The enduring impact of neurulation stage alcohol exposure: A combined behavioral and structural neuroimaging study in adult male and female C57BL/6J mice.

Prenatal alcohol exposure (PAE) can cause behavioral and brain alterations over the lifespan. In animal models, these effects can occur following PAE confined to critical developmental periods, equivalent to the third and fourth weeks of human gestation, before pregnancy is usually recognized. The current study focuses on PAE during early neurulation and examines the behavioral and brain structural consequences that appear in adulthood. On gestational day 8 C57BL/6J dams received two alcohol (2.8g/kg, i.p), or vehicle, administrations, four hours apart. Male and female offspring were reared to adulthood and examined for performance on the elevated plus maze, rotarod, open field, Morris water maze, acoustic startle, social preference (i.e. three-chambered social approach test), and the hot plate. A subset of these mice was later evaluated using magnetic resonance imaging to detect changes in regional brain volumes and shapes. In males, PAE increased exploratory behaviors on the elevated plus maze and in the open field; these changes were associated with increased fractional anisotropy in the anterior commissure. In females, PAE reduced social preference and the startle response, and decreased cerebral cortex and brain stem volumes. Vehicle-treated females had larger pituitaries than did vehicle-treated males, but PAE attenuated this sex difference. In males, pituitary size correlated with open field activity, while in females, pituitary size correlated with social activity. These findings indicate that early neurulation PAE causes sex specific behavioral and brain changes in adulthood. Changes in the pituitary suggest that this structure is especially vulnerable to neurulation stage PAE.

Acute alcohol exposure during neurulation: Behavioral and brain structural consequences in adolescent C57BL/6J mice.

Prenatal alcohol exposure (PAE) can induce physical malformations and behavioral abnormalities that depend in part on thedevelopmental timing of alcohol exposure. The current studies employed a mouse FASD model to characterize the long-term behavioral and brain structural consequences of a binge-like alcohol exposure during neurulation; a first-trimester stage when women are typically unaware that they are pregnant. Time-mated C57BL/6J female mice were administered two alcohol doses (2.8g/kg, four hours apart) or vehicle starting at gestational day 8.0. Male and female adolescent offspring (postnatal day 28-45) were then examined for motor activity (open field and elevated plus maze), coordination (rotarod), spatial learning and memory (Morris water maze), sensory motor gating (acoustic startle and prepulse inhibition), sociability (three-chambered social test), and nociceptive responses (hot plate). Regional brain volumes and shapes were determined using magnetic resonance imaging. In males, PAE increased activity on the elevated plus maze and reduced social novelty preference, while in females PAE increased exploratory behavior in the open field and transiently impaired rotarod performance. In both males and females, PAE modestly impaired Morris water maze performance and decreased the latency to respond on the hot plate. There were no brain volume differences; however, significant shape differences were found in the cerebellum, hypothalamus, striatum, and corpus callosum. These results demonstrate that alcohol exposure during neurulation can have functional consequences into adolescence, even in the absence of significant brain regional volumetric changes. However, PAE-induced regional shape changes provide evidence for persistent brain alterations and suggest alternative clinical diagnostic markers.

Proarrhythmic safety of repeat doses of mirabegron in healthy subjects: a randomized, double-blind, placebo-, and active-controlled thorough QT study.

Potential effects of the selective ß(3)-adrenoceptor agonist mirabegron on cardiac repolarization were studied in healthy subjects. The four-arm, parallel, two-way crossover study was double-blind and placebo- and active (moxifloxacin)-controlled. After 2 baseline ECG days, subjects were randomized to one of eight treatment sequences (22 females and 22 males per sequence) of placebo crossed over with once-daily (10 days) 50, 100, or 200 mg mirabegron or a single 400-mg moxifloxacin dose on day 10. In each period, continuous ECGs were recorded at two baselines and on the last drug administration day. The lower one-sided 95% confidence interval for moxifloxacin effect on QTcI was >5 ms, demonstrating assay sensitivity. According to ICH E14 criteria, mirabegron did not cause QTcI prolongation at the 50-mg therapeutic and 100-mg supratherapeutic doses in either sex. Mirabegron prolonged QTcI interval at the 200-mg supratherapeutic dose (upper one-sided 95% CI >10 ms) in females, but not in males.

