Polycomb contraction differentially regulates terminal human hematopoietic differentiation programs.

BACKGROUND: Lifelong production of the many types of mature blood cells from less differentiated progenitors is a hierarchically ordered process that spans multiple cell divisions. The nature and timing of the molecular events required to integrate the environmental signals, transcription factor activity, epigenetic modifications, and changes in gene expression involved are thus complex and still poorly understood. To address this gap, we generated comprehensive reference epigenomes of 8 phenotypically defined subsets of normal human cord blood. RESULTS: We describe a striking contraction of H3K27me3 density in differentiated myelo-erythroid cells that resembles a punctate pattern previously ascribed to pluripotent embryonic stem cells. Phenotypically distinct progenitor cell types display a nearly identical repressive H3K27me3 signature characterized by large organized chromatin K27-modification domains that are retained by mature lymphoid cells but lost in terminally differentiated monocytes and erythroblasts. We demonstrate that inhibition of polycomb group members predicted to control large organized chromatin K27-modification domains influences lymphoid and myeloid fate decisions of primary neonatal hematopoietic progenitors in vitro. We further show that a majority of active enhancers appear in early progenitors, a subset of which are DNA hypermethylated and become hypomethylated and induced during terminal differentiation. CONCLUSION: Primitive human hematopoietic cells display a unique repressive H3K27me3 signature that is retained by mature lymphoid cells but is lost in monocytes and erythroblasts. Intervention data implicate that control of this chromatin state change is a requisite part of the process whereby normal human hematopoietic progenitor cells make lymphoid and myeloid fate decisions.

Distinct patterns of DeltaFosB induction in brain by drugs of abuse.

The transcription factor DeltaFosB accumulates and persists in brain in response to chronic stimulation. This accumulation after chronic exposure to drugs of abuse has been demonstrated previously by Western blot most dramatically in striatal regions, including dorsal striatum (caudate/putamen) and nucleus accumbens. In the present study, we used immunohistochemistry to define with greater anatomical precision the induction of DeltaFosB throughout the rodent brain after chronic drug treatment. We also extended previous research involving cocaine, morphine, and nicotine to two additional drugs of abuse, ethanol and Delta(9)-tetrahydrocannabinol (Delta(9)-THC, the active ingredient in marijuana). We show here that chronic, but not acute, administration of each of four drugs of abuse, cocaine, morphine, ethanol, and Delta(9)-THC, robustly induces DeltaFosB in nucleus accumbens, although different patterns in the core vs. shell subregions of this nucleus were apparent for the different drugs. The drugs also differed in their degree of DeltaFosB induction in dorsal striatum. In addition, all four drugs induced DeltaFosB in prefrontal cortex, with the greatest effects observed with cocaine and ethanol, and all of the drugs induced DeltaFosB to a small extent in amygdala. Furthermore, all drugs induced DeltaFosB in the hippocampus, and, with the exception of ethanol, most of this induction was seen in the dentate. Lower levels of DeltaFosB induction were seen in other brain areas in response to a particular drug treatment. These findings provide further evidence that induction of DeltaFosB in nucleus accumbens is a common action of virtually all drugs of abuse and that, beyond nucleus accumbens, each drug induces DeltaFosB in a region-specific manner in brain.

Involvement of protein kinase A in ethanol-induced locomotor activity and sensitization.

RATIONALE: Mutant mice lacking the RIIbeta subunit of protein kinase A (regulatory subunit II beta(-/-)) show increased ethanol preference. Recent evidence suggests a relationship between heightened ethanol preference and susceptibility to ethanol-induced locomotor sensitization. It is currently unknown if protein kinase A signaling modulates the stimulant effects and/or behavioral sensitization caused by ethanol administration. To address this question, we examined the effects of repeated ethanol administration on locomotor activity RIIbeta(-/-) and littermate wild-type (RIIbeta(+/+)) mice on multiple genetic backgrounds. METHODS: Over three consecutive days, mice were given single i.p. saline injections and immediately placed in a locomotor activity apparatus to establish a composite baseline for locomotor activity. Next, mice maintained on a hybrid 129/SvEvxC57BL/6J or pure C57BL/6J genetic background were given 10 i.p. ethanol injections before being placed in the activity apparatus. Each ethanol injection was separated by 3-4 days. To determine if changes in behavior were specific to ethanol injection, naïve mice were tested following repeated daily saline injections. The effects of ethanol injection on locomotor behavior were also assessed using an alternate paradigm in which mice were given repeated ethanol injections in their home cage environment. RESULTS: Relative to RIIbeta(+/+) mice, RIIbeta(-/-) mice, regardless of genetic background, consistently showed significantly greater ethanol-induced locomotor activation. RIIbeta(-/-) mice also showed increased sensitivity to ethanol-induced locomotor sensitization resulting from repeated administration, an effect that was dependent on genetic background and testing paradigm. Increased locomotor activity by RIIbeta(-/-) mice was specific to ethanol injections, and was not related to altered blood ethanol levels. CONCLUSIONS: These data provide novel evidence implicating an influence of protein kinase A signaling on ethanol-induced locomotor activity and behavioral sensitization. The observation that RIIbeta(-/-) mice are more sensitive to the effects of repeated ethanol administration suggests that normal protein kinase A signaling limits, or is protective against, the stimulant effects of ethanol and the plastic alterations that underlie behavioral sensitization.

