Effects of a positive allosteric modulator of group II metabotropic glutamate receptors, LY487379, on cognitive flexibility and impulsive-like responding in rats.

Orthosteric group II metabotropic glutamate receptor (mGluR) agonists are regarded as novel, effective medications for all major symptom domains of schizophrenia, including cognitive disturbances. mGluR2s also can be affected in a more subtle way by positive allosteric modulators (PAMs) characterized by a unique degree of subtype selectivity and neuronal frequency-dependent activity. Because currently available treatments for schizophrenia do not improve cognitive dysfunction, the main aim of the present study was to examine the effects of a mGluR2 PAM, N-(4-(2-methoxyphenoxy)-phenyl-N-(2,2,2-trifluoroethylsulfonyl)-pyrid-3-ylmethylamine (LY487379), on rat cognitive flexibility and impulsive-like responding, assessed in an attentional set-shifting task (ASST) and a differential reinforcement of low-rate 72 s (DRL72) schedule of food reinforcement. In addition, in vivo microdialysis was used to assess the drug's impact on cortical levels of dopamine, norepinephrine, serotonin, and glutamate. Rats treated with LY487379 (30 mg/kg) required significantly fewer trials to criteria during the extradimensional shift phase of the ASST. Under a DRL72 schedule, LY487379 (30 mg/kg) decreased the response rate and increased the number of reinforcers obtained. These effects were accompanied by the shift of the frequency distribution of responses toward longer inter-response time durations. LY487379 significantly enhanced extracellular norepinephrine and serotonin levels in the medial prefrontal cortex. In summary, the present study demonstrates that a mGluR2 PAM, LY487379, promotes cognitive flexibility and facilitates behavioral inhibition. These procognitive effects may contribute to the therapeutic efficacy of agents stimulating mGluR2 in schizophrenia.

Amphetamine decreases behavioral inhibition by stimulation of dopamine D2, but not D3, receptors.

Behavioral disinhibition is a manifestation of impulsive behavior that is prominent in the psychopathology of various psychiatric disorders such as addiction, attention-deficit hyperactivity disorder, mania, and personality disorders. Impulsivity may be studied by measuring anticipatory responses made before the presentation of a food-predictive, brief light stimulus in a two-choice serial reaction time task. In such serial reaction time tasks, amphetamine has been shown to produce dose-dependent increases in premature responding in a manner dependent on dopamine D(2)-like receptor stimulation. So far, it is unknown whether it is the D(2) or D(3) receptor that is involved in this form of impulsivity. In this study, rats were trained in a two-choice serial reaction time task until baseline performance was stable. Next, effects of the dopamine D(2) preferring antagonist L-741,626 and selective D(3) antagonist SB-277011 were assessed alone and in the presence of amphetamine. Neither L-741,626 nor SB-277011 affected behavioral inhibition, although the latter significantly increased reaction time at 10 mg/kg. Amphetamine dose-dependently increased impulsivity. The effect of amphetamine was attenuated by L-741,626 (3 mg/kg), whereas SB-277011 (3 mg/kg) had no effect. Therefore, amphetamine-induced behavioral disinhibition depends on D(2), but not D(3), receptor stimulation.

Quantitative Systems Pharmacology Model for Alzheimer Disease Indicates Targeting Sphingolipid Dysregulation as Potential Treatment Option.

Alzheimer disease (AD) is a devastating neurodegenerative disorder with high unmet medical need. Drug development is hampered by limited understanding of the disease and its driving factors. Quantitative Systems Pharmacology (QSP) modeling provides a comprehensive quantitative framework to evaluate the relevance of biological mechanisms in the context of disease and to predict the efficacy of novel treatments. Here, we report a QSP model for AD with a particular focus on investigating the relevance of dysregulation of cholesterol and sphingolipids. We show that our model captures the modulation of several biomarkers in subjects with AD, as well as the response to pharmacological interventions. We evaluate the impact of targeting the sphingosine-1-phosphate 5 receptor (S1PR5) as a potential novel treatment option for AD, and model predictions increase our confidence in this novel disease pathway. Future applications for the QSP model are in validation of further targets and identification of potential treatment response biomarkers.

De Novo Design, Synthesis, and Biological Evaluation of 3,4-Disubstituted Pyrrolidine Sulfonamides as Potent and Selective Glycine Transporter 1 Competitive Inhibitors.

