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Opioid use disorder (OUD) has become a leading cause of death in the United States, yet current therapeutic strategies remain highly inadequate. To identify potential treatments for OUD, we screened a targeted selection of over 100 drugs using a recently developed opioid self-administration assay in zebrafish. This paradigm showed that finasteride, a steroidogenesis inhibitor approved for the treatment of benign prostatic hyperplasia and androgenetic alopecia, reduced self-administration of multiple opioids without affecting locomotion or feeding behavior. These findings were confirmed in rats; furthermore, finasteride reduced the physical signs associated with opioid withdrawal. In rat models of neuropathic pain, finasteride did not alter the antinociceptive effect of opioids and reduced withdrawal-induced hyperalgesia. Steroidomic analyses of the brains of fish treated with finasteride revealed a significant increase in dehydroepiandrosterone sulfate (DHEAS). Treatment with precursors of DHEAS reduced opioid self-administration in zebrafish in a fashion akin to the effects of finasteride. These results highlight the importance of steroidogenic pathways as a rich source of therapeutic targets for OUD and point to the potential of finasteride as a new treatment option for this disorder.

Opioid use disorder (OUD) has become a leading cause of death in the US, yet current therapeutic strategies remain highly inadequate. To identify novel potential treatments for OUD, we screened a targeted selection of over 100 drugs, using a recently developed opioid self-administration assay in zebrafish. This paradigm showed that finasteride, a steroidogenesis inhibitor approved for the treatment of benign prostatic hyperplasia and androgenetic alopecia, reduced self-administration of multiple opioids without affecting locomotion or feeding behavior. These findings were confirmed in rats; furthermore, finasteride did not interfere with the antinociceptive effect of opioids in rat models of neuropathic pain. Steroidomic analyses of the brains of fish treated with finasteride revealed a significant increase in dehydroepiandrosterone sulfate (DHEAS). Treatment with precursors of DHEAS reduced opioid self-administration in zebrafish, in a fashion akin to the effects of finasteride. Our results highlight the importance of steroidogenic pathways as a rich source of therapeutic targets for OUD and point to the potential of finasteride as a new option for this disorder.

Opioid signaling in the nucleus accumbens shell (sNAcc) has been implicated in hedonic feeding and binge eating behavior. The sNAcc projects to the lateral hypothalamus (LH), and this pathway has been suggested to modulate palatability-driven feeding behavior. In this study, we investigated the effects of sNAcc mu opioid receptor (MOR) stimulation on firing rates of LH neurons in previously sated rats. Neural firing in the LH was recorded while food-deprived rats performed an operant task to obtain sweetened Intralipid (a 4% fat emulsion containing 5% sucrose) before and after bilateral sNAcc infusion of either a MOR agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) or a saline control solution. During sessions in which saline was infused into the sNAcc, the number of trials completed after infusion were significantly lower than the number completed before infusion, likely reflecting animals' increased satiety state. During sessions in which DAMGO was infused into the sNAcc, the decrease in the number of trials completed (comparing post- vs. pre-infusion trials) was significantly attenuated. Electrophysiological recording showed that the percentage of LH neurons showing an excitatory response due to behavioral events (cue presentation, lever press, lever retraction, and consumption) was reduced in post vs. pre-saline infusion period. However, the percentage of LH neurons showing excitatory responses to the same behavioral events was similar in pre- and post-DAMGO infusion periods. These findings suggest that MOR stimulation in sNAcc leads to an increase in stimulus-evoked excitatory signaling in LH neurons which could contribute to preventing satiety-induced decline in palatable food intake.

Ethanol, like other drugs of abuse, has both rewarding and aversive properties. Previous work suggests that sensitivity to ethanol's aversive effects negatively modulates voluntary alcohol intake and thus may be important in vulnerability to developing alcohol use disorders. We previously found that rats with lesions of the lateral habenula (LHb), which is implicated in aversion-mediated learning, show accelerated escalation of voluntary ethanol consumption. To understand neural encoding in the LHb contributing to ethanol-induced aversion, we recorded neural firing in the LHb of freely behaving, water-deprived rats before and after an ethanol-induced (1.5 g kg−1 20% ethanol, IP) conditioned taste aversion (CTA) to saccharin taste. Ethanol-induced CTA strongly decreased motivation for saccharin in an operant task to obtain the tastant. Comparison of LHb neural firing before and after CTA induction revealed four main differences in firing properties. First, baseline firing after CTA induction was significantly higher. Second, firing evoked by cues signalling saccharin availability shifted from a pattern of primarily inhibition before CTA to primarily excitation after CTA induction. Third, CTA induction reduced the magnitude of lever press-evoked inhibition. Finally, firing rates were significantly higher during consumption of the devalued saccharin solution after CTA induction. Next, we studied sham- and LHb-lesioned rats in our operant CTA paradigm and found that LHb lesion significantly attenuated CTA effects in the operant task. Our data demonstrate the importance of LHb excitation in regulating expression of ethanol-induced aversion and suggest a mechanism for its role in modulating escalation of voluntary ethanol intake. This article is protected by copyright. All rights reserved

Rationale Ethanol has rewarding and aversive properties, and the balance of these properties influences voluntary ethanol consumption. Preclinical and clinical evidence show that the aversive properties of ethanol limit intake. The neural circuits underlying ethanol-induced aversion learning are not fully understood. We have previously shown that the lateral habenula (LHb), a region critical for aversive conditioning, plays an important role in ethanol-directed behaviors. However, the neurocircuitry through which LHb exerts its actions is unknown. Objective In the present study, we investigate a role for the rostromedial tegmental nucleus (RMTg), a major LHb projection target, in regulating ethanol-directed behaviors. Methods Rats received either sham or RMTg lesions and were studied during voluntary ethanol consumption; operant ethanol self-administration, extinction, and yohimbine-induced reinstatement of ethanol-seeking; and ethanol-induced conditioned taste aversion (CTA). Results RMTg lesions increased voluntary ethanol consumption and accelerated extinction of ethanol-induced CTA. Conclusions The RMTg plays an important role in regulating voluntary ethanol consumption, possibly by mediating ethanol-induced aversive conditioning.

The goal of the present research was to assess the degree to which a pattern completion process operates in cue-induced relapse to cocaine-seeking behavior. Using a novel cue-preference version of the place preference task, rats were administered cocaine or saline, which resulted in a preference for the cocaine-paired cues. After 21 days of abstinence and prior to the preference test, for one group, PBS or naloxone was injected into the CA3 subregion of the hippocampus and for a second group, saline or naloxone was injected systemically. The results indicated that infusions of naloxone into CA3 or systemic injections produced a marked disruption for one and two cues, but had minimal disruptive effect for three or four cues, suggesting that naloxone injections disrupt CA3 function and trigger a deficit in a pattern completion process. Thus, it appears that cue-based activation of the dorsal CA3 might be a critical trigger via a pattern completion process. Based on additional analyses it appears that there is a disruption primarily for object touches for one cue naloxone injections into the CA3 or systemic injections, but no effect on time (spatial context). © 2016 Wiley Periodicals, Inc.