Regional specificity of ethanol and NMDA action in brain revealed with FOS-like immunohistochemistry and differential routes of drug administration.

BACKGROUND: Inhibition of NMDA receptor function in brain is believed to be an important action of ethanol (EtOH). To investigate EtOH inhibition of NMDA receptor responses in vivo, the interaction of these agents in brain after different routes of administration were investigated by using transcription factor Fos protein expression to follow NMDA receptor activation and EtOH inhibition of this response. METHODS: The induction of Fos-like immunoreactivity (Fos-LI) in 38 regions of the rat brain was measured 2 hr after treatment with NMDA, EtOH, or both. To determine the relative contribution of abdominal drug effects on Fos induction, rats received either intraperitoneal (ip) or intragastric (ig) EtOH and ip or intravenous (iv) NMDA. Rats received EtOH (2.5 g/kg ip or 4 g/kg ig) or vehicle 15 min before NMDA (125 mg/kg ip or 60 mg/kg iv) or vehicle. RESULTS: For the 38 forebrain regions examined, ip and iv NMDA significantly induced Fos-LI in 13 and 32 regions, respectively. These effects occurred without elicitation of tonic-clonic seizure activity and were strong after iv NMDA in the frontal, prefrontal, and cingulate cortices, supraoptic nucleus, anterior lateral septum, and dentate gyrus. For EtOH, prominent Fos-LI induction was found in the central amygdala, dorsolateral bed nucleus of the stria terminalis, Edinger-Westphal nucleus, and paraventricular hypothalamus. Despite ip and ig EtOH induction of Fos-LI in these regions, the major effect of EtOH was to block NMDA-induced Fos-LI in 8 of 13 (ip) and 27 of 32 (ig) of the NMDA-positive regions, respectively, including retrosplenial, cingulate, and medial prefrontal cortices, central amygdala, and taenia tecta. CONCLUSIONS: These results provide new evidence for the regionally specific functional interactions of EtOH on NMDA receptors in vivo. Moreover, these results support efforts to identify brain region-specific targets for EtOH and EtOH-induced changes in gene expression.

Differential modulation of GABA- and NMDA-gated currents by ethanol and isoflurane in cultured rat cerebral cortical neurons.

Ethanol and the volatile anesthetics share many features including effects on both GABA and NMDA receptors. To determine the degree of similarity between these compounds, we examined the concentration-response curves for ethanol and isoflurane on currents gated by GABA or NMDA. The effects of isoflurane and ethanol on the righting reflex of rats were also observed. The concentration of ethanol causing loss of the righting reflex of rats was 82.3+/-2.9 mM, whereas median concentration of isoflurane exerting that effect was 0.125 mM. Both isoflurane and ethanol inhibited NMDA-gated currents in cultured cerebral cortical neurons at concentrations well below those associated with loss of the righting reflex or anesthesia. However, the effect of isoflurane was greater than that of ethanol and the slope of the concentration-response curve for isoflurane less steep than that for ethanol. Isoflurane enhanced GABA-gated currents at anesthetic concentrations but there was a sharp concentration-response curve with only minimal effects of isoflurane on GABA-gated currents at concentrations associated with loss of the righting reflex. In contrast, ethanol had no effect on GABA-gated currents even at lethal concentrations, i.e. 300 mM or 1.2%. Comparison of the concentration-response curves for the effects of isoflurane on NMDA- and GABA-gated currents has revealed both EC50 and Hill slope for the potentiation of GABA-gated currents were significantly greater than those for inhibition of NMDA-gated currents. These results support the hypothesis that isoflurane has actions on both the GABA and NMDA systems that are not shared by ethanol.

