Intravenous cocaine self-administration: individual differences in male and female C57BL/6J mice.

This study examined individual differences in male and female C57BL/6J (C57) mice responding for intravenous cocaine reinforcement. The experiment used 4 groups of mice, distinguished by sex and cocaine unit dose (0.3 or 1 mg/kg/infusion). Mice trained to lever respond for IV cocaine were given the drug initially on an FR2 schedule and then on a Progressive Ratio 2(PR2) schedule. Hierarchical linear modeling (HLM) techniques were used to examine data generated across four FR2 and four PR2 sessions, as well as within session data when cocaine was delivered on the PR2 schedule. HLM techniques, although uncommon in the animal literature, characterize individual differences in human studies and are likely to be useful in more complex preclinical studies. Analysis established distinct patterns of self-administration both across and within sessions. Responses for cocaine delivered on the FR2 schedule was dose-dependent, but did not differ according to sex. Response output was greater when either dose of cocaine was delivered on the PR2 than the FR2 schedule. Although response output for the more rewarding 1 mg/kg unit dose was similar for the two sexes, males responded more and had greater cocaine intake than females when the less reinforcing 0.3 mg/kg dose was delivered at the more behaviorally challenging PR2 schedule. HLM analysis of response patterns and cocaine intake within the PR2 sessions corroborated this sex difference and also indicated that trajectories differed for individual mice after accounting for the sex and dose factors. The reduced response output by females for cocaine in the present experiment is consistent with previous reports that sex differences in the rewarding effects of either alcohol or food reinforcement were revealed for C57 mice only when delivered on more behaviorally demanding schedules (e.g. PR2 or FR100).

Effects of the benzodiazepine antagonist flumazenil in PTSD.

OBJECTIVE: Evidence from preclinical and clinical studies suggests a role for alterations in the benzodiazepine/GABAA receptor complex in stress and anxiety. Flumazenil is a relatively pure benzodiazepine/GABAA antagonist with limited intrinsic activity. In panic disorder patients, but not healthy controls, flumazenil has been demonstrated to provoke panic attacks. METHOD: Vietnam combat veterans with PTSD (n = 14) received 90-second intravenous infusions of flumazenil 2 mg or placebo in a double-blind, crossover study design. PTSD symptomology was assessed using the PTSD Symptom Scale, and anxiety symptoms were measured with visual analogue rating scales. RESULTS: There was no significant difference in PTSD and anxiety symptoms between administration of flumazenil and placebo. CONCLUSION: Flumazenil administration does not produce an increase in anxiety and PTSD symptoms in patients with PTSD. This suggests that PTSD and panic disorder are dissimilar in terms of benzodiazepine/GABAA system function.

Improved models for pharmacological null experiments: calculation of drug efficacy at recombinant D1A dopamine receptors stably expressed in clonal cell lines.

