Acute Amphetamine Treatment Research Articles

Abdominal Vagal Afferents Modulate the Brain Transcriptome and Behaviors Relevant to Schizophrenia.

Reduced activity of vagal efferents has long been implicated in schizophrenia and appears to be responsible for diminished parasympathetic activity and associated peripheral symptoms such as low heart rate variability and cardiovascular complications in affected individuals. In contrast, only little attention has been paid to the possibility that impaired afferent vagal signaling may be relevant for the disorder's pathophysiology as well. The present study explored this hypothesis using a model of subdiaphragmatic vagal deafferentation (SDA) in male rats. SDA represents the most complete and selective vagal deafferentation method existing to date as it leads to complete disconnection of all abdominal vagal afferents while sparing half of the abdominal vagal efferents. Using next-generation mRNA sequencing, we show that SDA leads to brain transcriptional changes in functional networks annotating with schizophrenia. We further demonstrate that SDA induces a hyperdopaminergic state, which manifests itself as increased sensitivity to acute amphetamine treatment and elevated accumbal levels of dopamine and its major metabolite, 3,4-dihydroxyphenylacetic acid. Our study also shows that SDA impairs sensorimotor gating and the attentional control of associative learning, which were assessed using the paradigms of prepulse inhibition and latent inhibition, respectively. These data provide converging evidence suggesting that the brain transcriptome, dopamine neurochemistry, and behavioral functions implicated in schizophrenia are subject to visceral modulation through abdominal vagal afferents. Our findings may encourage the further establishment and use of therapies for schizophrenia that are based on vagal interventions.SIGNIFICANCE STATEMENT The present work provides a better understanding of how disrupted vagal afferent signaling can contribute to schizophrenia-related brain and behavioral abnormalities. More specifically, it shows that subdiaphragmatic vagal deafferentation (SDA) in rats leads to (1) brain transcriptional changes in functional networks related to schizophrenia, (2) increased sensitivity to dopamine-stimulating drugs and elevated dopamine levels in the nucleus accumbens, and (3) impairments in sensorimotor gating and the attentional control of associative learning. These findings may encourage the further establishment of novel therapies for schizophrenia that are based on vagal interventions.

Effects of amphetamine and methylphenidate on attentional performance and impulsivity in the mouse 5-Choice Serial Reaction Time Task.

Few studies have investigated the effects of conventional attention deficit-hyperactivity disorder (ADHD) medication in the mouse 5-choice serial reaction time task (5-CSRTT), and rat studies have yielded inconsistent results. We aimed to examine the effects of acute methylphenidate (MPH) and amphetamine (AMPH) treatment in the mouse 5-CSRTT. Trained male C57Bl/6J mice were tested in a variable stimulus duration schedule. Effects of AMPH (0.25, 0.5, and 1 mg/kg) and MPH (0.5, 1.0, and 2.0 mg/kg) on discriminative accuracy, omissions, and premature responses were assessed. Saline treatment data determined high- and low-attentive (LA), and high- and low-impulsive (LI) subgroups according to the upper and lower 30th percentiles, respectively. In the LA subgroup accuracy was improved by 0.5 mg/kg AMPH and 2 mg/kg MPH, while no effect was found in the high-attentive (HA) subgroup. Premature responses were increased by 1 mg/kg AMPH and 0.5 mg/kg MPH for all animals, and by 1 mg/kg AMPH for the LI subgroup. The use of variable stimulus duration, along with the division into high- and LA, and high-and LI subgroups, may improve the sensitivity of the 5-CSRTT when investigating drug effects on attention and impulsivity.

