Effects of context-drug learning on synaptic connectivity in the basolateral nucleus of the amygdala in rats.

Context-drug learning produces structural and functional synaptic changes in the circuitry of the basolateral nucleus of the amygdala (BLA). However, how the synaptic changes translated to the neuronal targets was not established. Thus, in the present study, immunohistochemistry with a cell-specific marker and the stereological quantification of synapses was used to determine if context-drug learning increases the number of excitatory and inhibitory/modulatory synapses contacting the gamma-aminobutyric acid (GABA) interneurons and/or the pyramidal neurons in the BLA circuitry. Amphetamine-conditioned place preference increased the number of asymmetric (excitatory) synapses contacting the spines and dendrites of pyramidal neurons and the number of multisynaptic boutons contacting pyramidal neurons and GABA interneurons. Context-drug learning increased asymmetric (excitatory) synapses onto dendrites of GABA interneurons and increased symmetric (inhibitory or modulatory) synapses onto dendrites but not perikarya of these same interneurons. The formation of context-drug associations alters the synaptic connectivity in the BLA circuitry, findings that have important implications for drug-seeking behavior.

Aberrant restoration of spines and their synapses in L-DOPA-induced dyskinesia: involvement of corticostriatal but not thalamostriatal synapses.

We examined the structural plasticity of excitatory synapses from corticostriatal and thalamostriatal pathways and their postsynaptic targets in adult Sprague-Dawley rats to understand how these striatal circuits change in l-DOPA-induced dyskinesias (LIDs). We present here detailed electron and light microscopic analyses that provide new insight into the nature of the structural and synaptic remodeling of medium spiny neurons in response to LIDs. Numerous studies have implicated enhanced glutamate signaling and persistent long-term potentiation as central to the behavioral sensitization phenomenon of LIDs. Moreover, experience-dependent alterations in behavior are thought to involve structural modifications, specifically alterations in patterns of synaptic connectivity. Thus, we hypothesized that in the striatum of rats with LIDs, one of two major glutamatergic pathways would form new or altered contacts, especially onto the spines of medium spiny neuron (MSNs). Our data provide compelling evidence for a dramatic rewiring of the striatum of dyskinetic rats and that this rewiring involves corticostriatal but not thalamostriatal contacts onto MSNs. There is a dramatic increase in corticostriatal contacts onto spines and dendrites that appear to be directly linked to dyskinetic behaviors, since they were not seen in the striatum of animals that did not develop dyskinesia. There is also an aberrant increase in spines receiving more than one excitatory contact(i.e., multisynaptic spines) in the dyskinetic animals compared with the 6-hydroxydopamine-treated and control rats. Such alterations could substantially impair the ability of striatal neurons to gate cortically driven signals and contribute to the loss of bidirectional synaptic plasticity.

Different roles of BDNF in nucleus accumbens core versus shell during the incubation of cue-induced cocaine craving and its long-term maintenance.

Brain-derived neurotrophic factor (BDNF) contributes to diverse types of plasticity, including cocaine addiction. We investigated the role of BDNF in the rat nucleus accumbens (NAc) in the incubation of cocaine craving over 3 months of withdrawal from extended access cocaine self-administration. First, we confirmed by immunoblotting that BDNF levels are elevated after this cocaine regimen on withdrawal day 45 (WD45) and showed that BDNF mRNA levels are not altered. Next, we explored the time course of elevated BDNF expression using immunohistochemistry. Elevation of BDNF in the NAc core was detected on WD45 and further increased on WD90, whereas elevation in shell was not detected until WD90. Surface expression of activated tropomyosin receptor kinase B (TrkB) was also enhanced on WD90. Next, we used viral vectors to attenuate BDNF-TrkB signaling. Virus injection into the NAc core enhanced cue-induced cocaine seeking on WD1 compared with controls, whereas no effect was observed on WD30 or WD90. Attenuating BDNF-TrkB signaling in shell did not affect cocaine seeking on WD1 or WD45 but significantly decreased cocaine seeking on WD90. These results suggest that basal levels of BDNF transmission in the NAc core exert a suppressive effect on cocaine seeking in early withdrawal (WD1), whereas the late elevation of BDNF protein in NAc shell contributes to incubation in late withdrawal (WD90). Finally, BDNF protein levels in the NAc were significantly increased after ampakine treatment, supporting the novel hypothesis that the gradual increase of BDNF levels in NAc accompanying incubation could be caused by increased AMPAR transmission during withdrawal.

'Rejuvenation' protects neurons in mouse models of Parkinson's disease.