Modern drug discovery demands accurate knowledge of the drug properties of affinity and efficacy at specific receptor proteins. Furthermore, drugs with well defined properties make better tools with which to explore and understand receptor regulation. The use of clonal cell lines stably expressing a given recombinant receptor may provide a highly useful model in which drug effects may be studied on one receptor subtype at a time. The present report was designed to evaluate the utility of a general method in which a clonal cell line stably expressing a recombinant D1A dopamine receptor was used as a model system for studying drug actions by null models. The null model for receptor occlusion (to calculate agonist Ka) and the null model for relative efficacy (to calculate test agonist affinity and epsilon r) were evaluated in these studies. To initiate these studies, rat C6 glioma cells that do not normally express DA receptors have been modified by stable transfection with the primate D1A DA receptor [Machida et al., 1992 (Molec. Pharmacol. 41: 652-659)] to a density of approximately equal to fmol/mg protein. The recombinant receptors show robust stimulation of cAMP in the stably transfected C6 cells. Calculation of agonist Ka from dose-response data requires that a portion of the cell's receptors be occluded in the absence of changes in post-receptor events leading to the response. Receptor reserve is typically reduced by alkylation, thereby lowering maximal response. Unfortunately, most of the currently available alkylating agents are not selective either for a particular receptor or for receptors vs other proteins within a signaling pathway. Short-term agonist treatment offers a possible complement to the use of non-selective or poorly characterized alkylating drugs for reducing maximum response in appropriate cell systems. The null method of receptor occlusion was used to determine the Ka for dopamine when maximum response was decreased by alkylation vs short-term agonist treatment. Direct non-linear curve fitting was used to analyze the data. In addition to DA, two other compounds were used to reduce receptor reserve to validate the method: fenoldopam (relatively high efficacy) and SKF38393 (low efficacy). Analyses indicated that the affinity of DA was similar whether calculated by alkylation (1.1 +/- 0.58 microM), 75 min DA treatment (0.57 +/- 0.16 microM) or 45 min treatment with DA (0.86 +/- 0.11 microM). Short-term agonist treatment experiments using multiple concentrations of DA, fenoldopam, or SKF38393 to decrease receptor reserve provided additional support for the validity of the Ka determinations using this procedure. Other experiments were conducted according to the null model for relative efficacy in which the affinity for DA is calculated by comparing the DA response before and after receptor occlusion, and the affinity and relative intrinsic efficacy of the test agonist are determined as a function of its actions relative to DA. We used the following four test drugs: + Br-APB, a novel agent with potential dopamine agonist properties, and three high-affinity DA agonists, fenoldopam, R-(-)-apomorphine (APO), and SKF38393. Intrinsic efficacy values relative to that of DA (1.0) were as follows: fenoldopam, 0.46 +/- 0.11; APO, 0.19 +/- 0.13; SKF38393, 0.07 +/- 0.01; and +Br-APB, 0.26 +/- 0.40. The agonist affinities (Ka) were: fenoldopam, 0.018 +/- 0.008 microM; APO, 0.80 +/- 0.18 microM; SKF38393, 0.16 +/- 0.04 microM; BR-APB, 0.43 +/- 0.29 microM; and DA, 0.58 +/- 0.17 microM. EC50/Ka ratios were consistent with relative intrinsic efficacies and Ka values were similar to KL values reported for membrane binding studies. Finally, Monte Carlo simulations were conducted to determine the precision of the parameter estimates...

Genotypic influences on pituitary responsiveness to haloperidol in mice.

Previous studies from this laboratory demonstrated that CBA/J mice have impaired striatal dopaminergic supersensitivity in response to subchronic haloperidol administration. Others have speculated that the peripheral hyperprolactinemia produced by haloperidol is necessary for the striatal dopamine receptor supersensitization produced by dopamine antagonists. In the present experiments, we tested the hypothesis that the impaired supersensitization response to haloperidol in CBA/J mice was secondary to an impaired hyperprolactinemic response by comparing the CBA/J mice with other mice that show normal supersensitization responses: the BALB/cJ and C57BL/6J strains. Acute haloperidol treatments increased serum prolactin levels 60 min later in all three strains, with the greatest response in CBA/J mice. After longer haloperidol treatment (2 or 21 days), serum prolactin remained elevated in CBA/J and, to a lesser extent, in C57BL/6J mice; levels remained low throughout treatment in BALB/cJ mice. Although, the basal density of pituitary dopamine receptors [( 3H]spiperone or D-2 binding sites) was greater in CBA/J than BALB/cJ mice, only BALB/cJ mice showed increased pituitary D-2 binding sites following chronic haloperidol administration. Taken together with previous studies of dopamine and noradrenaline receptors in these mouse strains, we conclude that CBA/J mice have a generalized impairment in their supersensitization responses to pharmacologic blockade of receptors. These data do not support the involvement of prolactin in haloperidol-induced dopamine receptor up-regulation.

Acute ethanol affects phosphorylation state of the NMDA receptor complex: implication of tyrosine phosphatases and protein kinase A.