The development of glycine transporter 1 (GlyT1) inhibitors may offer putative treatments for schizophrenia and other disorders associated with hypofunction of the glutaminergic N-methyl-d-aspartate (NMDA) receptor. Herein, we describe the synthesis and biological evaluation of a series of 3,4-disubstituted pyrrolidine sulfonamides as competitive GlyT1 inhibitors that arose from de novo scaffold design. Relationship of chemical structure to drug-drug interaction (DDI) and bioactivation was mechanistically investigated. Murine studies were strategically incorporated into the screening funnel to provide early assessments of in vivo target occupancy (TO) by ex vivo binding studies. Advanced compounds derived from iterative structure-activity relationship (SAR) studies possessed high potency in ex vivo binding studies and good brain penetration, promising preliminary in vivo efficacy, acceptable preclinical pharmacokinetics, and manageable DDI and bioactivation liabilities.

Discovery of Novel Aminotetralines and Aminochromanes as Selective and Competitive Glycine Transporter 1 (GlyT1) Inhibitors.

The glycine transporter 1 (GlyT1) has emerged as a key novel target for the treatment of schizophrenia. Herein, we report the synthesis and biological evaluation of aminotetralines and aminochromanes as novel classes of competitive GlyT1 inhibitors. Starting from a high-throughput screening hit, structure-activity relationship studies led first to the discovery of aminotetralines displaying high GlyT1 potency and selectivity, with favorable pharmacokinetic properties. Systematic investigations of various parameters (e.g., topological polar surface area, number of hydrogen bond donors) guided by ex vivo target occupancy evaluation resulted in lead compounds possessing favorable brain penetration properties as for (7 S,8 R)-27a. Further optimization revealed compounds with reduced efflux liabilities as for aminochromane 51b. In an in vivo efficacy model (7 S,8 R)-27a, dose-dependently reversed L-687,414 induced hyperlocomotion in mice with an ED50 of 0.8 mg/kg. All these results suggest (7 S,8 R)-27a and 51b as new GlyT1 inhibitors worthy of further profiling.

The guinea pig forced swim test as a new behavioral despair model to characterize potential antidepressants.

RATIONALE: Behavioral despair is a model of high predictivity for antidepressant activity in murids. For some drug targets, guinea pigs exhibit a higher homology to their human counterparts compared to murids. OBJECTIVES: In this paper, we established a model of behavioral despair namely, the forced swim test (FST) in guinea pigs. MATERIALS AND METHODS: Male guinea pigs underwent the FST similar to rats. Animals received intraperitoneal injections of either vehicle or drugs 24, 4, and 0.5 h before testing. We tested the tricyclic antidepressants desipramine and amitriptyline, the monoamine oxidase inhibitor tranylcypromine, the selective serotonin reuptake inhibitors fluoxetine and paroxetine, and the neurokinin 1 (NK(1)) receptor antagonist, L-733,060, and for comparison the antipsychotic clozapine and the stimulant methamphetamine. RESULTS: Desipramine (> or =3 mg/kg) and amitriptyline (>10 mg/kg) increased the latency to immobility (LTI) to greater than 230 s, and tranylcypromine (10 mg/kg) it to greater than 190 s. Paroxetine (>0.3 mg/kg) and fluoxetine (>10 mg/kg) also increased LTI significantly but only to greater than 120 s. Methamphetamine (3 mg/kg) completely eliminated immobility, whereas clozapine (5-20 mg/kg) had no effect. L-733,060 (10 mg/kg) increased LTI to 270 s. Doses producing significant effects in FST were investigated in the open field. Antidepressants did not affect locomotion, whereas methamphetamine induced hyperlocomotion. CONCLUSIONS: We demonstrate the suitability of a modified procedure of the FST for a nonmurid species: the guinea pig. Known antidepressants showed similar effects as in rats and mice. It is interesting to note that the NK(1) antagonist L-733,060 increased forced swimming, suggesting its antidepressant potential. Thus, the guinea pig FST allows the study of antidepressant activity also in NK(1) antagonists that cannot be studied appropriately in murids.

Discovery of Novel and Highly Selective Inhibitors of Calpain for the Treatment of Alzheimer's Disease: 2-(3-Phenyl-1H-pyrazol-1-yl)-nicotinamides.