Preclinical studies demonstrate that repeated, high-dose methamphetamine administrations rapidly decrease plasmalemmal dopamine uptake, which may contribute to aberrant dopamine accumulation, reactive species generation, and long-term dopaminergic deficits. The present study extends these findings by demonstrating a heretofore unreported, epitope-specific modification in the dopamine transporter caused by a methamphetamine regimen that induces these deficits. Specifically, repeated, high-dose methamphetamine injections (4 × 10 mg/kg/injection, 2-h intervals) rapidly decreased immunohistochemical detection of striatal dopamine transporter as assessed 1 h after the final methamphetamine exposure. In contrast, neither a single high dose (1 × 10 mg/kg) nor repeated injections of a lower dose (4 × 2 mg/kg/injection) induced this change. The high-dose regimen-induced alteration was only detected using antibodies directed against the N-terminus. Immunohistochemical staining using antibodies directed against the C-terminus did not reveal any changes. The high-dose regimen also did not alter dopamine transporter expression as assessed using [(125) I]RTI-55 autoradiography. These data suggest that the repeated, high-dose methamphetamine regimen alters the N-terminus of the dopamine transporter. Further, these data may be predictive of persistent dopamine deficits caused by the stimulant. Future studies of the signaling cascades involved should provide novel insight into potential mechanisms underlying the physiological and pathophysiological regulation of the dopamine transporter.

Instrumental actions are initially goal-directed and driven by their associated outcome. However, with repeated experience habitual actions develop which are automated and efficient, as they are instead driven by antecedent stimuli. Dopamine is thought to facilitate the transition from goal-directed to habitual actions. This idea has been largely derived from evidence that psychostimulants accelerate the development of habitual actions. In the current study, we examined the impact of L-dopa (levodopa or L-dihydroxyphenylalanine), which also potentiates dopamine activity, on habitual learning. L-dopa was systemically administered prior to training rats to press a lever for a food outcome. When tested, L-dopa exposed animals were insensitive to changes in the value of the food outcome, and hence demonstrated accelerated habitual behavioral control compared to control animals that remained goal directed. We also showed that when N-acetylcysteine (NAC), an antioxidant and regulator of glutamate activity, was co-administered with L-dopa, it prevented the transition to habitual behavior; an effect demonstrated previously for cocaine. Therefore, this study establishes similarities between L-dopa and psychostimulants in both the development and prevention of habitual actions, and supports the notion that excess dopamine potentiates habitual learning. This finding extends the limited existing knowledge of the impact of L-dopa on learning and behavior, and has implications for neurological disorders where L-dopa is the primary treatment.

Objective. To assess the impact of a Doctor of Pharmacy (PharmD) capstone project on students’ ability to conduct research and quality improvement, and to assess the feasibility of requiring projects in the core curriculum. Methods. Project proposals were solicited from faculty members and local colleagues, and students matched with an individual project and mentor. After developing a written research proposal in their third professional year, students completed the project with mentor oversight in their third and fourth professional years, culminating with a poster session and completion of a manuscript prior to graduation. Students’ knowledge of biostatistics, research confidence, and attitudes regarding research were evaluated using a validated survey instrument. Students and mentors were surveyed for feedback, and students’ publications and presentations were tracked. Results. Sixty-one students (97%) completed their projects on time. Students’ confidence in their ability to understand and participate in research increased, but improvement in statistical knowledge and interest in conducting future research projects was minimal. Fifty-eight percent of students presented posters at national conferences. Thirteen (21%) published manuscripts in peer-reviewed journals. Students and mentors responded positively overall about the program and the associated time requirements. Conclusion. Requiring PharmD students to complete a capstone project prior to graduation was feasible and increased student confidence in their ability to participate in research and the number of student and faculty poster presentations and peer-reviewed publications. These findings support the consideration of the Academy that analysis, synthesis, and creation of new knowledge can be successfully implemented into the core PharmD curricula.

Production of nitric oxide (NO) has been implicated in methamphetamine (METH)-induced dopamine (DA) neurotoxicity. The source of this NO has not been clearly delineated, but recent evidence suggests that it arises from activation of neuronal nitric oxide synthase (nNOS), which is selectively expressed in a subpopulation of striatal interneurons. Our objective was to determine whether inhibiting activation of nNOS-containing interneurons in the striatum blocks METH-induced neurotoxicity. These interneurons selectively express the neurokinin-1 (NK-1) receptor, which is activated by substance P. One particular toxin, a conjugate of substance P to the ribosome-inactivating protein saporin (SSP–-SAP), selectively destroys neurons expressing the NK-1 receptor. Thus, we examined the extent to which depletion of the nNOS-containing interneurons alters production of NO and attenuates METH-induced neurotoxicity. The SSP–SAP lesions resulted in significant loss of nNOS-containing interneurons throughout striatum. Surprisingly, this marked deletion did not confer resistance to METH-induced DA neurotoxicity, even in areas devoid of nNOS-positive cells. Furthermore, these lesions did not attenuate NO production, even in areas lacking nNOS. These data suggest that nNOS-containing interneurons either are not necessary for METH-induced DA neurotoxicity or produce NO that can diffuse extensively through striatal tissue and thereby still mediate neurotoxicity.

The lateral habenula (LHb) is an epithalamic brain region implicated in aversive processing via negative modulation of midbrain dopamine (DA) and serotonin (5-HT) systems. Given the role of the LHb in inhibiting DA and 5-HT systems, it is thought to be involved in various psychiatric pathologies, including drug addiction. In support, it has been shown that LHb plays a critical role in cocaine- and ethanol-related behaviors, most likely by mediating drug-induced aversive conditioning. In our previous work, we showed that LHb lesions increased voluntary ethanol consumption and operant ethanol self-administration and blocked yohimbine-induced reinstatement of ethanol self-administration. LHb lesions also attenuated ethanol-induced conditioned taste aversion suggesting that a mechanism for the increased intake of ethanol may be reduced aversion learning. However, whether afferents to the LHb are required for mediating effects of the LHb on these behaviors remained to be investigated. Our present results show that lesioning the fiber bundle carrying afferent inputs to the LHb, the stria medullaris (SM), increases voluntary ethanol consumption, suggesting that afferent structures projecting to the LHb are important for mediating ethanol-directed behaviors. We then chose two afferent structures as the focus of our investigation. We specifically studied the role of the inputs from the lateral hypothalamus (LH) and ventral pallidum (VP) to the LHb in ethanol-directed behaviors. Our results show that the LH-LHb projection is necessary for regulating voluntary ethanol consumption. These results are an important first step towards understanding the functional role of afferents to LHb with regard to ethanol consumption.