Opposite effects of acute and chronic amphetamine on Nurr1 and NF-κB p65 in the rat ventral tegmental area

Dopamine neurons are overstimulated by drugs of abuse and suffer molecular alterations that lead to addiction behavior. Nurr1 is a transcription factor crucial for dopamine neurons survival and dopamine production, activating the transcription of key genes like tyrosine hydroxylase (TH). Interestingly, nuclear factor-kappa B (NF-κB) has emerged as a new Nurr1 partner in response to inflammatory stimulus. In this study we evaluated the effects of single and repeated amphetamine administration in the expression of Nurr1 and the NF-κB p65 subunit in the rat ventral tegmental area (VTA). We found that acute amphetamine treatment increased Nurr1, p65 and TH protein levels in the VTA. On the other hand, chronic amphetamine treatment decreased Nurr1 and p65 protein levels, but TH was unchanged. Mammalian reporter assays in cell lines showed that p65 represses Nurr1 transcriptional activity in an artificial promoter driven by Nurr1 response elements and in the native rat TH promoter. These results indicate that Nurr1 and NF-κB p65 factors are involved in the adaptive response of dopamine neurons to psychostimulants and that both transcription factors could be regulating Nurr1-dependent transactivation in the VTA.

Vitamin D3: A Role in Dopamine Circuit Regulation, Diet-Induced Obesity, and Drug Consumption.

The influence of micronutrients on dopamine systems is not well defined. Using mice, we show a potential role for reduced dietary vitamin D3 (cholecalciferol) in promoting diet-induced obesity (DIO), food intake, and drug consumption while on a high fat diet. To complement these deficiency studies, treatments with exogenous fully active vitamin D3 (calcitriol, 10 µg/kg, i.p.) were performed. Nondeficient mice that were made leptin resistant with a high fat diet displayed reduced food intake and body weight after an acute treatment with exogenous calcitriol. Dopamine neurons in the midbrain and their target neurons in the striatum were found to express vitamin D3 receptor protein. Acute calcitriol treatment led to transcriptional changes of dopamine-related genes in these regions in naive mice, enhanced amphetamine-induced dopamine release in both naive mice and rats, and increased locomotor activity after acute amphetamine treatment (2.5 mg/kg, i.p.). Alternatively, mice that were chronically fed either the reduced D3 high fat or chow diets displayed less activity after acute amphetamine treatment compared with their respective controls. Finally, high fat deficient mice that were trained to orally consume liquid amphetamine (90 mg/L) displayed increased consumption, while nondeficient mice treated with calcitriol showed reduced consumption. Our findings suggest that reduced dietary D3 may be a contributing environmental factor enhancing DIO as well as drug intake while eating a high fat diet. Moreover, these data demonstrate that dopamine circuits are modulated by D3 signaling, and may serve as direct or indirect targets for exogenous calcitriol.

Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine

Acute amphetamine (AMPH) exposure elevates extracellular dopamine through a variety of mechanisms that include inhibition of dopamine reuptake, depletion of vesicular stores, and facilitation of dopamine efflux across the plasma membrane. Recent work has shown that the DAT substrate AMPH, unlike cocaine and other nontransported blockers, can also stimulate endocytosis of the plasma membrane dopamine transporter (DAT). Here, we show that when AMPH enters the cytoplasm it rapidly stimulates DAT internalization through a dynamin-dependent, clathrin-independent process. This effect, which can be observed in transfected cells, cultured dopamine neurons, and midbrain slices, is mediated by activation of the small GTPase RhoA. Inhibition of RhoA activity with C3 exotoxin or a dominant-negative RhoA blocks AMPH-induced DAT internalization. These actions depend on AMPH entry into the cell and are blocked by the DAT inhibitor cocaine. AMPH also stimulates cAMP accumulation and PKA-dependent inactivation of RhoA, thus providing a mechanism whereby PKA- and RhoA-dependent signaling pathways can interact to regulate the timing and robustness of AMPH's effects on DAT internalization. Consistent with this model, the activation of D1/D5 receptors that couple to PKA in dopamine neurons antagonizes RhoA activation, DAT internalization, and hyperlocomotion observed in mice after AMPH treatment. These observations support the existence of an unanticipated intracellular target that mediates the effects of AMPH on RhoA and cAMP signaling and suggest new pathways to target to disrupt AMPH action.