Effect of olanzapine on functional responses from sensitized D1-dopamine receptors in rats with neonatal dopamine loss.

Previous work has suggested that the therapeutic efficacy of olanzapine might be partially dependent on action at the D(1)-dopamine (DA) receptor site. Because early DA loss can lead to supersensitive D(1)-DA receptors, effects of olanzapine were investigated in adult rats given lesions to DA-containing neurons as neonates. In these animals, locomotor effects of SKF-38393 (a D(1)-DA agonist) were attenuated by olanzapine, but at doses (5 and 10 mg/kg) that decreased activity when given alone. Olanzapine prevented induction of striatal Fos protein by SKF-38393 and partially attenuated the long-term "priming" effect of repeated SKF-38393 treatment. Olanzapine also antagonized the stimulant effects of quinpirole (a D(2)-type DA agonist) in animals lesioned as young adults, at doses lower than those necessary to antagonize SKF-38393-induced activity. In addition, olanzapine antagonized apomorphine-induced self-injurious behavior in neonate-lesioned rats in a dose-related fashion. Attenuation of self-injury in this animal model suggests that olanzapine should be tested against this symptom in patient populations.

Interaction of nutrition and binge ethanol treatment on brain damage and withdrawal.

To determine if nutrition plays a role in ethanol withdrawal and alcohol-induced brain damage, the effects of a 4-day ethanol binge treatment using ethanol in a nutritionally complete liquid diet compared to ethanol mixed with water were studied. The nutritionally complete diet group (ETOH-diet) received a complete diet of sugars, proteins and fats with vitamins and minerals with approximately 53% of calories from ethanol while the nutritionally deprived group (ETOH-H2O) received 100% of calories from ethanol. No difference in withdrawal behavior was found between the ETOH-diet and ETOH-H2O groups during the 72-hour period studied. In addition, no difference was seen for serum levels of magnesium and zinc taken at last dose or following 72 h of withdrawal. Serum alanine aminotransferase (ALT) and ammonia were increased in both groups with ETOH-diet showing a greater increase in ALT than ETOH-H2O. Both groups showed damage in the olfactory bulb, perirhinal, agranular insular, piriform and lateral entorhinal cortical areas as well as hippocampal dentate gyrus and CA-3. Interestingly, the ETOH-diet group displayed more damage at last dose in the posterior dentate and CA-3 of hippocampus than did the ETOH-H2O group. This study suggests that nutritional components and total caloric intake do not effect behavior during ethanol withdrawal and that a nutritionally complete diet may increase ethanol-induced brain damage.

Binge ethanol consumption causes differential brain damage in young adolescent rats compared with adult rats.

BACKGROUND: Adolescents respond differently to alcohol than adults. Furthermore, binge drinking in young adolescents is becoming increasingly common. METHODS: To determine if the effects of binge drinking on brain damage are different in juveniles compared with adults, the effects of a 4 day binge ethanol treatment (e.g., 4 days of 4 times per day 15% ethanol intragastrically, approximately 9-10 g/kg/day ethanol) were investigated in adolescent-juvenile rats (JVN) 35 days old and compared with adult (ADT) rats 80 to 90 days old. Brain damage was measured by using the amino cupric silver stain of de Olmos et al. (1994). RESULTS: Significant brain damage was found in both groups. The olfactory bulbs were equally damaged in both groups; however, the associated frontal cortical olfactory regions were damaged only in JVN. The anterior portions of the piriform and perirhinal cortices also were damaged only in JVN rats. Quantitation of silver-stained frontal areas in binge ethanol-treated JVN rats ranged from 400% to 1,260% of control values. For example, in anterior perirhinal cortex, silver stain increased from 48 +/- 14 to 444 +/- 114 (mm2 x 10(3) argyrophilic area; p < 0.01) in JVN control and binge ethanol-treated animals, respectively. In contrast, posterior perirhinal cortex showed greater damage in adults, being 236 +/- 76 vs. 875 +/- 135 (mm2 x 10(3) argyrophilic area; p < 0.005) in JVN and ADT, respectively. CONCLUSIONS: The young-adolescent brain shows differential sensitivity to alcohol-induced brain damage compared with adults.

Enhanced ultrasonic vocalization and Fos protein expression following ethanol withdrawal: effects of flumazenil.