Regulation of dendritically localized mRNAs offers an important means by which neurons can sculpt precise signals at synapses. Arc is one such dendritically localized mRNA, and it has been shown to contain two exon-junction complexes (EJCs) within its 3′UTR. The EJC has been postulated to regulate cytoplasmic Arc mRNA availability through translation-dependent decay and thus contribute to synaptic plasticity. Core proteins of the EJC include eIF4A3, an RNA helicase, and Magoh, which stabilizes the interaction of eIF4A3 with target mRNAs. Arc mRNA expression is activity-regulated in numerous brain regions, including the dorsal striatum and hippocampus. Therefore in this study, the in vivo expression of these core EJC components was investigated in adult Sprague–Dawley rats to determine whether there are also behaviorally regulated changes in their expression. In the present work, there was no change in the expression of Magoh mRNA following spatial exploration, a paradigm previously reported to robustly and reliably upregulate Arc mRNA expression. Interestingly, however, there were increases in eIF4A3 mRNA levels in the dorsal striatum and hippocampus following spatial exploration, similar to previous reports for Arc mRNA. Furthermore, there were activity-dependent changes in eIF4A3 protein distribution and expression within the striatum following spatial exploration. Importantly, eIF4A3 protein colocalized with Arc mRNA in vivo . Like Arc mRNA expression, eIF4A3 mRNA expression in the dorsomedial striatum, but not dorsolateral striatum or hippocampus, significantly correlated with behavioral performance on a striatally-mediated, response-reversal learning task. This study provides direct evidence that a core EJC component, eIF4A3 , shows activity-dependent changes in both mRNA and protein expression in the adult mammalian brain. These findings thus further implicate eIF4A3 as a key mediator of Arc mRNA availability underlying learning and memory processes in vivo .

Neurotoxic doses of methamphetamine (METH) are known to cause depletions in striatal dopamine (DA) tissue content. However, the effects of METH-induced insults on dopaminergic neurotransmission are not fully understood. Here, we employed fast-scan cyclic voltammetry at a carbon-fiber microelectrode in the anesthetized rat striatum to assess the effects of a neurotoxic regimen of METH on phasic and tonic modes of dopaminergic signaling and underlying mechanisms of DA release and uptake. Extracellular DA was electrically evoked by stimulation of the medial forebrain bundle mimicking tonic and phasic firing patterns for dopaminergic cells and was monitored simultaneously in both the dorsomedial and dorsolateral striatum. Kinetic analysis of evoked recordings determined parameters describing DA release and uptake. Striatal DA tissue content was quantified by high performance liquid chromatography with electrochemical detection. METH-pretreatment (four doses of 7.5 or 10.0 mg/kg s.c.) induced DA depletions of ~40% on average, which are reported in both striatal subregions. METH pretreatment significantly decreased the amplitude of signals evoked by phasic, but not tonic, stimulation. Parameters for DA release and uptake were also similarly reduced by ~40%, consistent with effects on evoked phasic-like responses and DA tissue content. Taken together, these results suggest that METH-pretreatment selectively diminishes phasic, but not tonic, dopaminergic signaling in the dorsal striatum.

Repeated high-dose methamphetamine administrations can cause persistent dopaminergic deficits. As individuals abusing methamphetamine are often exposed to recurrent high-dose administration, the impact of its repeated exposure merits investigation. Accordingly, rats were pretreated with repeated high-dose injections of methamphetamine, and subsequently “challenged” with the same neurotoxic regimen 7 or 30 days later. Results revealed that the initial methamphetamine treatment caused persistent deficits in striatal dopamine levels, dopamine transporter function, and vesicular monoamine transporter-2 function. The subsequent methamphetamine challenge treatment was without further persistent effects on these parameters, as assessed 7 days after the challenge, regardless of the interval (7 or 30 days) between the initial and challenge drug exposures. Similarly, a methamphetamine challenge treatment administered 7 days after the initial drug treatment was without further acute effect on dopamine transporter or VMAT-2 function, as assessed 1 hour later. Thus, this study describes a model of resistance, possibly explained by: 1) the existence of dopaminergic neurons that are a priori refractory to deficits caused by methamphetamine; 2) the existence of dopaminergic neurons made persistently resistant consequent to a neurotoxic methamphetamine exposure; and/or 3) altered activation of post-synaptic basal ganglia systems necessary for the elaboration of methamphetamine-induced dopamine neurotoxicity.

Considerable research has examined regulation of striatal gene expression by neurotransmitters. A significant portion of this work has focused on unraveling interactions between neurotransmitters that regulate striatum, and the results strongly support the concept of broad regulation of striatal gene expression by interactions between multiple neurotransmitter systems. The studies reviewed in this chapter have contributed significantly to our foundational knowledge about the organization of the basal ganglia and how neurochemical systems therein may regulate its function. Further, studies examining gene expression have provided some correlative and causal links between gene expression in the striatum and behavior. With improved tools for evaluating the contribution of neurotransmitter systems to basal ganglia function, as well as improved understanding of the functional significance of changes in gene expression, research in this vein will continue to contribute significantly to our understanding of physiological and pathophysiological functions of the striatum.

The dorsal striatum is involved in motor-response learning, but the extent to which distinct populations of striatal efferent neurons are differentially involved in such learning is unknown. Activity-regulated, cytoskeleton-associated (Arc) protein is an effector immediate– early gene implicated in synaptic plasticity. We examined arc mRNA expression in striatopallidal vs. striatonigral efferent neurons in dorsomedial and dorsolateral striatum of rats engaged in reversal learning on a T-maze motor-response task. Male Sprague–Dawley rats learned to turn right or left for 3 days. Half of the rats then underwent reversal training. The remaining rats were yoked to rats undergoing reversal training, such that they ran the same number of trials but ran them as continued-acquisition trials. Brains were removed and processed using double-label fluorescent in situ hybridization for arc and preproenkephalin (PPE) mRNA. In the reversal, but not the continued-acquisition, group there was a significant relation between the overall arc mRNA signal in dorsomedial striatum and the number of trials run, with rats reaching criterion in fewer trials having higher levels of arc mRNA expression. A similar relation was seen between the numbers of PPE + and PPE – neurons in dorsomedial striatum with cytoplasmic arc mRNA expression. Interestingly, in behaviourally activated animals significantly more PPE – neurons had cytoplasmic arc mRNA expression. These data suggest that Arc in both striatonigral and striatopallidal efferent neurons is involved in striatal synaptic plasticity mediating motorresponse learning in the T-maze and that there is differential processing of arc mRNA in distinct subpopulations of striatal efferent neurons.

The expression of neuropeptide Y (NPY) and its co-messenger, agouti-related peptide (AgRP), in arcuate neurons of the hypothalamus is increased during lactation in rats. Our research has been addressing the questions of the physiological actions of these peptides during lactation and the physiological signals associated with lactation that result in increased expression of their genes. Our studies indicate that NPY and AgRP exert pleiotropic actions during lactation that help integrate neuroendocrine regulation of energy balance with controls over anterior and posterior pituitary hormone secretion. Further, reciprocal signaling to the NPY/AgRP system by leptin and ghrelin is responsible for the changes in expression of these hypothalamic peptides in lactating animals, and thus, may contribute to regulation of food intake and the various neuroendocrine adaptations of lactation.