Antipsychotic-induced vacuous chewing movements and extrapyramidal side effects are highly heritable in mice.

Pharmacogenomics is yet to fulfill its promise of manifestly altering clinical medicine. As one example, a predictive test for tardive dyskinesia (TD) (an adverse drug reaction consequent to antipsychotic exposure) could greatly improve the clinical treatment of schizophrenia but human studies are equivocal. A complementary approach is the mouse-then-human design in which a valid mouse model is used to identify susceptibility loci, which are subsequently tested in human samples. We used inbred mouse strains from the Mouse Phenome Project to estimate the heritability of haloperidol-induced activity and orofacial phenotypes. In all, 159 mice from 27 inbred strains were chronically treated with haloperidol (3 mg kg(-1) per day via subdermal slow-release pellets) and monitored for the development of vacuous chewing movements (VCMs; the mouse analog of TD) and other movement phenotypes derived from open-field activity and the inclined screen test. The test battery was assessed at 0, 30, 60, 90 and 120 days in relation to haloperidol exposure. As expected, haloperidol caused marked changes in VCMs, activity in the open field and extrapyramidal symptoms (EPS). Unexpectedly, factor analysis demonstrated that these measures were imprecise assessments of a latent construct rather than discrete constructs. The heritability of a composite phenotype was ∼0.9 after incorporation of the longitudinal nature of the design. Murine VCMs are a face valid animal model of antipsychotic-induced TD, and heritability estimates from this study support the feasibility of mapping of susceptibility loci for VCMs.

Social approach in genetically engineered mouse lines relevant to autism.

Profound impairment in social interaction is a core symptom of autism, a severe neurodevelopmental disorder. Deficits can include a lack of interest in social contact and low levels of approach and proximity to other children. In this study, a three-chambered choice task was used to evaluate sociability and social novelty preference in five lines of mice with mutations in genes implicated in autism spectrum disorders. Fmr1(tm1Cgr/Y)(Fmr1(-/y)) mice represent a model for fragile X, a mental retardation syndrome that is partially comorbid with autism. We tested Fmr1(-/y)mice on two genetic backgrounds, C57BL/6J and FVB/N-129/OlaHsd (FVB/129). Targeted disruption of Fmr1 resulted in low sociability on one measure, but only when the mutation was expressed on FVB/129. Autism has been associated with altered serotonin levels and polymorphisms in SLC6A4 (SERT), the serotonin transporter gene. Male mice with targeted disruption of Slc6a4 displayed significantly less sociability than wild-type controls. Mice with conditional overexpression of Igf-1 (insulin-like growth factor-1) offered a model for brain overgrowth associated with autism. Igf-1 transgenic mice engaged in levels of social approach similar to wild-type controls. Targeted disruption in other genes of interest, En2 (engrailed-2) and Dhcr7, was carried on genetic backgrounds that showed low levels of exploration in the choice task, precluding meaningful interpretations of social behavior scores. Overall, results show that loss of Fmr1 or Slc6a4 gene function can lead to deficits in sociability. Findings from the fragile X model suggest that the FVB/129 background confers enhanced susceptibility to consequences of Fmr1 mutation on social approach.

Advances in behavioral genetics: mouse models of autism.