Brain Region-Specific Trafficking of the Dopamine Transporter.

The dopamine transporter (DAT) is the key regulator of the dopamine neurotransmission in the CNS. In the present study, we developed a new approach for studying DAT localization and dynamics in intact neurons in acute sagittal brain slices from the knock-in mouse expressing epitope-tagged DAT. For the first time, the fluorescence imaging analysis of DAT was combined with the immunogold labeling of DAT and quantitative electron microscopy. In contrast to numerous studies of DAT trafficking in heterologous expression systems and dissociated cultured neurons, studies in intact neurons revealed a surprisingly low amount of endocytic trafficking of DAT at steady state and after acute amphetamine treatment and suggested that non-vesicular transport could be the main mechanism establishing DAT distribution within the dopaminergic neuron.

Protein Phosphatases but not Reactive Oxygen Species Play Functional Role in Acute Amphetamine-Mediated Dopamine Release

Drug abuse-induced neurodegeneration can be triggered by elevated production of reactive oxygen species (ROS). Involvement of oxidative stress in acute amphetamine (AMPH)-mediated dopamine (DA) release, however, has not been completely understood yet. In order to elucidate the dopaminergic response of PC12 cells to a single dose of 10 μM AMPH, ROS production was measured as related to the extracellular DA level. Due to the spontaneous oxidation of peroxide-sensitive fluorophore 2',7'-dichlorofluorescin diacetate (DCFH-DA) to 2',7'-dichlorofluorescein (DCF), the increase in fluorescence could not be unambiguously attributed to AMPH-triggered ROS production. Based on Amplex Red fluorescence, no ROS production was detected after acute AMPH application. Our data strongly suggest that ROS development was not the main triggering factor for immediate DA release after acute AMPH treatment. On the other hand, AMPH-induced elevation of DA levels in rat brain striatal slices was quenched by the water soluble antioxidant, N-acetylcysteine (NAC) at 10 mM. In this study, we also investigated the contribution of protein phosphatases to the AMPH-induced rat brain striatal dopaminergic response. The experimental protocol, double AMPH challenge was applied for screening the effect of NAC and cantharidin on AMPH-mediated DA release. Here we show that AMPH-mediated DA release increased nearly twofold in striatal rat brain slices pretreated for 30 min with 1000 μM cantharidin, a selective PP1 and PP2A inhibitor. These findings prove the lack of ROS inhibitory action on protein phosphatase activity in acute AMPH-mediated DA efflux.

Differential effects of acute amphetamine and phencyclidine treatment and withdrawal from repeated amphetamine or phencyclidine treatment on social interaction and social memory in rats

Although animal models based on amphetamine (AMPH) or phencyclidine (PCP) treatment have been used extensively to study the neurobiological and behavioral characteristics of schizophrenia, there are conflicting reports regarding their validity in modeling the negative symptoms and cognitive deficits of schizophrenia. The present study examined how acute AMPH or PCP treatment (Experiment 1) and withdrawal from repeated AMPH treatment (Experiment 2) or PCP treatment (Experiment 3) affects social behavior and social recognition memory in male Sprague-Dawley rats. Each subject was tested on two consecutive days. On the first day, the rats were tested four times (5 min/each) at 10-min intervals with the same partner rat (termed "AAAA" day). One day later, the rats were tested with the previous partner in the first three sessions and with a new partner rat in the final session (termed "AAAB" day). The results show that acute AMPH treatment (1.5 mg/kg, sc) significantly reduced the time spent on social interaction, but did not affect social recognition on the first day. Acute AMPH only disrupted social recognition on the second day of drug testing. In contrast, acute PCP treatment (2.0 mg/kg, sc) had no effect on time spent on social interaction, but did significantly disrupt social recognition on both days. Withdrawal from repeated AMPH (3.0 mg/kg/day for 7 days, ip) or PCP (5.0 mg/kg/twice daily for 7 days, ip) treatment did not affect social interaction or social recognition, indicating a lack of long-term detrimental effect of repeated AMPH or PCP treatment. These results suggest that acute AMPH treatment at a low dose (1.5 mg/kg) may be useful in modeling social withdrawal symptoms of schizophrenia, whereas acute PCP treatment at a similar dose range (2.0 mg/kg) may be useful in modeling the social cognitive deficit of schizophrenia.