The striatum, the primary input nucleus of the basal ganglia, is crucially involved in motor and cognitive function and receives significant glutamate input from the cortex and thalamus. Increasing evidence suggests fundamental differences between these afferents, yet direct comparisons have been lacking. We describe a slice preparation that allows for direct comparison of the pharmacology and biophysics of these two pathways. Visualization of slices from animals previously injected with BDA into the parafascicular nucleus revealed the presence of axons of thalamic origin in the slice. These axons were especially well-preserved after traversing the reticular nucleus, the location chosen for stimulation of thalamostriatal afferents. Initial characterization of the two pathways revealed both non-NMDA and NMDA receptor-mediated currents at synapses from both afferents and convergence of the afferents in 51% of striatal efferent neurons. Annihilation of action potentials was not observed in collision experiments, nor was current spread from the site of stimulation to striatum found. Differences in short-term plasticity suggest that the probability of release differs for the two inputs. The present work thus provides a novel rat brain slice preparation in which the effects of selective stimulation of cortical versus thalamic afferents to striatum can be studied in the same preparation.

Abused amphetamines, such as d-amphetamine (AMPH) and methamphetamine (METH), are highly addictive and destructive to health and productive lifestyles. The abuse of these drugs is associated with impulsive behavior, which is likely to contribute to addiction. The amphetamines also differentially damage dopamine (DA) and serotonin (5-HT) systems, which regulate impulsive behavior; therefore, exposure to these drugs may differentially alter impulsive behavior to effect the progression of addiction. We examined the impact of neurotoxicity induced by three amphetamines on impulsive action using a stop-signal task in rats. Animals were rewarded with a food pellet after lever pressing (i.e., a go trial), unless an auditory cue was presented and withholding lever press gained reward (i.e., a stop trial). Animals were trained on the task and then exposed to a neurotoxic regimen of either AMPH, p-chloroamphetamine (PCA), or METH. These regimens preferentially reduced DA transporter levels in striatum, 5-HT transporter levels in prefrontal cortex, or both, respectively. Assessment of performance on the stop-signal task beginning 1 week after the treatment revealed that AMPH produced a deficit in go-trial performance, whereas PCA did not alter performance on either trial type. In contrast, METH produced a deficit in stop-trial performance (i.e., impulsive action) but not go-trial performance. These findings suggest that the different neurotoxic consequences of substituted amphetamines are associated with different effects on inhibitory control over behavior. Thus, the course of addiction and maladaptive behavior resulting from exposure to these substances is likely to differ.

Preclinical studies demonstrate that repeated, high-dose methamphetamine administrations rapidly decrease plasmalemmal dopamine uptake, which may contribute to aberrant dopamine accumulation, reactive species generation, and long-term dopaminergic deficits. The present study extends these findings by demonstrating a heretofore unreported, epitope-specific modification in the dopamine transporter caused by a methamphetamine regimen that induces these deficits. Specifically, repeated, high-dose methamphetamine injections (4 × 10 mg/kg/injection, 2-h intervals) rapidly decreased immunohistochemical detection of striatal dopamine transporter as assessed 1 h after the final methamphetamine exposure. In contrast, neither a single high dose (1 × 10 mg/kg) nor repeated injections of a lower dose (4 × 2 mg/kg/injection) induced this change. The high-dose regimen-induced alteration was only detected using antibodies directed against the N-terminus. Immunohistochemical staining using antibodies directed against the C-terminus did not reveal any changes. The high-dose regimen also did not alter dopamine transporter expression as assessed using [(125) I]RTI-55 autoradiography. These data suggest that the repeated, high-dose methamphetamine regimen alters the N-terminus of the dopamine transporter. Further, these data may be predictive of persistent dopamine deficits caused by the stimulant. Future studies of the signaling cascades involved should provide novel insight into potential mechanisms underlying the physiological and pathophysiological regulation of the dopamine transporter.

Neurotensin is a neuropeptide associated with basal ganglia dopaminergic neurons. Because levels of neurotensin in striatal tissue are differentially affected by low or high doses of methamphetamine, we employed microdialysis to assess the dose-dependent effects of methamphetamine on neurotensin release from the terminals of striatonigral and striatopallidal neurons. A low (0.5 mg/kg), but not high (10 mg/kg), dose of methamphetamine significantly increased nigral extracellular levels of neurotensin. The low-dose effect on extracellular nigral neurotensin levels was blocked by pretreatment with either a dopamine D1 or D2 receptor antagonist. In the globus pallidus, only half of the animals demonstrated increased neurotensin release after the low dose of methamphetamine. These findings suggest that low and high doses of methamphetamine differentially affect the release of neurotensin from the terminals of striatonigral neurons and that both dopamine D1 and D2 receptor activation contributes to the low-dose methamphetamine effects in the substantia nigra.

Methamphetamine (METH) use results in depletion of monoamines in the striatum. The purpose of this study was to evaluate the relation between the degree of METH-induced monoamine depletion in the striatum and impairment on a striatally-dependent learning task in rats. Male Sprague-Dawley rats received four injections of METH (10 mg/kg) or saline at 2-h intervals. METH treatment produced a 38.5% (+/-5.6) and 46.7% (+/-6.7) dopamine (DA) depletion in the medial and lateral striatum, respectively. Serotonin (5-HT) was depleted 15.6% (+/-10.4) and 21.1% (+/-8.2) in the medial and lateral striatum, respectively. One month after treatment, rats were trained on a sequential-memory task on an 8-arm radial maze. METH-treated rats made significantly fewer direct movements between arms in the maze sequence across days of trials. The learning impairment was significantly correlated with the degree of DA depletion in the medial striatum, as well as serotonin tissue content in striatum. Only rats with a greater than 40% DA depletion in medial striatum showed significant impairments. These results provide additional evidence for METH-induced learning impairments and suggest that this impairment is dependent on the striatal monoamine loss, in general, and the degree of DA loss in medial striatum, in particular.

Cocaine and methamphetamine (METH) induce preprodynorphin (PPD) mRNA expression in the striatum. Cocaine induces PPD expression in both the patch and matrix compartments of the rostral striatum, whereas METH induces PPD expression in the patch compartment of the rostral striatum. In middle striatum, both stimulants increase PPD expression in the patch and matrix compartments. METH and cocaine treatment also increase extracellular serotonin (5-HT). Several studies have shown that 5-HT receptors are present on striatonigral neurons that express PPD mRNA, and that 5-HT is a positive regulator of striatal neuropeptide expression. The current study examined whether 5-HT plays a role in the patch/matrix expression of PPD mRNA induced by cocaine and METH in striatum. Male Sprague-Dawley rats were treated with p-chloroamphetamine (PCA; 8 mg/kg, i.p), a serotonin neurotoxin, 1 week prior to cocaine (30 mg/kg, i.p) and METH (15 mg/kg, s.c.) treatment. The 80% loss of 5-HT induced by PCA-pretreatment blocked cocaine-induced PPD expression in the rostral matrix compartment. Cocaine- and METH-induced PPD expression in the rostral patch compartment was unaffected by PCA-pretreatment. PCA-pretreatment also decreased both cocaine- and METH-induced PPD expression in the matrix, but not patch of middle striatum. PCA-induced 5-HT depletion did not affect stimulant-induced increases in PPT mRNA expression in the striatum. These data suggest that 5-HT plays a role in stimulant-induced PPD expression in the matrix compartment of rostral and middle striatum. Thus, 5-HT innervation may play a critical role in basal ganglia function.