Autism is a neurodevelopmental syndrome with markedly high heritability. The diagnostic indicators of autism are core behavioral symptoms, rather than definitive neuropathological markers. Etiology is thought to involve complex, multigenic interactions and possible environmental contributions. In this review, we focus on genetic pathways with multiple members represented in autism candidate gene lists. Many of these pathways can also be impinged upon by environmental risk factors associated with the disorder. The mouse model system provides a method to experimentally manipulate candidate genes for autism susceptibility, and to use environmental challenges to drive aberrant gene expression and cell pathology early in development. Mouse models for fragile X syndrome, Rett syndrome and other disorders associated with autistic-like behavior have elucidated neuropathology that might underlie the autism phenotype, including abnormalities in synaptic plasticity. Mouse models have also been used to investigate the effects of alterations in signaling pathways on neuronal migration, neurotransmission and brain anatomy, relevant to findings in autistic populations. Advances have included the evaluation of mouse models with behavioral assays designed to reflect disease symptoms, including impaired social interaction, communication deficits and repetitive behaviors, and the symptom onset during the neonatal period. Research focusing on the effect of gene-by-gene interactions or genetic susceptibility to detrimental environmental challenges may further understanding of the complex etiology for autism.

Significantly increased lifespan and improved behavioral performances by rAAV gene delivery in adult mucopolysaccharidosis IIIB mice.

Mucopolysaccharidosis (MPS) IIIB is an inherited lysosomal storage disease, caused by the deficiency of alpha-N-acetylglucosaminidase (NaGlu), resulting in severe global neurological involvement with high mortality. One major hurdle in therapeutic development for MPS IIIB is the presence of the blood-brain barrier, which impedes the global central nervous system (CNS) delivery of therapeutic materials. In this study, we used a minimal invasive strategy, combining an intravenous (i.v.) and an intracisternal (i.c.) injection, following an i.v. infusion of mannitol, to complement the CNS delivery of adeno-associated viral (AAV) vector for treating MPS IIIB in young adult mice. This treatment resulted in a significantly prolonged lifespan of MPS IIIB mice (11.1-19.5 months), compared with that without treatment (7.9-11.3), and correlated with significantly improved behavioral performances, the restoration of functional NaGlu, and variable correction of lysosomal storage pathology in the CNS, as well as in different somatic tissues. This study demonstrated the great potential of combining i.v. and i.c. administration for improving rAAV CNS gene delivery and developing rAAV gene therapy for treating MPS IIIB in patients.

Assessment of NMDA receptor activation in vivo by Fos induction after challenge with the direct NMDA agonist (tetrazol-5-yl)glycine: effects of clozapine and haloperidol.

Induction of Fos protein by the potent and direct NMDA agonist (tetrazol-5-yl)glycine (TZG) was examined in mice. Effects of antipsychotic drugs were assessed on this in vivo index of NMDA receptor activation. TZG induced the expression of Fos in a neuroanatomically selective manner, with the hippocampal formation showing the most robust response. In mice genetically altered to express low levels of the NR1 subunit of the NMDA receptor, TZG-induced Fos was reduced markedly in comparison to the wild type controls. TZG-induced Fos was also blocked by the selective NMDA antagonist MK-801. Pretreatment of mice with clozapine (3 and 10 mg/kg) reduced TZG-induced Fos in the hippocampal formation but not in other brain regions. Haloperidol at a dose of 0.5 mg/kg did not antagonize TZG induced Fos in any region. Haloperidol at a dose of 1.0 mg/kg did attenuate the induction of Fos by TZG in the hippocampus but not in other brain regions. The relatively high dose (1 mg/kg) of haloperidol required to block effects of TZG suggests that this action may not be related to the D(2) dopamine receptor-blocking properties, since maximal D(2) receptor blockade was probably achieved by the 0.5 mg/kg dose of haloperidol. The antidepressant drug imipramine (10 or 20 mg/kg) did not antagonize TZG induced Fos in any brain region. The data suggest that clozapine can reduce excessive activation of NMDA receptors by TZG administration in vivo at doses relevant to the drugs' actions in rodent models of antipsychotic activity. Whether or not this action of clozapine contributes to its therapeutic properties will require further study.

Sociability and preference for social novelty in five inbred strains: an approach to assess autistic-like behavior in mice.