Maternal immune activation in late gestation enhances locomotor response to acute but not chronic amphetamine treatment in male mice offspring: Role of the D1 receptor

Exposure to elevated levels of maternal cytokines can lead to functional abnormalities of the dopaminergic system in the adult offspring, including enhanced amphetamine (AMPH)-induced locomotion. Therefore, it seems reasonable to consider that offspring of challenged mothers would behave differently in models of addictive behavior, such as behavioral sensitization. Thus, we sought to evaluate the effects of prenatal exposure to lipopolysaccharide (LPS) on the locomotor response to acute and chronic AMPH treatment in male mice offspring. For this purpose, LPS (Escherichia coli 0127:B8; 120μg/kg) was administered intraperitoneally to pregnant Swiss mice on gestational day 17. At adulthood, male offspring were studied under one of the following conditions: (1) locomotor response to acute AMPH treatment (2.5 or 5.0mg/kg) in an open field test; (2) behavioral sensitization paradigm, which consists of a daily injection of AMPH (1.0mg/kg) for 10 days and observation of locomotion in the open field on days 1, 5, 10 (development phase), 15 and 17 (expression phase). The LPS stimulated offspring showed enhancement of the locomotor-stimulant effect after an acute AMPH challenge in comparison to baseline and saline pre-treated mice. They also showed development of behavioral sensitization earlier than the saline pre-treated group, although no changes between saline and LPS pre-treated groups were observed on development or expression of locomotor behavioral sensitization to AMPH. Furthermore, there was up-regulation of D1 receptor protein level within striatum in the LPS-stimulated offspring which was strongly correlated with increased grooming behavior. Taken together, our results indicate that motor and dopaminergic alterations caused by maternal immune activation are restricted to the acute AMPH challenge, mostly due to up-regulation of the D1 receptor within the mesolimbic and nigrostriatal pathways, but no locomotor differences were observed for behavioral sensitization to AMPH.

Brain region differences in regulation of Akt and GSK3 by chronic stimulant administration in mice

Acute amphetamine administration activates glycogen synthase kinase-3 (GSK3) by reducing its inhibitory serine-phosphorylation in mouse striatum and cerebral cortex. This results from Akt inactivation and is required for certain behavioral effects of amphetamine, such as increased locomotor activity. Here we tested if regulation of Akt and GSK3 was similarly affected by longer-term administration of amphetamine, as well as of methylphenidate, since each of these is administered chronically in patients with attention deficit hyperactivity disorder (ADHD). Akt is activated by post-translational phosphorylation on Thr308, and modulated by Ser473 phosphorylation, whereas phosphorylation on Ser21/9 inhibits the two GSK3 isoforms, GSK3α and GSK3β. After eight days of amphetamine or methylphenidate treatment, striatal Akt and GSK3 were dephosphorylated similar to reported changes after acute amphetamine treatment. Oppositely, in the cerebral cortex and hippocampus Akt and GSK3 phosphorylation increased after eight days of amphetamine or methylphenidate treatment. These opposite brain region changes in Akt and GSK3 phosphorylation matched opposite changes in the association of Akt with β-arrestin and GSK3, which after eight days of amphetamine treatment were increased in the striatum and decreased in the cerebral cortex. Thus, whereas the acute dephosphorylating effect of stimulants on Akt and GSK3 in the striatum was maintained, the response switched in the cerebral cortex after eight days of amphetamine or methylphenidate treatment to cause increased phosphorylation of Akt and GSK3. These results demonstrate that prolonged administration of stimulants causes brain region-selective differences in the regulation of Akt and GSK3.