Phosphorylation has been shown to regulate N-methyl-D-aspartic acid receptor (NMDAR) function. The inhibitory effect of ethanol on NMDAR function could be due, at least in part, to a change in NMDAR phosphorylation states. In order to investigate the effect of ethanol on phosphorylation of NR1 and NR2 subunits, NMDAR complexes were immunoprecipitated from cortical slices pre-exposed to ethanol. Acute ethanol, 100 and 200 mM, significantly decreased the tyrosine phosphorylation of NR2 subunits (Tyr-NR2). Treatment with a tyrosine phosphatase inhibitor reduced the inhibition of Tyr-NR2 phosphorylation caused by 100 mM ethanol. This suggests an involvement of tyrosine phosphatases in ethanol-induced inhibition of Tyr-NR2 phosphorylation. Slices pre-exposed to 100 and 200 mM ethanol exhibited a significant increase in the phosphorylation of NR1 by PKA at serine 897 (Ser897-NR1), which was blocked by a PKA inhibitor. Moreover, at 200 mM, ethanol produced a significant increase in PKA activity. Together, these results indicate that ethanol may increase Ser897-NR1 phosphorylation by activating PKA. However, ethanol did not affect phosphorylation of NR1 subunits by PKC at serine 896. We conclude that ethanol has the ability to modulate phosphorylation of both NR2 and NR1 subunits and these effects appear to implicate tyrosine phosphatases and PKA, respectively.

Mouse strain differences in apomorphine-induced behavior: an empirical and methodological study.

The behavioral response to 0.5-32.0 mg/kg apomorphine was evaluated in BALB/cJ, CBA/J, and C57BL/6J mice. Dose-response curves for each component of the drug-induced behavioral repertoire were derived, and ED50 (the dose at which 50% of the subjects meet or exceed a given rating) and slope were calculated for each. This method of analysis allows for a more quantitative estimate of drug sensitivity. Although some strain differences were observed in the qualitative characteristics of the response, differences in actual drug sensitivity (ED50s) were significant only at the higher ratings.

Dopamine agonist-induced stereotypic grooming and self-mutilation following striatal dopamine depletion.

Rats with bilateral 6-OHDA-induced striatal lesions exhibit altered apomorphine-induced behavior compared to the typical response seen in intact rats. This response is characterized by stereotypic grooming behavior which is transformed to compulsive and intense biting at higher doses. Other agonists, pergolide, bromocriptine, N-n-propylnorapomorphine and L-dopa/carbidopa, caused the same response. Direct intrastriatal infusion of apomorphine after 6-OHDA-induced lesions of the striata also produced this response, while similar SKF 38393 infusion caused more licking than biting, directed at the abdomen instead of the forepaws. The dopamine receptor antagonists haloperidol, SCH 23390 and sulpiride effectively blocked apomorphine-induced behaviors in dopamine-depleted animals. This altered behavior was not observed in rats with dopamine depletion in nucleus accumbens (NAS), nor did additional NAS lesions in rats with existing striatal lesions affect the behavior. Further, high doses of apomorphine (up to 60 mg/kg) failed to induce stereotypic grooming in unlesioned rats. We therefore propose that altered apomorphine-induced behavior is a unique consequence of striatal dopamine depletion and not just an intense form of normal stereotypic activity.

Diminished amphetamine anorexia and enhanced fenfluramine anorexia after midbrain 6-hydroxydopamine.

Rats were made hyperphagic by 6-hydroxydopamine (6-OHDA) injected bilaterally into the ventral midbrain; then they were restricted to a 6 h/day feeding schedule and tested for appetite suppression with amphetamine and fenfluramine in randomized order. Amphetamine anorexia was diminished while fenfluramine anorexia was enhanced (both P less than 0.001). The opposite effect on fenfluramine anorexia shows that the effect of 6-OHDA on amphetamine anorexia was not due to hyperphagia masking the anorexia. Norepinephrine in the forebrain was 90% depleted, but DA and serotonin levels were within 9% of normal. These results demonstrate a new way to dissociate amphetamine and fenfluramine anorexia, as others have done with lateral hypothalamic lesions or DA depletion. The 6-OHDA injections, which were of a type that cause hyperphagia, apparently destroyed a substrate for amphetamine anorexia and also facilitated a substrate for fenfluramine anorexia.

Startle and sensorimotor correlates of ventral thalamic dopamine and GABA in rodents.