Calpain overactivation has been implicated in a variety of pathological disorders including ischemia/reperfusion injury, cataract formation, and neurodegenerative diseases such as Alzheimer's disease (AD). Herein we describe our efforts leading to the identification of ketoamide-based 2-(3-phenyl-1H-pyrazol-1-yl)nicotinamides as potent and reversible inhibitors of calpain with high selectivity versus related cysteine protease cathepsins, other proteases, and receptors. Broad efficacy in a set of preclinical models relevant to AD suggests that inhibition of calpain represents an attractive approach with potential benefit for the treatment of AD.

Towards trans-diagnostic mechanisms in psychiatry: neurobehavioral profile of rats with a loss-of-function point mutation in the dopamine transporter gene.

The research domain criteria (RDoC) matrix has been developed to reorient psychiatric research towards measurable behavioral dimensions and underlying mechanisms. Here, we used a new genetic rat model with a loss-of-function point mutation in the dopamine transporter (DAT) gene (Slc6a3_N157K) to systematically study the RDoC matrix. First, we examined the impact of the Slc6a3_N157K mutation on monoaminergic signaling. We then performed behavioral tests representing each of the five RDoC domains: negative and positive valence systems, cognitive, social and arousal/regulatory systems. The use of RDoC may be particularly helpful for drug development. We studied the effects of a novel pharmacological approach metabotropic glutamate receptor mGluR2/3 antagonism, in DAT mutants in a comparative way with standard medications. Loss of DAT functionality in mutant rats not only elevated subcortical extracellular dopamine concentration but also altered the balance of monoaminergic transmission. DAT mutant rats showed deficits in all five RDoC domains. Thus, mutant rats failed to show conditioned fear responses, were anhedonic, were unable to learn stimulus-reward associations, showed impaired cognition and social behavior, and were hyperactive. Hyperactivity in mutant rats was reduced by amphetamine and atomoxetine, which are well-established medications to reduce hyperactivity in humans. The mGluR2/3 antagonist LY341495 also normalized hyperactivity in DAT mutant rats without affecting extracellular dopamine levels. We systematically characterized an altered dopamine system within the context of the RDoC matrix and studied mGluR2/3 antagonism as a new pharmacological strategy to treat mental disorders with underlying subcortical dopaminergic hyperactivity.

Effect of dopamine D3 antagonists on PPI in DBA/2J mice or PPI deficit induced by neonatal ventral hippocampal lesions in rats.

Schizophrenic patients typically exhibit impairment of sensorimotor gating, which can be modeled in animal models such as the test of prepulse inhibition of startle response (PPI) in rodents. It has been found that antipsychotics enhanced PPI in DBA mice and reversed the PPI deficit induced by neonatal ventral hippocampal (NVH) lesions in rats. However, the relative involvement of D(3) and D(2) receptors in these effects is unknown since all antipsychotics are D(2)/D(3) antagonists with limited binding preference at D(2) receptors. Therefore, in the current study, we investigated the influence of several dopamine antagonists with higher selectivity at D(3) vs D(2) receptors on PPI in DBA/2J mice and in NVH-lesioned rats. The PPI in DBA/2J mice was enhanced by the nonselective D(2)/D(3) antagonists, haloperidol at 0.3-3 mg/kg, or risperidone at 0.3-1 mg/kg, while PPI-enhancing effects were observed after the administration of higher doses of the preferential D(3)/D(2) antagonist, BP 897 at 8 mg/kg, and the selective D(3) antagonists, SB 277011 at 30 mg/kg and A-437203 at 30 mg/kg. No effect was observed following the treatment with the selective D(3) antagonist, AVE 5997 up to 30 mg/kg. The PPI deficits induced by NVH lesions were reversed by haloperidol but not by the more selective D(3) antagonists, A-437203 and AVE 5997. BP 897 enhanced PPI nonselectivity, that is, in both lesioned and nonlesioned rats. In summary, the present study indicates that PPI-enhancing effects induced by antipsychotics in DBA/2J mice and in NVH-lesioned rats are unlikely to be mediated by D(3) receptors.

Drug Tolerance: A Known Unknown in Translational Neuroscience.

In neuropsychiatric drug development, the rate of successful translation of preclinical to clinical efficacy has been disappointingly low. Tolerance, defined as a loss of efficacy with repeated drug exposure, is rarely addressed as a potential source of clinical failures. In this review, we argue that preclinical methods of tolerance development may have predictive validity and, therefore, inclusion of studies using repeated drug exposure early during the drug discovery and development process should serve to mitigate a proportion of clinical failures. Our analysis indicates that many published preclinical efficacy studies in the neuropsychiatry arena are conducted with acute drug administration only. Furthermore, specifically in the field of schizophrenia, there are several examples where tolerance development may be suspected as a factor contributing to translational failures. These and other examples highlight the need for built-for-purpose tolerance studies to be conducted, regardless of the target interaction mode of the drugs (i.e., agonist or antagonist, allosteric or orthosteric). We suggest that, for compounds that have failed in clinical studies, preclinical efficacy data sets need to be revisited to estimate the potential impact of tolerance development, one of the most significant known unknowns in the preclinical-to-clinical translation.