Amphetamine modulation of long‐term potentiation in the prefrontal cortex: dose dependency, monoaminergic contributions, and paradoxical rescue in hyperdopaminergic mutant

Amphetamine can improve cognition in healthy subjects and patients with schizophrenia, attention-deficit hyperactivity disorder, and other neuropsychiatric diseases; higher doses, however, can impair cognitive function, especially those mediated by the prefrontal cortex. We investigated how amphetamine affects prefrontal cortex long-term potentiation (LTP), a cellular correlate of learning and memory, in normal and hyperdopaminergic mice lacking the dopamine transporter. Acute amphetamine treatment in wild-type mice produced a biphasic dose-response modulation of LTP, with a low dose enhancing LTP and a high dose impairing it. Amphetamine-induced LTP enhancement was prevented by pharmacological blockade of D(1) - (but not D(2)-) class dopamine receptors, by blockade of β-adrenergic receptors, or by inhibition of cAMP-PKA signaling. In contrast, amphetamine-induced LTP impairment was prevented by inhibition of post-synaptic protein phosphatase-1, a downstream target of PKA signaling, or by blockade of either D(1) - or D(2)-class dopamine, but not noradrenergic, receptors. Thus, amphetamine biphasically modulates LTP via cAMP-PKA signaling orchestrated mainly through dopamine receptors. Unexpectedly, amphetamine restored the loss of LTP in dopamine transporter-knockout mice primarily by activation of the noradrenergic system. Our results mirror the biphasic effectiveness of amphetamine in humans and provide new mechanistic insights into its effects on cognition under normal and hyperdopaminergic conditions.

Differential laminar effects of amphetamine on prefrontal parvalbumin interneurons

The increase in excitatory outflow from the medial prefrontal cortex is critical to the development of sensitization to amphetamine. There is evidence that psychostimulant-induced changes in dopamine–GABA interactions are key to understanding the behaviorally sensitized response. The objective of this study was to characterize the effects of different amphetamine paradigms on the Fos activation of GABAergic interneurons that contain parvalbumin in the medial prefrontal cortex. Although a sensitizing, repeated regimen of amphetamine induced Fos in all cortical layers, only layer V parvalbumin-immunolabeled cells were activated in the infralimbic and prelimbic cortices. Repeated amphetamine treatment was also associated with a loss of parvalbumin immunoreactivity in layer V, but only in the prelimbic cortex. An acute amphetamine injection to naive rats was associated with an increase in Fos, but in parvalbumin-positive neurons of the prelimbic cortex, where it was preferentially induced in layer III. These data indicate that distinct substrates mediate the response to repeated or acute amphetamine treatment. They also suggest that a sensitizing amphetamine regimen directs medial prefrontal cortex (mPFC) outflow, via changes in inhibitory neuron activation, toward subcortical centers important in reward.

Omega-3 fatty acid deficiency augments amphetamine-induced behavioral sensitization in adult mice: Prevention by chronic lithium treatment