Deficits in social interaction are important early markers for autism and related neurodevelopmental disorders with strong genetic components. Standardized behavioral assays that measure the preference of mice for initiating social interactions with novel conspecifics would be of great value for mutant mouse models of autism. We developed a new procedure to assess sociability and the preference for social novelty in mice. To quantitate sociability, each mouse was scored on measures of exploration in a central habituated area, a side chamber containing an unfamiliar conspecific (stranger 1) in a wire cage, or an empty side chamber. In a secondary test, preference for social novelty was quantitated by presenting the test mouse with a choice between the first, now-familiar, conspecific (stranger 1) in one side chamber, and a second unfamiliar mouse (stranger 2) in the other side chamber. Parameters scored included time spent in each chamber and number of entries into the chambers. Five inbred strains of mice were tested, C57BL/6J, DBA/2J, FVB/NJ, A/J and B6129PF2/J hybrids. Four strains showed significant levels of sociability (spend- ing more time in the chamber containing stranger 1 than in the empty chamber) and a preference for social novelty (spending more time in the chamber containing stranger 2 than in the chamber containing the now-familiar stranger 1). These social preferences were observed in both male and female mice, and in juveniles and adults. The exception was A/J, a strain that demonstrated a preference for the central chamber. Results are discussed in terms of potential applications of the new methods, and the proper controls for the interpretation of social behavior data, including assays for health, relevant sensory abilities and motor functions. This new standardized procedure to quantitate sociability and preference for social novelty in mice provides a method to assess tendencies for social avoidance in mouse models of autism.

Automated apparatus for quantitation of social approach behaviors in mice.

Mouse models of social dysfunction, designed to investigate the complex genetics of social behaviors, require an objective methodology for scoring social interactions relevant to human disease symptoms. Here we describe an automated, three chambered apparatus designed to monitor social interaction in the mouse. Time spent in each chamber and the number of entries are scored automatically by a system detecting photocell beam breaks. When tested with the automated equipment, juvenile male C57BL/6J mice spent more time in a chamber containing a stranger mouse than in an empty chamber (sociability), similar to results obtained by the observer scored method. In addition, automated scoring detected a preference to spend more time with an unfamiliar stranger than a more familiar conspecific (preference for social novelty), similar to results obtained by the observer scored method. Sniffing directed at the wire cage containing the stranger mouse correlated significantly with time spent in that chamber, indicating that duration in a chamber represents true social approach behavior. Number of entries between chambers did not correlate with duration of time spent in the chambers; entries instead proved a useful control measure of general activity. The most significant social approach behavior took place in the first five minutes of both the sociability and preference for social novelty tests. Application of these methods to C57BL/6J, DBA/2J and FVB/NJ adult males revealed that all three strains displayed tendencies for sociability and preference for social novelty. To evaluate the importance of the strain of the stranger mouse on sociability and preference for social novelty, C57BL/6J subject mice were tested either with A/J strangers or with C57BL/6J strangers. Sociability and preference for social novelty were similar with both stranger strains. The automated equipment provides an accurate and objective approach to measuring social tendencies in mice. Its use may allow higher-throughput scoring of mouse social behaviors in mouse models of social dysfunction.

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.

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.

Enduring effects of prenatal cocaine administration on emotional behavior in rats.

The present studies sought to determine whether prenatal cocaine administration (15 mg/kg b.i.d. between gestational ages 1-20) had enduring effects on emotional behavior of rats. Rats prenatally treated with cocaine interacted less with other rats in the social interaction test of anxiety at both 30 and 120 days of age. However, there were no differences in the elevated plus maze test of anxiety. Rats prenatally treated with cocaine were significantly more immobile in the forced-swim test at 60 and 120 days of age. In addition, animals exposed to prenatal cocaine were more sensitive to the enhancing effect of phencyclidine (2.0 mg/kg) on startle responses to an acoustic stimulus. The cocaine-treated animals tested at 50 to 60 days of age showed higher levels of prepulse inhibition, in comparison to the saline group, after vehicle pretreatment, but not after phencyclidine. Although there were gender differences in the expression of some of these behavioral tasks, there were no gender differences in the effects of cocaine. These findings indicate that when emotional behavior is altered by prenatal cocaine administration, the effects are enduring.

