deficits in the acquisition of new information and blocks the induction of hippocampal long-term potentiation (LTP), a candidate neurophysiological correlate for learning and memory. We report that, in adult rats, local application of the dopamine (DA) D1 receptor antagonist SCH-23390 into the lateral septum (LS) blocks ethanol-induced suppression of LTP and alterations of paired-pulse responses in the dentate gyrus. This suggests a primary role for an extra-hippocampal circuit and neurotransmitter system mediating ethanol's ability to suppress LTP.

Sensitivity of nucleus accumbens neurons in vivo to intoxicating doses of ethanol.

The nucleus accumbens septi (NAcc) is considered an important component of the final common pathway involved in the reinforcing properties of ethanol. We studied the effects of intraperitoneal administration of ethanol on spontaneous, glutamate-activated, and fimbria-activated NAcc neurons in acute anesthetized and freely moving unanesthetized rats. Ethanol significantly reduced the firing rate of spontaneous and glutamate-activated NAcc neurons in both electrophysiological preparations. Stimulation of the ipsilateral fimbria evoked single-unit activity in NAcc neurons with two characteristic latencies (early, 7.21 +/- 0.74 msec; late, 18.24 +/- 0.66 msec). Intoxicating doses of ethanol inhibited the recruitment of late, but not of early, fimbria-activated NAcc neurons. These data demonstrate electrophysiological evidence for the existence of neurons in the core region of the NAcc that are sensitive and insensitive to acute systemic ethanol administration.