Objectives Emerging data suggests that omega-3 fatty acid deficiency may be a risk factor for bipolar disorder. In the present study, we determined the effects of chronic dietary-induced omega-3 fatty acid deficiency and/or concomitant chronic lithium chloride (LiCl) treatment on amphetamine (AMPH)-induced behavioral sensitization, a phenomenon that may recruit neuroplastic mechanisms relevant to the pathophysiology of bipolar disorder. Method Adult male C57BL/6J mice were randomly assigned to one four diets: Control (α-linolenic-fortified), Control + LiCl (0.255%), α-linolenic-Deficient, or Deficient + LiCl (0.255%), and behavioral testing initiated 65 days later. Locomotor activity was determined following 3 intermittent (separated by 7 d) injections of amphetamine (AMPH) (1 mg/kg). After behavioral testing, red blood cell (RBC) and regional brain (prefrontal cortex, hippocampus, ventral striatum) fatty acid composition was determined by gas chromatography. Results Each diet group exhibited comparable locomotor activity following acute AMPH treatment. However, the development of sensitization following repeated AMPH treatment was significantly augmented in Deficient mice relative to controls, and this augmented response was prevented by chronic LiCl treatment. Relative to controls, Deficient mice exhibited deficits in RBC and regional brain docosahexaenoic acid (DHA, 22:6 n−3) composition, reciprocal elevations in vaccenic acid (18:1 n−7), arachidonic acid (AA, 20:4 n−6), and docosapentaenoic acid (DPA, 22:5 n−6) compositions, and elevations in AA:DHA, oleic acid:DHA, and DPA:DHA ratios. The fatty acid abnormalities in Deficient mice were not altered by concurrent chronic lithium treatment. Mice fed the Control + LiCl diet exhibited a significant increase in AA composition in RBC and all brain regions, and an elevated AA:DHA ratio in the prefrontal cortex and hippocampus, relative to Controls. Fatty acid composition in RBC and different brain regions were predominantly positively correlated. Within the ventral striatum, DHA composition was inversely correlated, and AA:DHA and oleic acid:DHA ratios positively correlated, with total distance traveled following the final AMPH treatment. Conclusion These data indicate that alterations in fatty acid composition resulting from dietary-induced omega-3 fatty acid deficiency augment the development of AMPH-induced behavioral sensitization in a manner that is prevented by chronic lithium treatment. The implications of these findings for understanding the contribution of omega-3 fatty acid deficiency to the pathophysiology and progression of bipolar disorder are discussed.

Acute amphetamine treatment decreases the expression of 180–200 kDa isoform of polysialic acid linked neural cell adhesion molecule in mouse hippocampus

The behavioral sensitization produced by repeated treatment with amphetamine may represent neural adaptations underlying some of the features of psychosis and addiction in humans. Under some circumstances, learning contextual cues can gain powerful control over the ability of the sensitized neural substrate to influence behavior. Here, we investigated the expression levels of a neural cell adhesion molecule (NCAM) and a polysialylated form of the neuronal cell adhesion molecule (PSA-NCAM) as markers of synaptic plasticity, in the associative learning mechanisms related to behavioral sensitization. To achieve our goal we examined the effects of amphetamine treatment on the expression levels of PSA-NCAM and NCAM in mouse hippocampus, cortex and striatum in a context-specific behavioral sensitization model. We found that amphetamine (2.0 mg/kg, i.p.) produced robust behavioral sensitization and the expression of sensitization after the saline challenge was context-dependent. Immunoblotting analysis demonstrated that acute administration of amphetamine selectively and time-dependently decreases the expression of 180–200 kDa isoform of PSA-NCAM in hippocampus in both context associated (the Paired) as well as context non-associated (the Unpaired) groups. Thus, our results suggest that acute amphetamine administration time-dependently decreases the expression of 180–200 kDa isoform of PSA-NCAM in mouse hippocampus and PSA-NCAM is not involved in amphetamine-induced associated learning mechanism.

Increased oxidative stress in submitochondrial particles after chronic amphetamine exposure

Previous studies have suggested that reactive oxygen species (ROS) production may play a role in the pathophysiology of many neuropsychiatric disorders, such as bipolar disorder (BD) and schizophrenia (SCZ). In addition, there is an emerging body of data indicating that BD and SCZ may be associated with mitochondrial dysfunction. We studied the effects of acute and chronic d-amphetamine on ROS production in submitochondrial particles of rat brain. Male Wistar rats were divided in two experimental groups: acute and chronic treatment. In the acute treatment, rats received one single IP injection of d-amphetamine (1, 2 or 4 mg/kg) or saline (control group). In the chronic treatment, rats received one daily IP injection of d-amphetamine (1, 2 or 4 mg/kg) or saline for 7 days. Locomotor activity was assessed with the open field task, and thiobarbituric acid reactive substances (TBARS) and superoxide production were measured in submitochondrial particles of the prefrontal cortex and hippocampus. Both acute and chronic amphetamine treatment increased locomotor behavior. Chronic amphetamine exposure induced a 3- to 6-fold increase of TBARS and a 1.5- to 2-fold increase of superoxide production in submitochondrial particles of prefrontal cortex and hippocampus ( P < 0.05). No effects on superoxide or TBARS were observed with acute treatment. These findings suggest that amphetamine-induced mitochondrial ROS generation may be a useful model to investigate the hypothesis of altered brain energy metabolism associated with BD and SCZ. Further studies assessing the effects of mood stabilizers and antipsychotics in preventing mitochondrial oxidative stress are necessary.