RATIONALE: Administration of flumazenil, a benzodiazepine (BZD) antagonist, has therapeutic efficacy against some anxiogenic effects of ethanol withdrawal. This observation has led to the suggestion that anxiety associated with ethanol withdrawal is related to release in brain of an endogenous BZD inverse agonist. OBJECTIVE: The present studies further tested this hypothesis by assessing the effect of flumazenil on withdrawal-induced changes in a behavioral task and on the expression of the neuronal protein, Fos. METHODS: Male Sprague-Dawley rats were withdrawn from a chronic ethanol regimen and tested, with or without flumazenil pretreatment, for either ultrasonic vocalization in response to air puff or for the induction of Fos protein-like immunoreactivity (Fos-LI) in brain. In addition, flumazenil effects on Fos-LI were measured in a group of animals treated with the BZD inverse agonist DMCM (0.75 and 1.0 mg/kg). RESULTS: Flumazenil (5.0 mg/kg) significantly reduced the number of ultrasonic vocalizations observed following withdrawal from chronic ethanol. In contrast, flumazenil (5.0 mg/kg), given either 14 h before withdrawal from chronic ethanol, or during hours 3 and 5 following withdrawal, did not attenuate the effects of withdrawal on Fos-LI. Subsequent testing with DMCM confirmed that a benzodiazepine inverse agonist can induce Fos-LI in most of the same brain regions as observed following ethanol withdrawal, and that this change in Fos protein can be attenuated by pretreatment with flumazenil (5.0 mg/kg). CONCLUSIONS: Overall, these results demonstrate that specific behavioral indices of anxiety, but not measures of Fos-LI, support the contribution of an endogenous BZD inverse agonist in the ethanol withdrawal syndrome.

Selective breeding of 5-HT(1A) receptor-mediated responses: application to emotion and receptor action.

Rat lines that were selectively bred for high (high DPAT-sensitive, HDS) or low (low DPAT-sensitive, LDS) hypothermic responses to the specific 5-HT(1A) receptor agonist, 8-hydroxy-di-n-propylaminotetralin (8-OH-DPAT), differ in receptor binding and certain behaviors related to anxiety and depression. After reviewing this literature, the present communication summarizes new experiments designed to clarify and extend the nature of the pharmacological and biochemical differences between the lines. A challenge with the 5-HT(2) receptor agonist, DOI, produced similar degrees of head shakes and skin crawls in the HDS and LDS rats, suggesting similar sensitivity of 5-HT(2A) and 5-HT(2C) receptors. In contrast, DOI-induced flat body posture (FBP), which has been linked to 5-HT(1A) receptor stimulation, was observed more readily in the HDS rats. The HDS and LDS rats exhibited similar degrees of increase in 8-OH-DPAT-stimulated [35S]GTPgammaS binding in several brain regions. This result suggests that the dramatic differences in hypothermia in HDS and LDS rats cannot be related to 5-HT(1A) receptor-mediated action on G proteins. Overall, these findings indicate that the selective breeding for 5-HT(1A)-mediated hypothermia has been fairly selective, and that differences in emotionally relevant behaviors between these two rat lines can strongly be associated with an unidentified component of the 5-HT(1A) receptor signaling pathway.

Induction of cyclooxygenase-2 in brain during acute and chronic ethanol treatment and ethanol withdrawal.

Prostaglandins play an important role in the regulation of nervous system function including thermoregulation, autonomic nervous system function, hypothalamic regulation of pituitary function, and neuronal excitation. Prostaglandin synthesis is catalyzed by cyclooxygenase (COX; prostaglandin synthase) which occurs as two isozymes, COX-1 and COX-2. COX-1 and COX-2 are constitutively expressed in brain whereas COX-2 type is also inducible in brain by excitatory neurotransmission. Ethanol intoxication and the hyperexcitability of ethanol withdrawal may be influenced by inducible proteins, thus we investigated COX-2 in the rat brain during acute and chronic ethanol treatment, ethanol withdrawal, and after peripheral administration of excitatory amino acids. Kainic acid or NMDA treatment increased COX-2 immunoreactivity in the cortex, hippocampus, and amygdala. An acute dose of ethanol (5 g/kg, intragastric-i.g.) increased COX-2, particularly in the CA4 region of the hippocampus and agranular insular cortex. Chronic ethanol treatment (4 days-intragastric) robustly induced COX-2 in limbic cortex, isocortex, and amygdala. Particularly dense immunocytochemical staining was found in perirhinal and piriform cortices, dentate gyrus, and tenia tecta. During ethanol withdrawal, COX-2 expression increased further in some regions, peaking in most areas 16 hr after the last dose of ethanol. These results indicate that COX-2 immunoreactivity is: 1) increased in the brain during acute ethanol exposure that increases further during chronic treatment; 2) sensitive to excitatory amino acid receptor stimulation; and 3) dramatically increased during ethanol withdrawal. These studies suggest that COX-2 induction may be involved in the acute and chronic effects of ethanol.