The effects of temporary inactivation of the core and the shell subregions of the nucleus accumbens on prepulse inhibition of the acoustic startle reflex and activity in rats.

The nucleus accumbens can be dissociated into at least two subregions: a 'core' and a 'shell'. Using temporary chemical inactivation of these subregions, we investigated whether they are differentially involved in the regulation of prepulse inhibition (PPI) of the acoustic startle reflex and activity. For this purpose, rats were bilaterally implanted with guide cannulae aimed at either the core or the shell and infused with the GABA(A) receptor agonist muscimol (0.5 microg/0.2 microl per side). The control group consisted of vehicle infused and unoperated rats. To ascertain the region selectivity of the infusions, 0.2 microl of [3H]muscimol was infused into either the core or the shell of an additional group of rats. The behavioral results demonstrated that in comparison to the control group, inactivation of the core led to a loss of the prepulse intensity dependency of PPI. Moreover, core inactivation resulted in akinesia directly after infusion, but in hyperactivity 24 and 72 h thereafter in contrast to the control group. In both experiments, inactivation of the shell was ineffective compared to controls. Analysis of the autoradiograms revealed that the spread of drug into the other subregion was minimal, supporting the region selectivity of the inactivation. These results lend further support to the existence of a functional dissociation between the core and the shell, with the former being preferentially involved in PPI and locomotion. The persistent hyperactivity after the muscimol infusion into the core could be explained by compensatory mechanisms taking place in the nucleus accumbens.

Effects of prenatal methylazoxymethanol acetate (MAM) treatment in rats on water maze performance.

Prenatal methylazoxymethanol acetate (MAM) treatment has been shown to induce morphological abnormalities in cortical areas of the offspring. Based on the neuroanatomical and behavioural abnormalities, this treatment has been suggested as a useful animal model for schizophrenia. In a previous study (Jongen-Relo AL, Leng A, Luber M, Pothuizen HHJ, Weber L, Feldon J. The prenatal methylazoxymethanol acetate treatment: a neurodevelopmental animal model for schizophrenia? Behav Brain Res 2004;149:159-81) we have studied MAM-treated animals in a series of behavioural tests related to schizophrenia, such as latent inhibition and pre-pulse inhibition of the acoustic startle response to establish the validity of prenatal MAM treatment (20mg/kg i.p. on gestational days 9-15; MAM 9-MAM 15). We found that, apart from a marginal effect of increased activity in the open field, the MAM treatment on gestational day 15 was behaviourally ineffective. Here, we extended our previous study to a water maze experiment conducted in the same batch of animals as presented previously (MAM 12-MAM 15). MAM-treated animals showed similar water maze performance compared with control animals during the acquisition phase and the probe tests. However, during the reversal phase, MAM 15 animals showed impaired acquisition of the new platform location. This might indicate some cognitive deficits in MAM 15 animals in terms of working memory or behavioural flexibility. However, in combination with the lack of behavioural abnormalities of MAM 12-MAM 15 animals in several other tests related to schizophrenia in the previously reported study, the use of MAM treatment (MAM 12-MAM 15) as a valid model for schizophrenia still remains debatable.

Brain area- and isoform-specific inhibition of synaptic plasticity by apoE4.

The allele E4 of apolipoprotein E4 (apoE4), the most prevalent genetic risk factor of Alzheimer's disease (AD), inhibits the improvements in learning and memory which result from exposure of apoE transgenic mice to environmental stimulation (ES). In the present study, we investigated the extent to which these cognitive deficits are associated with distinct presynaptic, postsynaptic and axonal impairments and whether these effects are brain area-specific. Exposure to an enriched environment of young mice transgenic for human apoE3, which is the AD benign apoE allele, increased the levels of the presynaptic protein synaptophysin and of the dendritic marker MAP-2 in the hippocampus and entorhinal cortex, whereas the corresponding levels of these proteins in the apoE4 transgenic mice were unaffected by the enriched environment. In contrast, the levels of synaptophysin and MAP-2 in the motor cortex were elevated by environmental stimulation in both the apoE3 and the apoE4 transgenic mice. These findings show that apoE4 inhibits synaptic plasticity following environmental stimulation and that this effect is both isoform- and brain area-specific.