Regulation of genes involved in dopamine transporter modulation by acute cocaine in rat striatum

It is well established that acute administration of psychostimulants alters dopamine transport. However, the exact mechanism of this modulation is still unknown. In this study we examined the mRNA levels of several proteins involved in the various proposed processes following cocaine administration. The expression levels of several immediate early genes were also studied. This was performed in rat striatum using real-time quantitative PCR. As expected, a marked increase of the immediate early genes Fos, Egr1 and Egr3 was observed. Egr2 was also found up-regulated. Among the different genes studied only Synaptotagmin4 in the SNARE family and Synphilin1 in the synaptic vesicles binding family were modulated by acute cocaine treatment. Interestingly, acute amphetamine treatment did not increase either Synaptotagmin4 and Synphilin1 mRNA levels, although increases in early genes expression were noted.

Effects of Amphetamine on Immune-Mediated Lung Inflammatory Response in Rats

Objective: The present study analyzed the effects of acute amphetamine (AMPH) treatment on immune-mediated lung inflammatory response in rats. Methods: There were four experiments. In the first and second experiments, rats were treated with AMPH (1 mg/kg) or 0.9% NaCl, and locomotor activity (experiment 1) and serum AMPH concentrations (experiment 2) were measured 1 or 12 h after treatment. In the third experiment, rats which were immunized with ovalbumin (OVA) were treated 14 days later with 0.9% NaCl or AMPH (1 mg/kg). Twelve hours after these treatments, all animals were submitted to challenge by 1% OVA inhalation being analyzed afterwards for bronchoalveolar lavage fluid (BAL), peripheral blood and bone marrow cellularity. In the fourth and final experiment, rats were treated and studied as for experiment 3, except that half of the animals within each group were previously treated with metyrapone prior to the OVA challenge. Results: In the non- immunized rats, AMPH treatment induced an increase in locomotor activity synchronized to high serum AMPH concentrations 1 h after, but not 12 h after treatment. In OVA-challenged rats, AMPH treatment decreased the total number of inflammatory cells, recovered in both BAL and peripheral blood and increased the total number of bone marrow cells. These effects, observed 1 day after OVA challenge, were abrogated by previous metyrapone treatment. Conclusion: AMPH treatment changed HPA-axis responsiveness to the stress condition imposed by the OVA challenge decreasing lung and blood leukocytes cellularity most probably via corticosterone actions on bone marrow activity.

Cortical-subcortical dysconnectivity expressed by attenuated cortical c-fos induction by acute amphetamine treatment or acute swim stress in mice with reduced NMDA receptor1 expression

Reelin is a large glycoprotein that is secreted into the extracellular matrix. In the embryonic brain, the binding of Reelin to its receptors ApoER2 and VLDLR induces subcellular events that include the activation Fyn tyrosine kinase, and plays a crucial role in cortical formation. Reelin signaling is also involved in postnatal brain functions such as dendrite development and synaptic plasticity. However, the molecular events involved in Reelin signaling in the postnatal brain remain to be elucidated. Here, we evaluated the proteins downstream of Reelin signaling by comparing the tyrosine-phosphorylated proteins in the postnatal hippocampus of heterozygous and homozygous reeler and wild-type mice, by Western blot analyses. We found that the levels of several phosphoproteins were highest in the hippocampus of the heterozygous reeler mice. The most prominent increase was of two 180-kDa phosphoproteins, which were identified as the NR2A and NR2B subunits of NMDA-R. The amounts of these proteins also increased in the hippocampus of heterozygous reeler mice. However, the mRNA levels of the NMDA-R subunits, determined by quantitative RT-PCR, were the same as in wild-type mice. We also found that the increase in NR2A and NR2B proteins in heterozygous reeler was dependent on Fyn, because this change was absent in heterozygous reeler/homozygous Fyn-deficient double-mutant mice. Thus, the NMDA-R protein level is regulated by the Reelin protein level in a Fyn-dependent manner in the mouse brain.