Relatively little is known concerning the interaction of psychostimulants with hypothalamic neuropeptide systems or metabolic hormones implicated in regulation of energy balance. The present studies tested whether methamphetamine alters the expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP), two important orexigenic neuropeptides, or proopiomelanocortin (POMC), the precursor for the anorexigenic peptide alpha-melanocyte-stimulating hormone, or the secretion of leptin, insulin and ghrelin, concomitant with inhibition of food intake. Female rats were either fed ad libitum (AL) or placed on a scheduled feeding (SF) regimen, with access to food limited to 4 h/day. Administration of (+/-)-methamphetamine (7.5 mg/kg, i.p.) 2 h prior to food presentation significantly inhibited food intake in SF animals, but did not affect intake in AL animals. In a separate study, AL and SF animals were killed just prior to expected food presentation, and expression of NPY, AgRP and POMC mRNAs in hypothalamus was determined using in situ hybridisation; concentrations of leptin, insulin and ghrelin in serum were determined with radioimmunoassays. In saline-treated, SF controls, NPY and AgRP mRNA expression in arcuate nucleus and serum ghrelin were significantly elevated, and serum leptin and insulin were significantly reduced. Methamphetamine reversed the up-regulation of NPY mRNA expression observed in the SF condition, without affecting AgRP mRNA or the serum concentrations of metabolic hormones. However, in AL animals, NPY mRNA expression in arcuate and dorsomedial nuclei was significantly increased by methamphetamine, which also reduced serum leptin and insulin and increased serum ghrelin concentrations. These findings suggest that the inhibition of NPY expression in SF animals may be a mechanism underlying the anorexigenic effect of methamphetamine seen in this condition. The increase in NPY expression produced by methamphetamine in AL animals may be mediated by the ability of this drug to decrease secretion of leptin and insulin and increase secretion of ghrelin.

Substance P (SP) is a neuropeptide closely associated with basal ganglia dopaminergic neurons. Because some neuropeptide systems in the basal ganglia (i.e. neurotensin and metenkephalin) are differentially affected by treatment with low or high doses of methamphetamine, we determined if basal ganglia SP pathways were also differentially influenced in a dose-dependent manner by this psychostimulant. Employing in vivo microdialysis, it was observed that the lowdose (0.5 mg/kg) of methamphetamine increased the extracellular concentration of SP in the substantia nigra, but not in globus pallidus or striatum. In contrast, the high dose (10 mg/kg) of methamphetamine did not increase extracellular SP content in any of these structures. The effect of the low-dose methamphetamine treatment on nigral extracellular SP levels was blocked by pre-treatment with either a D1 or D2 antagonist. In addition, 12 h after similar methamphetamine treatments, a dose-dependent differential response in SP tissue levels occurred in some of the regions examined. When these changes occurred, the low dose of methamphetamine usually reduced, whereas the high dose increased, SP tissue content. This study demonstrated opposite responses of the basal ganglia SP system to low and high doses of methamphetamine and suggested that a combination of dopamine D1 and D2 receptor activity contributed to these effects.

High-dose methamphetamine treatment induces long-term deficits in central monoamine systems. However, the mechanisms underlying these effects are unknown. Previous work has shown that the Kappa-opioid receptor agonist U-69593 [(+)-(5alpha,7alpha,8b)-(+)-N-methyl-N[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl] benzeneacetamide] attenuates the neurotoxic effects of methamphetamine on extracellular dopamine levels in mice, suggesting that endogenous Kappa-opioid receptor ligands, such as dynorphin, may protect against methamphetamine-induced toxicity and play a role in mediating the long-term consequences of methamphetamine. To further examine the role that dynorphin systems play in methamphetamine-induced neurotoxicity, we administered to male rats a total of four injections of methamphetamine (7.5 mg/kg, s.c.), with a 2-h interval between each dose. Rats were pretreated with either the Kappa-agonist U-69593 (0.32 mg/kg, s.c.) or vehicle, 15 min prior to the first and third methamphetamine injection. Furthermore, cages containing the U-69593 + methamphetamine-treated rats were placed on heating pads for 30 min after the first U-69593 injection to prevent the drug from blocking methamphetamine-induced hyperthermia. Rats were sacrificed 7 days after treatment. Striatal dopamine and serotonin contents were decreased approximately 75% and 55%, respectively, in the methamphetamine-treated rats and approximately 88% and 65%, respectively, in rats receiving the U-69593 + methamphetamine combination. There was a approximately 20% mortality rate in the rats treated with methamphetamine compared to approximately 75% mortality rate in rats treated with both U-69593 and methamphetamine. A similar rate of mortality was observed when combining a different Kappa-agonist, U-50488 [trans-(-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamine], with methamphetamine. These data suggest that Kappa-agonists do not protect against methamphetamine-induced toxicity to monoamines in rats, and may potentiate mortality when co-administered with methamphetamine.

We and others previously reported that equimolar doses of methamphetamine and cocaine differentially increase preprodynorphin mRNA in striatum: methamphetamine causes a patchy increase, whereas cocaine produces a more homogenous one. The current study directly examined whether this effect reflects differential induction in the patch–matrix division of striatum, as identified by µ opioid receptor immunohistochemistry. In addition, we determined whether doses of cocaine (30 mg/kg) and methamphetamine (2 mg/kg) that produced equivalent increases in extracellular dopamine differentially affected preprodynorphin mRNA expression in striatum of male, Sprague–Dawley rats. In both experiments, methamphetamine and cocaine differentially affected preprodynorphin mRNA in striatum after 3 h. The high, equimolar dose of methamphetamine selectively increased preprodynorphin mRNA in the patch division of rostral striatum, whereas cocaine increased preprodynorphin mRNA throughout patch and matrix divisions of striatum. In contrast, a dose of methamphetamine (2.0 mg/kg) that caused an increase in extracellular dopamine similar to that produced by 30 mg/kg cocaine did not significantly affect preprodynorphin mRNA in any region of striatum. These data provide further evidence that cocaine and amphetamines exert distinct effects on the patch–matrix division of striatum and suggest further that the post-synaptic consequences of elevated extracellular dopamine produced by methamphetamine and cocaine are distinct.