Comparison of maternal separation and early handling in terms of their neurobehavioral effects in aged rats.

In the rat, relative to pup nonhandling (NH), early handling (EH) leads to old-adult offspring with a hyporesponsive HPA axis, superior spatial cognition, and greater hippocampal (HIPP) neuronal density. The present study compared the effects of EH and repeated maternal separation (MS), in the form of 6-hr separation on each of 4 days beginning at day 12, on spatial cognition, corticosterone (CORT) levels, and HIPP characteristics, in aged rats. Male Wistar rat pups were exposed to EH, MS, NH or our normal in-house husbandry (CON) and tested at 18-20 months. Relative to NH and CON, EH demonstrated superior spatial cognition, reduced CORT stress response, reduced CA-field volume and no change in HIPP neuronal number. MS demonstrated a trend to superior spatial cognition, an unaffected CORT stress response, reduced CA-field volume and no change in HIPP neuronal number. These findings are important in terms of the life-span mechanisms via which postnatal manipulations induce neurobehavioral effects, and the mechanisms via which CORT and HIPP structure relate to HIPP function.

A differential involvement of the shell and core subterritories of the nucleus accumbens of rats in memory processes.

The role of the core and the shell subterritories of the nucleus accumbens in conditioned freezing and spatial learning was investigated by means of selective N-methyl-D-aspartate lesions. Shell-lesioned rats showed reduced conditioned freezing to context and a tendency toward reduced freezing to the discrete stimulus compared with controls. However, lesions of the core did not modify the freezing response either to the context or to the discrete stimuli. Although spatial memory, as assessed by a water-maze paradigm, was not disrupted by the lesions, in a 4-arm baited, 4-arm unbaited radial-arm maze paradigm, the shell-lesioned rats showed selective deficits in working memory, but not in reference memory. In contrast, core-lesioned rats showed no memory deficits.

The prenatal methylazoxymethanol acetate treatment: a neurodevelopmental animal model for schizophrenia?

The prenatal methylazoxymethanol acetate (MAM) treatment has been proposed as a suitable model for the neurodevelopmental aspects of schizophrenia since the morphological abnormalities it induces in the brain are subtle and in line with most reports of neuropathology in schizophrenic brains. However, the functional aspects of this treatment have not been investigated with behavioural paradigms that are relevant for the psychopathology of the symptoms of schizophrenia. In the present study, we investigated the validity of the prenatal MAM treatment as a developmental model for schizophrenia with a prepulse inhibition of the acoustic startle reflex, latent inhibition, locomotor activity, and cognition and emotionality with freezing in fear conditioning paradigms. We have conducted two studies: in Study I, MAM was injected from E09 to E12, and in Study II MAM was administered at later stages in the embryonic development, from E12 to E15. Morphologically, the prenatal MAM treatment induced mild to severe reduction in brain weights and in the entorhinal cortex, prefrontal cortex and striatum volumes, the severity of the effects depending on the timing of administration. However, despite the morphological abnormalities induced by the MAM treatments, no behavioural deficits were observed in the MAM-treated animals when compared to Controls in prepulse inhibition, latent inhibition with the two-way active avoidance, and in the freezing paradigms. Therefore, due to the consistent lack of treatment effect observed in the present investigation, we conclude that the prenatal MAM treatment has no validity as a behavioural model for schizophrenia.

Effect of social isolation on stress-related behavioural and neuroendocrine state in the rat.

The present study investigated the effects of post-weaning social isolation (SI) on behavioural and neuroendocrine reactivity to stress of male and female rats. Innate aspects of fear and anxiety were assessed in the open field and elevated plus maze tests. Spontaneous startle reflex and conditioned fear response were further investigated. The neuroendocrine response of isolates was examined by measuring basal and stress release of ACTH and corticosterone and by evaluating the mRNA expression of mineralocorticoid (MR) and glucocorticoid (GR) receptors using in situ hybridization. Locomotor activity in the open field was not modified by chronic SI. In males, but not females, SI produced an anxiogenic profile in the elevated plus maze. Male isolates showed a trend towards increased startle reflex amplitude relative to socially-reared controls. Moreover, SI in males produced alterations of the HPA axis functioning as reflected by higher basal levels of ACTH, and enhanced release of ACTH and corticosterone following stress. In contrast, startle response or HPA axis functioning were not altered in female isolates. Social isolates from both genders showed reduced contextual fear-conditioning. Finally, the mRNA expression of MR and GR was not modified by SI. The results of the present study suggest that chronic SI increases emotional reactivity to stress and produces a hyperfunction of the HPA axis in adult rats, particularly in males.