Amphetamine administration does not alter protein levels of the GLT-1 and EAAC1 glutamate transporter subtypes in rat midbrain, nucleus accumbens, striatum, or prefrontal cortex

Our laboratory and others have previously shown that glutamate transmission is required for chronic amphetamine-induced neuroadaptations, and that glutamate transmission itself is altered by chronic amphetamine administration. For example, N-methyl- d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor subunit expression are altered in a region- and withdrawal-specific manner. The goal of this study was to determine whether repeated amphetamine administration influences the expression of two glutamate transporter subtypes, GLT-1 and EAAC1. Rats were treated with saline or 5 mg/kg amphetamine for 5 days (chronic saline and amphetamine groups, respectively), or saline for 4 days and 5 mg/kg amphetamine on day 5 (acute amphetamine group), and decapitated 24 h after the last injection. Tissue was dissected from brain regions involved in the psychomotor effects of amphetamine (nucleus accumbens, striatum, prefrontal cortex, ventral tegmental area, and substantia nigra). Levels of GLT-1 and EAAC1 were quantified by Western blotting and normalized to actin levels. We found no significant change in levels of GLT-1 or EAAC1 in response to either acute or chronic amphetamine treatment. These findings suggest that the transporter component of the glutamate system might not play a significant role in the alterations in glutamate transmission observed following repeated amphetamine administration.

Rat strain differences in responses of plasma prolactin and PRL mRNA expression after acute amphetamine treatment or restraint stress.

1. The aim of this study was to compare the effects of acute amphetamine (AMPH) treatment and restraint stress on plasma level of prolactin (PRL) and PRL mRNA expression in the adenohypophysis in Sprague-Dawley and Lewis male rats, the latter known to have a deficient hypothalamo-pituitary-adrenal (HPA) axis. 2. Both restraint stress and AMPH treatment (i.p. in a dose of 8 mg/kg of b.w.) were applied 15 or 30 min before termination of the experiment. Plasma PRL and corticosterone (CORT) were determined by radioimmunoassay. PRL mRNA expression was estimated by a dot-blot hybridization. 3. Restraint stress and AMPH treatment induced a significant increase in the CORT plasma level, as an indicator of stress response. Compared to Sprague-Dawley rats, the magnitude of CORT increase after both stimuli was significantly lower in Lewis rats. 4. Although restraint stress significantly increased the PRL plasma levels in both rat strains, AMPH treatment reduced the PRL levels in both rat strains. However, the changes of PRL plasma levels had another pattern in Lewis rats than in Sprague-Dawley rats. Control plasma PRL levels were significantly higher in Lewis rats, and in this rat strain AMPH treatment for 30 min increased the PRL levels as compared to the values obtained after AMPH treatment for 15 min. 5. Expression of PRL mRNA in adenohypophysis by restraint stress and AMPH treatment had a similar pattern. After a 15-min lasting restraint stress, the expression of PRL mRNA was decreased insignificantly in both rat strains. AMPH treatment induced in Sprague-Dawley rats a significant decrease of PRL mRNA after a 15-min interval while after 30 min there was a significant increase. However, in Lewis rats AMPH failed to significantly change PRL mRNA. 6. The results from the present study indicate that the mechanisms mediating the effects of acute restraint stress and acute AMPH treatment differ in PRL response in Sprague-Dawley and Lewis male rat strains. Differences in the observed responses in Lewis rats could be related to the deficient activity of HPA axis in this rat strain.