Fas ligand expression in the bone marrow in myelodysplastic syndromes correlates with FAB subtype and anemia, and predicts survival.

Increased apoptosis in the bone marrow (BM) may contribute to the cytopenias that occur in myelodysplastic syndromes (MDS). The Fas receptor, Fas ligand (FasL) pathway is a major mechanism of apoptosis. Since hematopoietic progenitors can express the Fas receptor, they may be susceptible to apoptosis induced by FasL-expressing cells. We examined FasL expression in the BM of patients with MDS (n = 50), de novo acute myeloid leukemia (AML; n = 10), AML following prior MDS (n = 6), and normal controls (n = 6). Compared to controls, FasL expression was increased in MDS, and was highest in AML. In MDS, FasL expression was seen in myeloid blasts, erythroblasts, maturing myeloid cells, megakaryocytes and dysplastic cells, whereas in AML, intense expression was seen in the blasts. FasL expression correlated with the FAB subtype groups of MDS, and also correlated directly with the percentage of abnormal metaphases on cytogenetic analysis. The FasL expressed in MDS BM inhibited the growth of clonogenic hematopoietic progenitors. This inhibition could be blocked by a soluble recombinant FasFc protein. In MDS, FasL expression in the initial diagnostic BM was higher in patients who were more anemic, correlated directly with red cell transfusion requirements over the subsequent course of the disease, and was predictive of survival. These studies indicate that FasL expression in MDS is of prognostic significance, and suggest that pharmacological blockade of the Fas-FasL pathway may be of clinical benefit.

Action of ethanol and zolpidem on gamma-aminobutyric acid responses from cerebellar Purkinje neurons: relationship to beta-adrenergic receptor input.

The observation that cerebellar Purkinje cells contain type-I benzodiazepine-sensitive GABA(A) receptors is consistent with findings in the present work that the majority of Purkinje neurons are sensitive to enhancement of GABA by the type-1 benzodiazepine agonist, zolpidem. Previous work has demonstrated a relation between zolpidem and ethanol enhancement of GABA responses in several brain regions, but had not tested Purkinje neurons. Therefore, given that a majority of Purkinje neurons were found to be sensitive to zolpidem, ethanol would have been expected to enhance GABA responses from this cell type. However, in agreement with earlier electrophysiological studies, ethanol enhanced GABA inhibitory responses from only a small proportion of these cerebellar Purkinje neurons. Rather than enhancement of GABA, local application of ethanol either inhibited or did not affect responses to GABA from a majority of cerebellar-Purkinje neurons. Nonetheless, as previously reported, a portion of the Purkinje neurons initially insensitive to ethanol enhancement of GABA became sensitive to this action of ethanol with co-application of the beta-adrenergic agonist, isoproterenol. Thus, these results collectively implicate a beta-adrenergic input dependency for ethanol enhancement of GABA from some, but not all, cerebellar Purkinje neurons sensitive to zolpidem. Because a beta-adrenergic input did not allow ethanol enhancement of GABA from all Purkinje neurons, future studies should explore the possibility that other auxiliary neural inputs to zolpidem-sensitive cerebellar Purkinje neurons may be required for ethanol enhancement of GABA responsiveness when a beta-adrenergic input does not have this action. Likewise, knowing that the action of zolpidem can predict ethanol enhancement of GABA in other brain regions, the present findings suggest that a future determination be made concerning whether zolpidem-sensitive neurons in these other regions of brain require a beta-adrenergic or an alternative neural input for ethanol enhancement of GABA responses.

Sensitivity to ethanol across development in rats: comparison to [3H]zolpidem binding.