Recent studies have implicated the thalamus as a possible site for neuroanatomical and neurochemical changes in schizophrenia. In the present study, we investigated thalamic neurochemical correlates of behaviors potentially linked to schizophrenia. Whole thalamic DOPAC levels were elevated in rats that had poor extinction of the acoustic startle response. The dopamine agonist apomorphine microinjected into the ventromedial thalamus (VmT) disrupted prepulse inhibition of startle. Catalepsy was induced by VmT microinjections of the GABA-A agonist muscimol. A previous study revealed attentional disturbances and suppression of frontal cortical metabolic activity after muscimol microinjections into the mediodorsal thalamic nucleus. Together with recent findings of neuron cell loss and elevated DA levels in the thalamus of schizophrenics, these data suggest the involvement of disturbances of thalamic neurotransmission in schizophrenia.

Striatal depth EEG reveals postsynaptic activity of striatal neurons following dopamine receptor stimulation and blockade.

This paper demonstrates a technique for measuring depth electroencephalographic (EEG) recordings from the freely moving mouse. This technique minimizes electrical artifact associated with gross movements by amplifying the current of the EEG signal directly at permanently indwelling electrodes. Stable EEG signals, with high signal-to-noise ratios, can be obtained from these animals while their movement inside the testing cage remains relatively unrestricted. We used this technique to examine the effects of dopamine (DA) receptor agonist and antagonist treatments on depth EEG signals generated within the striatum. Baseline measures of spontaneous striatal EEG activity were obtained prior to drug administration and post-drug measures of striatal activity were subsequently obtained. Apomorphine treatment resulted in desynchronization of striatal EEG signals while haloperidol or sulpiride treatment induced slow wave synchronization. Fast Fourier analysis of EEG signals revealed that DA agonist and antagonist treatment altered spontaneous striatal EEG activity in an opposite manner--relative to baseline signals, apomorphine attenuated low frequency components and augmented higher frequency components of the signal while haloperidol augmented low frequency components and attenuated higher frequency components of the signal. Moreover, mice pretreated with unilateral intracerebral injections of sulpiride and subsequently administered systemic apomorphine simultaneously demonstrated EEG synchronization on the side ipsilateral to the injection of sulpiride and EEG desynchronization on the contralateral side. The population of neurons examined in the medial striatum appear to have the properties of being excitatory to DA agonist stimulation and show decreased activity following DA antagonist treatment. These results suggest that striatal EEG activity may be used as measure of postsynaptic activity of dopaminergic neurons.

Repeated perfusion with elevated potassium in in vivo microdialysis--A method for detecting small changes in extracellular dopamine.

As a great deal of variability between subjects is often seen when using the microdialysis technique to measure the effects of depolarizing agents on extracellular neurotransmitter levels, we have developed a technique to account for the variability inherent in this method. High potassium (50 or 100 mM) artificial cerebrospinal fluid (ACSF), perfused through the probe for 10 min, significantly increased extracellular dopamine (DA) concentration during both an initial and second perfusion, and the two responses were highly correlated. However, extracellular DA returned to normal following the first perfusion with 50 mM K+ but not 100 mM K+ perfusion. The slope of the regression line obtained by plotting the response of the second K+ perfusion as a function of the first K+ perfusion for all K+ concentrations was 1.03 (not significantly different from unity). Similarly, when the time between two 50 mM potassium perfusions was varied from 30-150 min, the responses were highly correlated. This technique was used to demonstrate an interaction between N-methyl-D-aspartate (NMDA) and 50 mM K+. Perfusion of 0.1 mM NMDA alone had no effect on extracellular DA, but NMDA paired with a 50 mM K+ perfusion significantly increased extracellular DA over that increase by 50 mM K+ alone. We propose that a first stimulation with 50 mM potassium may characterize an individual animal's responsiveness to a depolarizing stimulus, and may be used as a control for testing drug effects by coupling drug treatments with a second 50 mM potassium stimulation to give a more accurate measure of small changes in extracellular dopamine.

Dose and time response analysis of apomorphine's effect on prepulse inhibition of acoustic startle.