Phasic dopamine (DA) signaling, during which burst firing by DA neurons generates short-lived elevations in extracellular DA in terminal fields called DA transients, is implicated in reinforcement learning. Disrupted phasic DA signaling is proposed to link DA depletions and cognitive-behavioral impairment in methamphetamine (METH)-induced neurotoxicity. Here, we further investigated this disruption by assessing effects of METH pretreatment on DA transients elicited by a drug cocktail of raclopride, a D2 DA receptor antagonist, and nomifensine, an inhibitor of the dopamine transporter (DAT). One advantage of this approach is that pharmacological activation provides a large, high-quality data set of transients elicited by endogenous burst firing of DA neurons for analysis of regional differences and neurotoxicity. These pharmacologically evoked DA transients were measured in the dorsomedial (DM) and dorsolateral (DL) striatum of urethane-anesthetized rats by fast-scan cyclic voltammetry. Electrically evoked DA levels were also recorded to quantify DA release and uptake, and DAT binding was determined by means of autoradiography to index DA denervation. Pharmacologically evoked DA transients in intact animals exhibited a greater amplitude and frequency and shorter duration in the DM compared to the DL striatum, despite similar pre- and post-drug assessments of DA release and uptake in both sub-regions as determined from the electrically evoked DA signals. METH pretreatment reduced transient activity. The most prominent effect of METH pretreatment on transients across striatal sub-region was decreased amplitude, which mirrored decreased DAT binding and was accompanied by decreased DA release. Overall, these results identify marked intrastriatal differences in the activity of DA transients that appear independent of presynaptic mechanisms for DA release and uptake and further support disrupted phasic DA signaling mediated by decreased DA release in rats with METH-induced neurotoxicity.

A single administration of a low (0.5 mg/kg) or high (10 mg/kg) dose of methamphetamine (METH) significantly altered the met-enkephalin (M-Enk) systems associated with some, but not all, limbic structures examined. Neither treatment influenced M-Enk levels 3 h after drug exposure in any limbic region studied; however, 12 h after drug administration, 0.5 mg/kg of METH reduced the tissue content of this peptide in both the nucleus accumbens shell (NAs) and the frontal cortex (FrCx). This was similar to the effect of this treatment on the anterior striatal region. In contrast, the high dose of METH increased M-Enk content in the frontal cortex and anterior striatum (AS), but had no effect in the nucleus accumbens shell. By 24 h, the effects of METH in the anterior striatum subsided, but decreases in M-Enk levels were still observed after both the low- and the high-dose METH treatments in the nucleus accumbens shell. The levels of M-Enk were not changed at any of the time points examined in the core of the nucleus accumbens (NAc). In general, treatment with a low or high dose of METH causes distinct and regional selective changes in the tissue levels of M-Enk in the limbic system. These changes appear to be mediated by dopamine (DA) D2 and D1 receptor activation.

Striatal efferent neurons receive dopamine- and glutamate-utilizing afferents. Previous studies have shown that dopamine depletion increases gene expression in striatopallidal neurons and decreases it in striatonigral neurons. Previous work has also reported increased expression of the 2A subunit of the N -methyl- d -aspartate receptor in the dopamine-depleted striatum. The purpose of this study therefore was to determine whether dopamine depletion differentially alters the expression of the 2A subunit of the N -methyl- d -aspartate receptor in rat striatal neurons. 6-Hydroxydopamine (8 μg/2 μl) was infused unilaterally into the medial forebrain bundle. Rats were killed three weeks later. Double-label in situ hybridization was performed using an 35 S-labeled ribonucleotide probe directed against the messenger RNA of the 2A subunit and a digoxigenin-labeled ribonucleotide probe directed towards preproenkephalin messenger RNA to mark striatopallidal neurons. Analysis of single-labeled film autoradiograms revealed a significant increase in the expression of 2A subunit messenger RNA in the ipsilateral, but not the contralateral, striatum of dopamine-depleted animals, consistent with other studies in the literature. Cellular analysis of 2A subunit expression indicated that as a consequence of dopamine depletion there is a significant increase in the expression of this subunit in both enkephalin-positive and enkephalin-negative neurons. From this study we conclude that dopamine depletion increases messenger RNA expression of the 2A subunit of the N -methyl- d -aspartate receptor in striatopallidal and presumed striatonigral (enkephalin-negative) neurons. Such alterations may affect the pharmacology and function of the resultant receptor, and thus alter glutamate transmission in both populations of medium spiny neurons after dopamine depletion.

This study employed in situ hybridization to directly compare the effects of cocaine and methamphetamine on neurotensin/neuromedin N and preprotachykinin messenger RNAs in distinct striatal regions. Male, Sprague-Dawley rats received a single administration of 15mg/kg methamphetamine (s.c.) or 30mg/kg cocaine (i.p.) and were killed 30min or 3h later. Methamphetamine and cocaine produced significant increases in preprotachykinin messenger RNA in the striatum after 3h, but often in different subregions. Both drugs produced similar effects on preprotachykinin messenger RNA in the rostral striatum. However, methamphetamine produced significant increases in all regions of the caudal striatum, whereas cocaine-induced preprotachykinin messenger RNA expression was limited to dorsal regions of this striatal area. Methamphetamine also produced a significant increase in preprotachykinin messenger RNA in the caudal striatum after 30min, whereas cocaine had no significant effect on preprotachykinin messenger RNA at this early time-point. The pattern of changes in neurotensin/neuromedin N messenger RNA caused by methamphetamine and cocaine after 3h was even more distinct. Cocaine produced significant increases in neurotensin/neuromedin N messenger RNA in all regions of the rostral striatum, whereas methamphetamine had no effect in these areas. Furthermore, in more caudal sections, cocaine predominantly affected neurotensin/neuromedin N expression in dorsal aspects of the striatum, whereas methamphetamine significantly increased neurotensin/neuromedin N messenger RNA in all regions. There was much less effect of either drug on neuropeptide expression in the nucleus accumbens. The only significant effect was an increase in neurotensin/neuromedin N messenger RNA in the core region 3h after methamphetamine administration. These results indicate that methamphetamine and cocaine increase preprotachykinin and neurotensin/neuromedin N messenger RNAs in distinct regions of the striatum. The ability of methamphetamine and cocaine to alter neuropeptide messenger RNA expression in unique regions of the striatum may be important for the long-term effects of these drugs, such as sensitization, since the striatum is not homogeneous in its connections and function.

Cortical afferents excite striatal efferent neurons through activation of N-methyl-D-aspartate (NMDA) receptors, which can be modulated by D2 dopamine receptors. It is suggested that activation of PKA by D2 receptor blockade leads to NMDA receptor phosphorylation in the dendrites or phosphorylation of transcription factors in the nucleus. Thus, the levels and cellular localization of activated PKA may determine if D2 antagonist-mediated gene expression is dependent on NMDA receptor activation. We have previously demonstrated that NMDA receptor antagonists block gene expression induced by a high dose of eticlopride in medial and central but not lateral striatum. Here, we examined the effects of NMDA receptor antagonists on striatal gene expression after administration of a low dose of eticlopride. The results showed that NMDA receptor antagonists blocked gene induction by eticlopride throughout striatum. Less PKA activation by the low dose of eticlopride might explain why the expression was more sensitive in the lateral striatum to NMDA receptor blockade than in our previous study. To increase levels of PKA activation to the extent that NMDA receptor blockade would have less effect on eticlopride-mediated gene induction in all regions of striatum, we administered the phosphodiesterase inhibitor IBMX to animals treated with eticlopride. The combined administration of IBMX and eticlopride induced gene expression that was only partially attenuated (c-fos) or unaffected (zif268) by NMDA receptor blockade. These data support the suggestion that the degree of second messenger activation by D2 receptor blockade determines whether D2 dopamine receptor antagonist-mediated gene expression is dependent on NMDA receptor activation.