The use of stereological counting methods to assess immediate early gene immunoreactivity.

The issue of whether profile and stereological counting methods are interchangeably accurate when assessing immediate early gene expression still needs to be resolved. To compare these two counting techniques, we quantified the expression of c-fos in the nucleus accumbens core and shell, and in the lateral septum as a control structure, of rats treated with neuroleptics. With the profile counting method, which relies on selective placement of a counting grid within a structure, we evaluated the density of c-fos labeled cells within a box of fixed dimension. With stereology, which applies random and systematic sampling methods, we used the optical fractionator method and counted the absolute number of c-fos labeled cells within the contours of each structure examined. Our results showed that the substantial increase in c-fos expression in the shell and core induced by haloperidol treatment was detected by both stereological and profile counting methods; in contrast, the weaker effect of clozapine on c-fos expression was detected differentially by the two methods. Whereas the profile counting method reported a reduction of c-fos in the core by clozapine, and an increase in c-fos in the lateral septum, these effects were not replicated using stereology. These findings suggest that stereological and profile counting methods do not always produce equivalent results. This may be particularly relevant when a measured effect is relatively small, and it is not distributed homogeneously within a structure. In this respect, the random and systematic sampling methods of stereology may yield more accurate and unbiased results than the profile counting method, and therefore may be preferred for a more accurate and thorough investigation of a treatment effect on immediate early gene expression in a specific brain region.

Comparison of central corticosteroid receptor expression in male Lewis and Fischer rats.

The histocompatible inbred Lewis (LEW) and Fischer (F344) rat strains exhibit marked phenotypic differences in the hormonal activity of the hypothalamic-pituitary-adrenal (HPA) axis. As such they provide an important comparative model for the study of HPA regulation including the central feedback regulation by the glucocorticoid transcription factors, the mineralocorticoid receptor (MR) and the glucocortiocoid receptor (GR). In adult male rats, basal nadir plasma corticosterone values were similar in the two strains whereas the amplitude and duration of the corticosterone response to restraint were significantly reduced in LEW. MR and GR mRNA expression were compared in LEW and F344 throughout the rostrocaudal extent of the hippocampus. Hippocampal MR expression was consistent throughout the rostrocaudal extent and similar in F344 and LEW males. Hippocampal GR expression was consistent throughout the rostrocaudal extent, but significantly greater in the rostral dentate gyrus and Cornu ammonis subfields in LEW males compared with F344 males. The LEW phenotype of relatively high hippocampal GR expression and HPA hypoactivity is shared by outbred rats exposed to neonatal handling and GR-overexpressing transgenic mice. Whether or not this relationship is causal, as well as the functional significance of the rostral-extent specific GR difference, remains to be elucidated.

Sensitized Fos expression in subterritories of the rat medial prefrontal cortex and nucleus accumbens following amphetamine sensitization as revealed by stereology.

Behavioral sensitization to the locomotor activating effects of amphetamine refers to the progressive, long lasting increase in locomotor activity that occurs with repeated injections. This phenomenon is thought to result from neuroadaptations occurring in the projection fields of mesocorticolimbic dopaminergic neurons. In the present study, we investigated the effects of amphetamine sensitization on Fos immunoreactivity (Fos-IR) in subterritories of the nucleus accumbens (core and shell) and medial prefrontal cortex (mPFC; dorsal and ventral) using stereology. Rats received five daily injections of amphetamine (1.5 mg/kg, i.p.) or saline. Behavioral sensitization was measured 48 h following the last injection, in response to a challenge injection of 1.5 mg/kg amphetamine. Sensitized rats showed a greater enhancement of locomotor activity upon drug challenge compared with their saline counterparts. Densities of Fos-positive nuclei were enhanced more in the dorsal than the ventral mPFC subterritory, whereas in the nucleus accumbens, densities of Fos-positive nuclei were increased more in the core than the shell of amphetamine-sensitized rats compared to controls. These results represent, to our knowledge, the first published report using stereological methods to quantify Fos-IR in the brain and suggest functional specialization of cortical and limbic regions in the expression of behavioral sensitization to amphetamine.