Previous studies have suggested a dopaminergic modulation of the acoustic startle response. Using a 73 dB tone as a prestimulus to 100 dB white noise (PP trials), and 100 dB white noise alone (WN trials) during a 5-30 min or 5-90 min session, we found that IP injections of apomorphine (APO; 1.6 and 3.2 mg/kg) initially elevated PP trial means to levels statistically indistinguishable from WN trial means. These results suggest that APO can dose-dependently disrupt prepulse inhibition of the acoustic startle response. After the initial startle amplitude elevation 5-10 min post-injection in the 5-90 min protocol, startle response amplitude of the 3.2 mg/kg APO dose group decreased during successive testing periods, in contrast to the pattern of vehicle controls, which did not decrease during the session. The results of this extended time course study suggest complex time and dose effects of APO on reflex modification mechanisms. The finding that systemic APO administration produces a loss of prepulse inhibition in rodents underscores the pivotal role of DA neurotransmission in the mediation of startle behavior.

A quantitative estimate of the role of striatal D-2 receptor proliferation in dopaminergic behavioral supersensitivity: the contribution of mesolimbic dopamine to the magnitude of 6-OHDA lesion-induced agonist sensitivity in the rat.

Rats with unilateral depletions of neostriatal dopamine display increased sensitivity to dopamine agonists estimated to be 30 to 100 x in the 6-hydroxydopamine (6-OHDA) rotational model. Given that mild striatal dopamine D-2 receptor proliferation occurs (20-40%), it is difficult to explain the extent of behavioral supersensitivity by a simple increase in receptor density. This study was designed to investigate the quantitative aspects of the rotational behavior model utilizing constrained non-linear curve fitting routines. A dose-response curve for the rotational response arising from apomorphine stimulation of the normosensitive striatum was obtained in animals bearing unilateral lesions of striatal efferents (predominantly the striato-nigral pathway as previously described). After the control dose-response experiment, rats received a dopamine- (DA) depleting lesion in the contralateral hemisphere. In one group, 6-OHDA was infused into the medial forebrain bundle (MFB), a placement which is common in the literature and is known to deplete DA in both the striatum and nucleus accumbens. In a second group of rats, 6-OHDA was infused into the globus pallidus at a site which depletes caudate DA, but leaves n. accumbens DA relatively intact. The two experimental groups were tested in identical apomorphine-induced rotation dose-response experiments. The ED50's of the MFB- and caudate-lesioned rats were reduced by 36 and 5.8 fold, respectively, as compared to the control dose-response curve. The MFB and caudate lesions depleted striatal DA and produced a 30 and 36% increase in striatal D-2 binding sites, respectively. Modeling the behavioral and biochemical data with the null model for receptor occlusion indicated that increased striatal D-2 receptor density could account for the magnitude of behavioral supersensitivity in neither the MFB-lesioned group, nor even in the caudate-lesioned group. Thus simple up-regulation or D-2 receptors is unlikely to account for supersensitization as measured in the rotational model. Further, we suggest that quantitative modeling of such hypotheses is a valuable experimental technique for assessing relationships between biochemical and behavioral variables.

Lesion-induced DA supersensitivity in aging C57BL/6J mice.

Lesion-induced dopaminergic supersensitivity was investigated in 4-, 10-, and 28-month-old C57BL/6J mice. Apomorphine-induced rotational behavior was examined 5, 10, and 20 days after destruction of the dopamine-containing nigro-striatal pathway by intrastriatal infusion of 6-OHDA. No major differences between ages were observed in the extent or rate of development of contralateral rotation. It is concluded that age-differences in dopaminergic supersensitization are dependent upon the nature and/or severity of the sensitizing stimulus.

Localization of [3H]N-propylnorapomorphine binding in mouse striatum.

The anatomic localization of specific striatal [3H]N-propylnorapomorphine ( [3H]PNA) binding was determined in male C57BL/6J mice. Striatal [3H]PNA binding was high affinity and sensitive to guanine nucleotides. Frontal cortical ablation did not alter striatal [3H]PNA binding, but reduced [3H]spiperone binding by 36%. Kainic acid reduced and 6-hydroxydopamine elevated [3H]PNA binding. A combined frontal cortical ablation and striatal kainic acid lesion was similar to that of kainate alone. These data are consistent with a localization of [3H]PNA binding sites on neurons intrinsic to the mouse striatum.

Quantification of lesion-induced dopaminergic supersensitivity using the rotational model in the mouse.