Why dopamine-containing neurons of the brain's substantia nigra pars compacta die in Parkinson's disease has been an enduring mystery. Our studies suggest that the unusual reliance of these neurons on L-type Ca(v)1.3 Ca2+ channels to drive their maintained, rhythmic pacemaking renders them vulnerable to stressors thought to contribute to disease progression. The reliance on these channels increases with age, as juvenile dopamine-containing neurons in the substantia nigra pars compacta use pacemaking mechanisms common to neurons not affected in Parkinson's disease. These mechanisms remain latent in adulthood, and blocking Ca(v)1.3 Ca2+ channels in adult neurons induces a reversion to the juvenile form of pacemaking. Such blocking ('rejuvenation') protects these neurons in both in vitro and in vivo models of Parkinson's disease, pointing to a new strategy that could slow or stop the progression of the disease.

Amphetamine-associated contextual learning is accompanied by structural and functional plasticity in the basolateral amygdala.

Drug seeking and the vulnerability to relapse occur when individuals are exposed to an environment with sensory cues in which drug taking has occurred. Memory formation is thought to require plasticity in synaptic circuits, and so we examined whether the memory for a drug-paired environment correlates with changes in the synaptic circuits of the basolateral amygdala (BLA), in which emotional learning is a recognized phenomenon. We used amphetamine (AMPH) as the unconditioned stimulus in the conditioned place preference (CPP) paradigm. Rats were conditioned with 1.0 mg/kg AMPH and tested, drug free, 72 h after the last conditioning session. Controls included a saline-conditioned group and a home cage AMPH injection group, whose exposure to the CPP apparatus was delayed by 4 h, long enough to clear the AMPH from the brain. We counted excitatory synapses in the BLA using the electron microscope and the physical disector design (stereology). Rats that expressed AMPH CPP had an increase in excitatory synapses compared with controls. Excitatory synaptic activity was measured using in vivo intracellular recordings from the BLA in anesthetized rats. We found that AMPH CPP, but not drug alone, increased measures of synaptic drive, including the frequency of synaptic events, and the paired-pulse ratio of synaptic inputs to BLA pyramidal neurons. The in vivo findings suggest that the increase in BLA neuronal excitatory drive reflects the change in excitatory synapse number. Thus, context-drug associations are accompanied by structural and functional plasticity in the BLA, findings that have important implications for drug-seeking behavior.

Increased synapses in the medial prefrontal cortex are associated with repeated amphetamine administration.

Psychostimulant drug experience leads not only to long-lasting changes in behavior but also modifications in the activity and morphology of pyramidal neurons in the medial prefrontal cortex (mPFC). The objective of this study was to establish whether repeated treatment of rats with amphetamine (AMPH) is accompanied by changes in the pattern or types of synapses in the mPFC and, specifically, onto neurons that project to the lateral hypothalamus, where our earlier work has shown increased markers of neuronal activity after repeated AMPH treatment (Morshedi and Meredith [2008] Psychopharmacology (Berl) 197:179-189). Rats were treated with a behaviorally sensitizing regimen of AMPH, following which synapses in the infralimbic and prelimbic cortices of the mPFC, were analyzed with unbiased stereology (physical disector and electron microscopy). All synapses were counted and their targets were identified by standard methodological criteria. Repeated AMPH administration was associated with a significant increase in the number of asymmetric axospinous synapses, no change in axodendritic or axosomatic contacts, and no change in the total number of synapses on corticolateral hypothalamic pyramidal neurons compared to vehicle-treated rats. Therefore, behavioral sensitization as a result of repeated exposure to AMPH is accompanied by the increased formation of spine, but not dendritic, synapses onto pyramidal neurons in the mPFC.

Repeated amphetamine administration induces Fos in prefrontal cortical neurons that project to the lateral hypothalamus but not the nucleus accumbens or basolateral amygdala.

RATIONALE: The development of sensitization to amphetamine (AMPH) is dependent on increases in excitatory outflow from the medial prefrontal cortex (mPFC) to subcortical centers. These projections are clearly important for the progressive enhancement of the behavioral response during drug administration that persists through withdrawal. OBJECTIVES: The objective of this study was to identify the mPFC subcortical pathway(s) activated by a sensitizing regimen of AMPH. MATERIALS AND METHODS: Using retrograde labeling techniques, Fos activation was evaluated in the predominant projection pathways of the mPFC of sensitized rats after a challenge injection of AMPH. RESULTS: There was a significant increase in Fos-immunoreactive cells in the mPFC, nucleus accumbens (NAc), basolateral amygdala (BLA), and lateral hypothalamus (LH) of rats treated repeatedly with AMPH when compared to vehicle-treated controls. The mPFC pyramidal neurons that project to the LH but not the NAc or BLA show a significant induction of Fos after repeated AMPH treatment. In addition, we found a dramatic increase in Fos-activated orexin neurons. CONCLUSIONS: The LH, a region implicated in natural and drug reward processes, may play a role in the development and persistence of sensitization to repeated AMPH through its connections with the mPFC and possibly through its orexin neurons.