Previous research has suggested that rats tested at 28 to 30 days of age show a marked subsensitivity to the sedative effects of ethanol. In the present study, rats of different ages were tested for aerial righting following acute ethanol (3 g/kg) treatment. These results were compared with the effects of the atypical benzodiazepine zolpidem (3 and 5 mg/kg) and pentobarbital (10 and 15 mg/kg). Animals tested at 25, 28, or 35 days of age were significantly less impaired by ethanol than preweanling rats (age 20 days) or older rats (age 65 to 75 days), whereas animals tested at 25 or 28 days of age were less impaired by the higher dose of zolpidem. With pentobarbital, the most distinct age-related trend was greater impairment in 20-day-old rats. Because ethanol may be active at the same type I GABA(A) receptor site selectively labeled by [3H]zolpidem, levels of [3H]zolpidem binding were determined for rats of different ages. Although some brain regions showed progressive increases in binding of [3H]zolpidem across development, other regions demonstrated increased binding from day 12 or 17 to day 20, then a plateau of binding levels across days 20, 25, and 28, with further increases occurring by day 36 or day 60. This pattern was observed in the cingulate cortex, medial septal nucleus, globus pallidus, inferior colliculus, red nucleus, and cerebellum. Overall, the results indicate that the period of subsensitivity to the sedative effects of ethanol is coincident with a change in the developmental pattern of GABA(A) receptor sites targeted by [3H]zolpidem.

Species differences in regional patterns of 3H-8-OH-DPAT and 3H-zolpidem binding in the rat and human brain.

The rat has proven to be a valuable preclinical model for characterizing effects of psychotrophic drugs and for identifying new psychotherapeutic agents in pharmacological screens. However, substantial differences have been described between the rat and human brain in regard to the neuroanatomical distribution of some drug and neurotransmitter receptor binding sites. To assess the utility of the rat as a model for the neuroanatomical topography of 5-HT1A and type 1 benzodiazepine (BDZ) receptors in humans, the distribution of binding sites for 3H-8-OH-DPAT (5-HT1A agonist) and 3H-zolpidem (type 1 BDZ agonist) was compared with autoradiography in select regions of the rat and human brain. Concordance in the binding patterns for the two ligands was observed in several brain regions for the two species. However, substantial differences were also found in the topography of binding sites for the ligands in the rat and human brain. High 3H-8-OH-DPAT binding was seen in the dorsal raphe nucleus and hippocampal formation in both the rat and human brain. However, species differences were observed in the relative distribution of ligand binding among hippocampal subregions. In the cerebral cortex, the laminar distribution of 3H-8-OH-DPAT binding sites was notably different for rats and humans. In humans, outer cortical layers were most densely labeled with 3H-8-OH-DPAT, whereas in the rat cortex, the highest binding was in the inner layers. A striking difference between rats and humans was observed for 3H-8-OH-DPAT binding in the lateral septal nucleus, which was densely labeled in the rat but weakly labeled in humans. Substantial differences between rats and humans were also observed for 3H-zolpidem binding. In the rat brain, high densities of binding sites were found in the medial septum, inferior colliculus, and substantia nigra reticulata. These regions showed very low 3H-zolpidem binding in the human brain. Intermediate binding was seen in the rat cerebral cortex, and low binding was found in the hippocampus. By contrast, in humans, cerebral cortical regions were the most densely labeled of all regions studied, and certain hippocampal subregions exhibited relatively high binding. The striking neuroanatomical differences in 3H-8-OH-DPAT and 3H-zolpidem binding observed between rats and humans suggest that different functional consequences may be produced within specific brain regions after administration of drugs that influence 5-HT1A and type 1 BZD receptors.

Differential effects of chronic antidepressant treatment on swim stress- and fluoxetine-induced secretion of corticosterone and progesterone.

Hypersecretion of cortisol occurs in numerous patients with major depression and normalizes with clinical recovery during the course of chronic antidepressant treatment. These clinical data suggest that investigation of the effects of antidepressant treatments on the regulation of the brain-pituitary-adrenal axis may assist in elucidating the therapeutic basis of antidepressant actions. In the present investigation, both swim stress and acute fluoxetine challenge increased release of corticosterone and progesterone to reflect an activation of the brain pituitary-adrenal axis. The effects of chronic antidepressant treatment (21 days) on corticosterone and progesterone secretion induced by these challenges were investigated. Chronic fluoxetine treatment (5 mg/kg/day) completely blocked the increased secretion of corticosterone and progesterone in response to the acute fluoxetine challenge. Chronic treatment with desipramine, imipramine or amytriptyline (15 mg/kg/day) also markedly attenuated fluoxetine-induced corticosterone and progesterone secretion. However, chronic treatment with the monoamine oxidase inhibitors, phenelzine (5 mg/kg) and tranylcypromine (5 mg/kg), did not affect this hormonal response to acute fluoxetine challenge. Plasma levels of fluoxetine after acute challenge were not significantly different for the various chronic antidepressant treatment conditions from the chronic saline controls; therefore, an increase in the metabolism of fluoxetine can not explain the antagonism of the fluoxetine-induced hormonal response after chronic antidepressant treatment. In contrast to the effects of selected antidepressants on acute fluoxetine-induced steroid release, chronic treatment with imipramine (20 mg/kg/day), fluoxetine (5 mg/kg/day) or phenelzine (5 mg/kg) did not significantly alter this swim stress-induced corticosterone or progesterone secretion. Because chronic fluoxetine and tricyclic antidepressant drugs blocked the acute action of fluoxetine to increase adrenal cortical secretion, but did not alter swim stress-induced secretion of these steroids, we propose that distinct neurochemical mechanisms control fluoxetine and swim stress-induced steroid release. We speculate that the substantial adaptive response to those chronic antidepressant treatments, which minimize the effect of acute fluoxetine challenge to increase in corticosterone and progesterone secretion, may be relevant to the therapeutic actions of these drugs.