Administration of selective agonists of D1 dopamine receptors increases immediate early gene expression in striatal neurons, a response which is particularly robust in the dopamine-depleted striatum. Although interactions between dopamine and glutamate receptor-mediated responses in striatal neurons have been demonstrated in a number of experimental paradigms, our previous findings indicate that N-methyl-D-aspartate antagonists do not block D1 receptor-mediated induction of immediate early genes in the dopamine-depleted striatum. In the present study, we therefore examined interactions between D1 dopamine receptors and the (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate subtypes of glutamate receptor by determining whether striatal infusion of the (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione would block D1 receptor-mediated induction of the immediate early genes c-fos and zif268 in the dopamine-depleted striatum. Striatal infusion of 6-cyano-7-nitroquinoxaline-2,3-dione (1 mM) completely blocked (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate-induced c-fos and zif268 expression. However, 6-cyano-7-nitroquinoxaline-2,3-dione (1 microM-1 mM) did not significantly affect induction of c-fos and zif268 by D1 receptor stimulation (SKF 38393, 2 mg/kg, i.p.) in the dopamine-depleted striatum. To more generally block excitatory input, tetrodotoxin (10 microM) was infused into the striatum of rats receiving a D1 agonist. Local infusion of tetrodotoxin had minimal effect on induction of c-fos and zif268 in the dopamine-depleted striatum. In contrast, tetrodotoxin abolished induction of c-fos and zif268 messenger RNAs by the D2 antagonist eticlopride (0.5 mg/kg, i.p.) in both intact rats and dopamine-depleted rats receiving continuous D2 agonist treatment (quinpirole, 0.5 mg/kg/day). The results indicate that D1 receptor-mediated induction of immediate early genes in the dopamine-depleted striatum occurs by mechanisms that are independent of excitatory input through (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors.

Changes in extrapyramidal dopamine (DA) function significantly alter the activity of striatal neurotensin (NT) systems. Specifically, stimulation of DA D-1 or D-2 receptors increases or decreases striatal NT tissue levels, respectively. In contrast, removal of D-2 receptor basal activity with either an antagonist or lesion of the nigrostriatal DA projection increases striatal NT content. To understand better the significance of these changes in the levels of NT peptide, we determined the effects of treatment with the selective D-1 agonist, SKF 82958, alone or in combination with a lesion of the nigrostriatal DA pathway, on the levels of NT mRNA in various regions of the caudate nucleus. Removal of at least 90% of this DA pathway significantly increased NT mRNA in most, but not all, regions throughout the caudate nucleus. In contrast, four, but not one, administrations of SKF 82958 (2 mg kg-1 dose-1) increased NT mRNA levels in principally middle, but not rostral, caudate regions. Lesioning the nigrostriatal DA pathway enhanced the effects of SKF 82958 so that a lower, single dose (1 mg/kg) of this D-1 agonist also increased NT mRNA levels predominantly in the middle caudate sections. These findings demonstrate that DA D-1 receptors profoundly regulate the striatal expression of NT mRNA in a regionally selective fashion, which appears to be unique from that principally influenced by DA D-2 regulation.

Dopamine D1 receptor regulation of striatal gene expression is dependent on N-methyl-D-aspartate (NMDA) receptors. To determine whether pharmacologically distinct NMDA receptors are differentially involved, we examined the effects of different NMDA receptor antagonists on D1-induced immediate early gene expression. Systemic administration of the channel blocking antagonists MK-801 and dextrorphan and the competitive antagonist CGS 19755 blocked gene expression induced by the D1 agonist SKF 82958. The NMDA polyamine site antagonist ifenprodil, however, potentiated the effect of SKF 82958. Since ifenprodil is selective for receptors containing the NR2B subunit, the data suggest that subtypes of NMDA receptors may be differentially involved in regulating striatal function.

Methamphetamine (METH) exposure results in long-term damage to the dopamine system in both human METH abusers and animal models. One factor that has been heavily implicated in this METH-induced damage to the dopaminergic system is the activation of D1 dopamine (DA) receptors. However, a significant caveat to the studies investigating the role of the receptor in such toxicity is that genetic and pharmacological manipulations of the D1 DA receptor also mitigate METH-induced hyperthermia. Importantly, METH-induced hyperthermia is tightly associated with the neurotoxicity, such that simply cooling animals during METH exposure protects against the neurotoxicity. Therefore, it is difficult to determine whether D1 DA receptors per se play an important role in METH-induced neurotoxicity or whether the protection observed simply resulted from a mitigation of METH-induced hyperthermia. To answer this important question, the current study infused a D1 DA receptor antagonist into striatum during METH exposure while controlling for METH-induced hyperthermia. Here we found that even when METH-induced hyperthermia is maintained, the coadministration of a D1 DA receptor antagonist protects against METH-induced neurotoxicity, strongly suggesting that D1 DA receptors play an important role in METH-induced neurotoxicity apart from the mitigation of METH-induced hyperthermia.

Methamphetamine (METH) abuse results in long-term damage to the dopaminergic system, manifesting as decreases in dopamine (DA) tissue content, DA transporter (DAT) binding, as well as tyrosine hydroxylase (TH) and vesicular monoamine transporter (VMAT) immunostaining. However, the exact cascade of events that ultimately results in this damage has not been clearly elucidated. One factor that has been heavily implicated in METH-induced DA terminal degeneration is the production of nitric oxide (NO). Unfortunately, many of the studies attempting to clarify the role of NO in METH-induced neurotoxicity have been confounded by issues such as the disruption of METH-induced hyperthermia, preventing the formation of strong conclusions. As a result, there is a body of work suggesting that NO is sufficient for METH-induced neurotoxicity, while other studies suggest that NO does not play a role in METH-induced degeneration of DA nerve terminals. This review summarizes the existing studies investigating the role of NO in METH-induced neurotoxicity, and argues that while NO may be necessary for METH-induced neurotoxicity, it is not sufficient. Finally, important areas of future investigation are highlighted and discussed.