A dual lesion technique was used to determine the degree of supersensitivity resulting from nigrostriatal lesions in C57BL/6J mice. Internal capsule lesions encroaching on globus pallidus resulted in reliable ipsilateral rotation both to apomorphine and amphetamine. Dose-response curves to apomorphine were determined before and 21 days after 6-hydroxydopamine lesion of the contralateral nigrostriatal pathway. A 31.5-fold shift to the left was observed following the nigrostriatal lesion, with no change in slope. The extent and placement of the internal capsule lesion, as well as the magnitude of supersensitization correspond closely to those previously reported in the rat.

Body weight and regulatory deficits following unilateral nigrostriatal lesions.

Unilateral nigrostriatal lesions were performed in rats by punctate injection of 6-hydroxydopamine. Lesioned animals exhibited anorexia and weight loss for 7-11 days after surgery. These animals then displayed normal rate of weight gain. However, there was a chronic depression of body weight in the lesioned animals relative to sham controls, evident for over two months after 6-hydroxydopamine administration. Unilateral nigrostriatal damage also produced major deficits in the animals' water intake in tests of prandial drinking, response to water deprivation, and response to osmotic challenge. The relationship of nigrostriatal damage to the "lateral hypothalamic syndrome" and the role of dopaminergic neurons in regulation and ingestive behaviors are discussed.

Genotypic influences on striatal dopaminergic regulation in mice.

Genotypic influences on dopaminergic-induced behaviors and striatal dopaminergic receptors were evaluated in CBA/J, C57BL/6J and BALB/cJ male mice. CBA/J mice were less behaviorally sensitive to apomorphine (stereotypic behavior) but more sensitive to haloperidol (catalepsy) than C57BL/6J and BALB/cJ mice. Striatal dopaminergic receptors, assayed by binding of [3H]spiroperidol (antagonist) and [3H]ADTN (agonist), were 50% fewer in CBA/J compared to BALB/cJ mice; C57BL/6J mice had low to intermediate numbers of receptors. Striatal dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations were similar in all strains. However, a 20% higher DOPAC/dopamine ratio in CBA/J mice suggests greater dopamine turnover. Median eminence dopamine was similar in all strains, but norepinephrine was 30% higher in BALB/cJ mice. CBA/J mice failed to show antagonist-induced supersensitivity-type responses to chronic haloperidol treatment: enhanced stereotypic response to apomorphine and a 30% increase of dopaminergic receptors occurred in C57BL/6J and BALB/cJ mice, but not in CBA/J mice. These data suggest that CBA/J mice either cannot respond to chronic haloperidol treatment or have an elevated threshold for induction of supersensitivity response. Chronic treatment with the dopamine agonist bromocriptine (7d) depressed apomorphine-induced stereotypic behavior in C57BL/6J mice and eliminated stereotypy in BALB/cJ mice, but caused no change in stereotypic behavior in CBA/J mice. Dopaminergic receptors were 15% lower after bromocriptine treatment in all strains. These results suggest that some striatal dopaminergic functions are impaired in CBA/J mice relative to BALB/cJ and C57BL/6J mice. The impaired haloperidol-induced supersensitivity responses in the CBA/J mouse may be a useful model for analyzing similar impairments of supersensitivity responses in old rodents.

Effect of duration of haloperidol treatment on DA receptor supersensitization in aging C57BL/6J mice.

Apomorphine-induced behavior, striatal [3H]spiperone binding, and striatal choline acetyltransferase (ChAT) activity were assessed in 6 1/2, 13, and 27-30 month-old male C57BL/6J mice following 0, 30, 60 or 90 days treatment with the dopaminergic (DA) antagonist haloperidol. Both apomorphine-induced behavior and [3H]spiperone binding (Bmax) increased linearly with duration of haloperidol treatment, with no detectable age difference in the degree of supersensitization, although basal receptor density declined with age. Middle- and old-aged mice showed prolonged stereotypic behavior relative to young mice, suggesting slower apomorphine clearance. No differences in ChAT activity were detected with either age or duration of haloperidol treatment. Although the group means of binding and behavior were highly related, the within group correlations were poor. Overall, the results suggest that aged animals are capable of DA receptor supersensitization when given a sufficient stimulus--in this case, relatively long treatment regimes. Previously reported deficits in neuroleptic-induced supersensitization in old mice may be confined to relatively short treatment periods at low doses.