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.

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.

Differential effects of bilateral dopamine depletion in neonatal and adult rats.

Both Lesch-Nyhan syndrome and Parkinson's disease are associated with decreased brain dopamine, yet each disorder is characterized by a different set of motor symptoms. Lesch-Nyhan syndrome is manifested in early childhood, while parkinsonism usually does not appear until adulthood, suggesting that age at the time of dopamine loss is one determinant of the effects of neurotransmitter deficiency. Support for this view is found in studies of animals given dopamine-depleting lesions at different ages and then tested in adulthood. Animals lesioned as neonates show a supersensitivity to dopamine agonists, especially D1-dopamine receptor agonists, and to MK-801, an NMDA receptor antagonist. In addition, neonatally treated animals show a 'priming' effect following repeated exposure to D1-dopamine agonists. Animals depleted of dopamine as adults are more supersensitive to agonists acting on the D2-dopamine receptor, and do not evidence priming to dopamine agonists or an enhanced response to MK-801. These differential pharmacological profiles suggest that the changes in neurotransmitter systems following dopamine depletion are, at least in part, determined by age at the time of the lesion.

Flumazenil blockade of anxiety following ethanol withdrawal in rats.

In previous research, the drug flumazenil has been categorized both as a pure benzodiazepine antagonist and as a benzodiazepine partial agonist. The following studies used an elevated plus maze to test whether flumazenil would exert any antianxiety action in rats. While chlordiazepoxide (3.0 mg/kg), ethanol (0.75 g/kg), and the atypical benzodiazepine zolpidem (1.0 mg/kg) all significantly increased time spent on the open arms and percent open arm entries, flumazenil (1-10 mg/kg) alone did not produce any anxiolytic effects on the maze. Withdrawal from chronic ethanol treatment led to a decrease in open arm time and percent open arm entries. Flumazenil (3.0 mg/kg) blocked these changes, suggesting that the effects of flumaxenil are at least partially dependent upon the levels of stress or anxiety in the subjects. An anxiolytic action of flumazenil was not seen following the central administration of the neuropeptide corticotropin-releasing factor (CRF), which reduced open arm time on the elevated plus maze. These results support the hypothesis that the mechanism of action for flumazenil effects on the anxiety observed during ethanol withdrawal involves antagonism of an endogenous benzodiazepine inverse agonist, rather than activity as a partial agonist or blockade of CRF-mediated effects.

Action of zolpidem on responses to GABA in relation to mRNAs for GABA(A) receptor alpha subunits within single cells: evidence for multiple functional GABA(A) isoreceptors on individual neurons.