Induction of Fos-like proteins and ultrasonic vocalizations during ethanol withdrawal: further evidence for withdrawal-induced anxiety.

The ethanol withdrawal syndrome includes anxiety as a prominent symptom. Because the extent that specific regions of brain are critical to the generation of this emotional state is unknown, Fos-like immunoreactivity (Fos-LI) was used to associate specific regions of the rat brain with the anxiety component of the ethanol withdrawal syndrome exacerbated by an air puff challenge in rats. Chronic ethanol liquid diet was administered intragastrically for 4 days or by having the rats consume the diet for 14 days. During withdrawal from either treatment protocol, Fos-LI was induced most prominently in forebrain areas, although the midbrain and hindbrain were also represented. Included in these Fos-LI positive regions were many cortical regions, septum, accumbens, claustrum, amygdala, paraventricular nucleus of the thalamus and hypothalamus, hippocampus, locus coeruleus, and central gray. Fos-LI expression differed mostly in intensity between the two treatment and withdrawal protocols, with the gastric protocol producing the greatest Fos-LI induction in most brain regions. The threshold for air puff-induced ultrasonic vocalizations was decreased, and the number of vocalizations was increased and the period of vocalization was extended. These behavioral data indicate that aversively motivated responding in rats during ethanol withdrawal can be readily quantified with the ultrasonic vocalizations test without precipitating convulsive activity. Furthermore, a comparison of the effects of the air puff challenge versus withdrawal on Fos-LI indicated that the behavioral state induced in these two situations share functional neuroanatomical features. Some regions--such as the accumbens core, medial septum, subregions of the amygdala, hippocampus, substantia nigra, and cerebellum--exhibited little Fos-LI during withdrawal and also did not exhibit strong increases after the addition of the air puff challenge. However, other regions-such as the cerebral cortex (medial prefrontal, frontal, cingulate and ventrolateral orbital, claustrum, and tenia tecta), hypothalamus, and locus ceoruleus- exhibited Fos-LI at levels higher than that seen after either the ethanol withdrawal or puff challenge alone. These overlapping patterns of Fos-LI in specific regions of the brain, activated by both ethanol withdrawal and an anxiety provoking behavioral challenge, suggest that specific neuroanatomical sites in brain are associated with the symptom of anxiety observed during the "ethanol withdrawal syndrome."

Metabolic mapping of the rat brain after subanesthetic doses of ketamine: potential relevance to schizophrenia.

Subanesthetic doses of ketamine have been shown to exacerbate symptoms in schizophrenia and to induce positive, negative, and cognitive schizophrenic-like symptoms in normal subjects. The present investigation sought to define brain regions affected by subanesthetic doses of ketamine, using high resolution autoradiographic analysis of 14C-2-deoxyglucose (2-DG) uptake and immunocytochemical staining for Fos-like immunoreactivity (Fos-LI). Both functional mapping approaches were used because distinct and complementary information is often obtained with these two mapping methods. Ketamine, at a subanesthetic dose of 35 mg/kg, substantially increased 2-DG uptake in certain limbic cortical regions, including medial prefrontal, ventrolateral orbital, cingulate, and retrosplenial cortices. In the hippocampal formation, the subanesthetic dose of ketamine induced prominent increases in 2-DG uptake in the dentate gyrus, CA-3 stratum radiatum, stratum lacunosum moleculare, and presubiculum. Increased 2-DG uptake in response to 35 mg/kg ketamine was also observed in select thalamic nuclei and basolateral amygdala. Ketamine induced Fos-LI in the same limbic cortical regions that exhibited increased 2-DG uptake in response to the subanesthetic dose of the drug. However, no Fos was induced in some brain regions that showed increased 2-DG uptake, such as the hippocampal formation, anterioventral thalamic nucleus, and basolateral amygdala. Conversely, ketamine induced Fos in the paraventricular nucleus of the hypothalamus and central amygdala, although no effect of the drug on 2-DG uptake was apparent in these regions. In contrast to the increase in 2-DG uptake observed in select brain regions after the subanesthetic dose, an anesthetic dose of ketamine (100 mg/kg) produced a global suppression of 2-DG uptake. By contrast, a robust induction of Fos-LI was observed after the anesthetic dose of ketamine that was neuroanatomically identical to that produced by the subanesthetic dose. Results of the present investigation show that anesthetic and subanesthetic doses of ketamine have pronounced effects on regional brain 2-DG uptake and induction of Fos-LI. The alterations in regional brain metabolism induced by the subanesthetic dose may be relevant to effects of ketamine to induce schizophrenic-like symptoms.