Methamphetamine-induced partial dopamine depletions are associated with impaired basal ganglia function, including decreased preprotachykinin mRNA expression and impaired transcriptional activation of activity-regulated, cytoskeleton-associated (Arc) gene in striatum. Recent work implicates deficits in phasic dopamine signaling as a potential mechanism linking methamphetamine-induced dopamine loss to impaired basal ganglia function. The present study thus sought to establish a causal link between phasic dopamine transmission and altered basal ganglia function by determining whether the deficits in striatal neuron gene expression could be restored by increasing phasic dopamine release. Three weeks after pretreatment with saline or a neurotoxic regimen of methamphetamine, rats underwent phasic- or tonic-like stimulation of ascending dopamine neurons. Striatal gene expression was examined using in situ hybridization histochemistry. Phasic-like, but not tonic-like, stimulation induced immediate-early genes Arc and zif268 in both groups, despite the partial striatal dopamine denervation in methamphetamine-pretreated rats, with the Arc expression occurring in presumed striatonigral efferent neurons. Phasic-like stimulation also restored preprotachykinin mRNA expression. These results suggest that disruption of phasic dopamine signaling likely underlies methamphetamine-induced impairments in basal ganglia function, and that restoring phasic dopamine signaling may be a viable approach to manage long-term consequences of methamphetamine-induced dopamine loss on basal ganglia functions.

Neurotoxic regimens of methamphetamine (METH) result in reactive microglia and astrocytes in striatum. Prior data indicate that rats with partial dopamine (DA) loss resulting from prior exposure to METH are resistant to further decreases in striatal DA when re-exposed to METH 30 days later. Such resistant animals also do not show an activated microglia phenotype, suggesting a relation between microglial activation and METH-induced neurotoxicity. To date, the astrocyte response in such resistance has not been examined. Thus, this study examined glial-fibrillary acidic protein (GFAP) and CD11b protein expression in striata of animals administered saline or a neurotoxic regimen of METH on post-natal days 60 and/or 90 (Saline:Saline, Saline:METH, METH:Saline, METH:METH). Consistent with previous work, animals experiencing acute toxicity (Saline:METH) showed both activated microglia and astocytes, whereas those resistant to the acute toxicity (METH:METH) did not show activated microglia. Interestingly, GFAP expression remained elevated in rats exposed to METH at PND60 (METH:Saline), and was not elevated further in resistant rats treated for the second time with METH (METH:METH). These data suggest that astrocytes remain reactive up to 30 days post-METH exposure. In addition, these data indicate that astrocyte reactivity does not reflect acute, METH-induced DA terminal toxicity, whereas microglial reactivity does.

Drug-seeking behavior elicited by drug-associated cues contributes to relapse in addiction; however, whether relapse elicited by drug-associated conditioned reinforcers (CR) versus discriminative stimuli (DS) involves distinct or overlapping neuronal populations is unknown. To address this question, we developed a novel cocaine self-administration and cue-induced reinstatement paradigm that exposed the same rats to distinct cocaine-associated CR and DS. Rats were trained to self-administer cocaine in separate sessions. In one, a DS signaled cocaine availability; in the other, cocaine delivery was paired with a different CR. After extinction training and reinstatement testing, where both cues were presented in separate sessions, rats were sacrificed and processed for cellular analysis of temporal activity by fluorescent in situ hybridization (CatFISH) for activity regulated cytoskeleton-associated protein (Arc) mRNA and for radioactive in situ hybridization for Arc and zif268 mRNAs. CatFISH did not reveal significant changes in Arc mRNA expression. Similar results were obtained with radioactive in situ hybridization. We have shown that while rats reinstate drug seeking in response to temporally discrete presentations of distinct drug-associated cues, such reinstatement is not associated with increased transcriptional activation of Arc or zif268 mRNAs, suggesting that expression of these genes may not be necessary for cue-induced reinstatement of drug-seeking behavior.

D1- and D2-dopamine receptor-mediated regulation of immediate early gene levels in identified populations of neurons in the striatum was examined with quantitative in situ hybridization histochemical techniques. Levels of messenger RNA (mRNA) encoding the immediate early genes zif268 and c-fos were examined in two experiments in rats with unilateral lesions of the nigrostriatal dopamine pathway. In a dose- response study, animals were treated with doses of 0.5, 1.0, and 1.5 mg/kg of the D1 agonist SKF-38393 either alone or in combination with the D2 agonist quinpirole (1 mg/kg). Levels of immediate early gene mRNAs 60 min following drug treatments showed a dose-related increase to the D1 agonist alone and a potentiation to combined D1 and D2 against treatment. In a second experiment, in animals receiving 1 mg/kg SKF-38393 either alone or in combination with 1 mg/kg quinpirole, the level of zif268 mRNA was measured with a double-labeling method in striatal neurons containing enkephalin mRNA, a marker of D2-containing neurons, and in neurons not containing enkephalin, putative D1- containing neurons. In the dopamine-depleted striatum, D1 agonist treatment alone did not affect enkephalin-positive neurons but significantly elevated zif268 mRNA levels in nearly all enkephalin- negative neurons. Combined D1 and D2 agonist treatment further increased zif268 mRNA levels in this population of enkephalin-negative neurons and decreased zif-268 mRNA levels in enkephalin-positive neurons. These data indicate that the synergistic response to combined D1- and D2-receptor stimulation is mediated by interneuronal interactions involving the activation of D1 and D2 receptors on separate populations of striatal neurons.

Advances in basic neuroscience have increased our knowledge about the neural processes underlying learning and memory and the cortical reorganization that occurs in response to environmental demands and cortical injury. This article provides a selective review of published studies conducted in animals that examine functional and structural substrates of neural plasticity in the adult mammalian brain, and discusses the implications of this knowledge for aphasia therapy. The processes and constraints identified in the studies reviewed can be used to refine and justify current aphasia therapies, as well as to design additional behavioral interventions.

Nitric oxide is implicated in methamphetamine (METH)-induced neurotoxicity; however, the source of the nitric oxide has not been identified. Previous work has also revealed that animals with partial dopamine loss induced by a neurotoxic regimen of methamphetamine fail to exhibit further decreases in striatal dopamine when re-exposed to methamphetamine 7–30 days later. The current study examined nitric oxide synthase expression and activity and protein nitration in striata of animals administered saline or neurotoxic regimens of methamphetamine at postnatal days 60 and/or 90, resulting in four treatment groups: Saline:Saline, METH:Saline, Saline:METH, and METH:METH. Acute administration of methamphetamine on postnatal day 90 (Saline:METH and METH:METH) increased nitric oxide production, as evidenced by increased protein nitration. Methamphetamine did not, however, change the expression of endothelial or inducible isoforms of nitric oxide synthase, nor did it change the number of cells positive for neuronal nitric oxide synthase mRNA expression or the amount of neuronal nitric oxide synthase mRNA per cell. However, nitric oxide synthase activity in striatal interneurons was increased in the Saline:METH and METH:METH animals. These data suggest that increased nitric oxide production after a neurotoxic regimen of methamphetamine results from increased nitric oxide synthase activity, rather than an induction of mRNA, and that constitutively expressed neuronal nitric oxide synthase is the most likely source of nitric oxide after methamphetamine administration. Of interest, animals rendered resistant to further methamphetamine-induced dopamine depletions still show equivalent degrees of methamphetamine-induced nitric oxide production, suggesting that nitric oxide production alone in response to methamphetamine is not sufficient to induce acute neurotoxic injury.