The relationship between zolpidem sensitivity and GABA(A) receptor alpha subunits was studied in individual dissociated neurons from rat brain. Using whole-cell recording, similar EC50 values were demonstrated for the effect of gamma-aminobutyric acid (GABA) on gated-chloride currents from substantia nigra reticulata (SNR) and lateral septal neurons. Subsequently, many neurons from both the SNR or lateral septum were found to exhibit enhanced GABA-gated chloride currents across concentrations of zolpidem ranging from 10 to 300 nM. Some neurons exhibited a greater than 20% increase in responsiveness to GABA at 30 nM of zolpidem without further increase at higher concentrations of zolpidem. Conversely, zolpidem enhancement of GABA from another group of neurons was not observed at 30 nM zolpidem, but between 100 and 300 nM the response to GABA increased greater than 20%. Finally, a third group of neurons reached both of these criteria for zolpidem enhancement of GABA. This latter spectrum of responses to GABA after varying concentrations of zolpidem was consistent with the presence of either two GABA(A) receptors or a single receptor with differing affinities for zolpidem on an individual neuron. Following determination of the sensitivity of neurons from SNR or lateral septum to zolpidem, cytoplasm was extracted from some individual cells to allow identification of cellular mRNAs for the alpha1, alpha2 and alpha3 GABA(A) receptor subunits with RT-PCR. Those neurons that responded to the 30 nM zolpidem concentration invariably expressed the alpha1-GABA(A) receptor subunit. This result is consistent with the GABA(A) alpha1-receptor subunit being an integral part of a functional high-affinity zolpidem type 1-BZD receptor complex on neurons in brain. Those neurons which showed enhancement of GABA from 100 to 300 nM zolpidem contained mRNAs for the alpha2 and/or the alpha3 receptor subunits, a finding consistent with these alpha subunits forming type 2-BZD receptors. Some individual dissociated SNR neurons were sensitive to both low and high concentrations of zolpidem and contained mRNAs for all three alpha-receptor subunits. These latter individual neurons are proposed to have at least two functional GABA(A) receptor subtypes. Thus, the present investigation emphasizes the importance of characterizing the relationship between endogenous GABA(A) receptor function and the presence of specific structural components forming GABA(A) receptor subtypes on neurons.

Impaired acquisition and operant responding after neonatal dopamine depletion in rats.

The effect of neonatal dopamine depletion in rats was examined using operant conditioning. Rat pups were given 6-hydroxydopamine (6-OHDA) or sham lesions at 3 days of age. When tested as adults, 6-OHDA treated subjects were impaired in the acquisition of lever pressing for reward and displayed stereotyped sniffing patterns not observed in the control subjects. In addition, significantly lower rates of responding were measured for the lesion group during continuous reinforcement (CRF), dilution of reinforcer efficacy, and with progressively increasing fixed ratio requirements. Alpha-methyl-p-tyrosine (AMPT), given before one CRF session, attenuated responding in over half the lesion animals and in none of the controls. Dopamine content in caudate nucleus was found to significantly correlate with number of trials to acquisition and rates of responding following AMPT in the lesion group, but not in the control group. Overall, the results of this experiment showed that neonatal dopamine depletion does not lead to severe motor impairment or the inability to learn, but does disrupt the normal patterns of behavior associated with operant conditioning.

Effects of ethanol, MK-801, and chlordiazepoxide on locomotor activity in different rat lines: dissociation of locomotor stimulation from ethanol preference.

Several lines of research have suggested a link between the reward value of a drug and its ability to stimulate locomotion. One goal of the present study was to determine whether ethanol preferentially stimulates locomotor activity in lines of rat that show a preference for ethanol. A secondary goal was to determine the extent to which the benzodiazepine-like and NMDA antagonistic action of ethanol accounted for its effect on locomotor activity. To meet these goals, the effects of varying doses of ethanol (0.125-1.0 g/kg), MK-801 (0.1-0.3 mg/kg), and chlordiazepoxide (0.3-3 mg/kg) on locomotor activity were studied in several lines of rats that had been habituated to the testing procedure. The effect of low doses of ethanol on motor activity in the Alcohol-Preferring (P) and Fawn-Hooded rats, which show a strong ethanol preference, were similar to those of the alcohol-nonpreferring (NP), Flinders Sensitive Line, and Flinders Resistant Line rats. Only the Flinder Resistant Line rats showed a small, but significant increase in locomotor activity after the administration of ethanol. The highest dose of ethanol (1.0 g/kg) produced locomotor depression in all lines except the P and NP lines, which were not tested at this dose. These findings do not support a link between locomotor stimulation by ethanol and ethanol preference. In contrast, all lines exhibited locomotor stimulation after moderate (0.1-0.3 mg/kg) doses of MK-801, but did not exhibit increases in activity following any dose of chlordiazepoxide. These data indicate that the profiles of activity after MK-801 and chlordiazepoxide were distinct from that of ethanol in the various rat lines.(ABSTRACT TRUNCATED AT 250 WORDS)