Brain 5-HT1A receptor autoradiography and hypothermic responses in rats bred for differences in 8-OH-DPAT sensitivity.

Three rat lines were selectively bred for high (HDS), random (RDS), or low (LDS) hypothermic responses to the specific 5-HT1A receptor agonist 8-OH-DPAT. Forty-five minutes after 8-OH-DPAT administration (0.5 mg/kg), body temperatures dropped 3-5 degrees C in HDS rats, yet this dose produced only about 1.2 degrees C and 0.7 degree C drops in RDS and LDS rats, respectively. To investigate the relationship of body temperature of 5-HT1A receptor binding sites, autoradiographic analyses of [3H]8-OH-DPAT binding to 5-HT1A receptors in brains of these rats were conducted. Significant differences in binding were found in specific limbic cortical projection sites, with the HDS line having the greatest density of sites. Body temperature responses correlated significantly with [3H]8-OH-DPAT receptor binding in only a few areas of frontal cortex. Binding in many other brain regions, including the anterior and posterior hypothalami (regions long associated with body temperature regulation) and the raphe showed no significant differences among the lines. [3H]Ketanserin binding to cortical 5-HT2 receptors did not differ among the lines, except in the cingulate and superficial frontal cortices where HDS exhibited higher binding. These data suggest that differences in 5-HT1A receptor number may contribute to the exaggerated hypothermic response to 8-OH-DPAT in HDS rats. These studies also suggest that genetic regulation of receptor density may be brain region specific which should encourage future studies of the mechanisms of 5-HT1A receptor activity in brain and the action of drugs affecting this receptor.

Ultrasonic vocalization behavior differs between lines of ethanol-preferring and nonpreferring rats.

To further understand the relationship between emotional state and alcohol intake in rats, the tendency to emit ultrasonic vocalizations in response to an aversive, but nonpainful, air puff stimulus was tested in several rat lines. Included in this group were Maudsley Reactive (MR) and Non-Reactive (MNR) rats, and several lines of rats with either high ethanol preference or a low ethanol preference: Preferring, (P), Alko-Alcohol (AA), and Fawn-Hooded (FH) animals; and Non-Preferring (NP), Alko-Non-Alcohol (ANA), and Flinders Resistant Line (FRL). MR rats emitted fewer ultrasonic vocalizations (USVs) and showed less preference for ethanol than did MNR animals. An overall analysis that included the P, NP, FH, FRL, AA, and ANA groups demonstrated a significant negative correlation between the total number of USVs emitted and ethanol consumption. NP, FRL, and especially ANA rats (low ethanol-preferring) emitted the most USVs--to an extent similar to that typically found for normal rats. The duration of vocalizing was higher only in the NP and the FRL rats the relative to their P and FH comparison groups, respectively. In the ethanol-preferring and nonpreferring lines, the numbers of USVs emitted correlated positively with the duration of vocalizing, but not with the latency to vocalize, which in turn did not correlate strongly with ethanol intake. The latency to vocalize did not correlate significantly with ethanol intake across all drinking lines or MR or MNR rats, but was found to be higher in FH and AA rats relative to their nondrinking comparison groups. These associations suggest that the relationship between emotional state and ethanol drinking is complex and cannot be attributed to a simple elevated state of anxiety or emotionality. Further examination of the central nervous system mechanisms mediating the difference in USVs between paired lines of ethanol-preferring and nonpreferring rats may identify neurochemical factors that predict ethanol preference.