Modeling PD pathogenesis in mice: advantages of a chronic MPTP protocol.

Formidable challenges for Parkinson's disease (PD) research are to understand the processes underlying nigrostriatal degeneration and how to protect dopamine neurons. Fundamental research relies on good animal models that demonstrate the pathological hallmarks and motor deficits of PD. Using a chronic regimen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p) in mice, dopamine cell loss exceeds 60%, extracellular glutamate is elevated, cytoplasmic inclusions are formed and inflammation is chronic. Nevertheless, isradipine, an L-type calcium-channel blocker, attenuates the degeneration. These data support the validity of the MPTP/p model for unravelling the degenerative processes in PD and testing therapies that slow their progress.

Amphetamine-induced place preference and conditioned motor sensitization requires activation of tyrosine kinase receptors in the hippocampus.

The environmental context in which abused drugs are taken contribute to the drug experience and is a powerful and persistent stimulus to elicit memories of that experience even in the abstinent addict. Using amphetamine (AMPH) as the unconditioned stimulus, the present study compared two popular context-dependent paradigms in rats, conditioned motor sensitization (CMS) and conditioned place preference (CPP), to ascertain whether particular brain regions were differentially involved. The neuronal substrates underlying these context-dependent behaviors are poorly understood, but regulators of the neuronal plasticity that accompany learning, such as neurotrophic factors and their cognate tyrosine kinase receptors (e.g., TrkB), are credible candidates. We found a significant elevation of TrkB-like immunoreactivity specifically in CA3/dentate gyrus (DG) subregions of the hippocampus after AMPH (0.3 mg/kg)-induced CPP, but not in the delayed-paired (control) AMPH condition. A higher AMPH dose (1.0 mg/kg) induced both CPP and CMS and elevated TrkB in the CA3/DG as well as in the nucleus accumbens shell. The development of both conditioned behaviors was blocked by intra-CA3/DG infusion of the Trk inhibitor K-252a. These findings reveal that CPP and CMS are induced by different doses of AMPH and are associated with TrkB changes in particular brain regions. Moreover, Trk receptors in the hippocampus are critical mediators of the neuronal changes necessary for inducing both forms of conditioning. Thus, although these two conditioning models are distinct, because they are commonly regulated by the hippocampal Trk system, these receptors may be a therapeutic target for attenuating the significance of contextual cues that otherwise strengthen the addictive properties of abused drugs.

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.

MPTP administration in mice changes the ratio of splice isoforms of fosB and rgs9.

Most cases of Parkinson's disease (PD) are sporadic, suggesting an environmental influence on individuals affected by this neurodegenerative disorder. Environmental stresses often lead to changes in the regulation of splicing of pre-mRNA transcripts and this may lead to the pathogenesis of the disease. A 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/probenecid mouse model was used to examine the changes in the splicing of the fosB and rgs9 transcripts. The ratio of DeltafosB/fosB transcript was decreased in the substantia nigra and unchanged in the striatum after acute MPTP treatment. The DeltafosB/fosB transcript ratio decreased initially and then increased in the striatum of chronically MPTP-treated animals due to different degrees of reduction for the splice variants over time, whereas the ratio was unchanged in the substantia nigra. The ratio of rgs9-2/rgs9-1 transcript decreased in the substantia nigra of mice after acute MPTP treatment and increased temporarily in the striatum after chronic MPTP treatment. There was an increase in the DeltaFosB/FosB and RGS9-2/RGS9-1 protein ratios 3 weeks and 3 days post-treatment, respectively, in chronically treated mice. The data indicate that the pattern of splice isoforms of fosB and rgs9 reflects the brain's immediate and long-term responses to the physiological stress associated with Parkinsonism.

The role of oxidative stress in the dysregulation of gene expression and protein metabolism in neurodegenerative disease.

There are few examples for which the genetic basis for neurodegenerative disease has been identified. For the majority of these disorders, the key to their understanding lies in knowledge of the molecular changes that contribute to altered gene expression and the translational modification of the protein products. Environmental factors play a role in the development and chronicity of neurodegenerative disorders. Environmental stimuli such as hypoxia, toxins, or heavy metals, increase production of reactive oxygen species and lower energy reserves. Chronic exposure to oxidative radicals can adversely affect gene expression and proteolysis. This review summarizes what is currently known about some of the changes in gene expression and protein metabolism that occur after oxidative stress which contribute to neurodegeneration, and reveals areas where more research is clearly needed.

Amphetamine increases tyrosine kinase-B receptor expression in the dorsal striatum.

Neurotrophic signaling is thought to be important for neuroplasticity in certain forebrain regions following psychostimulant exposure. In this study, we found that repeated administration of amphetamine (5 mg/kg, once daily, 5 days) to rats significantly increased tyrosine kinase-B receptor mRNA levels in the striatum, ventral bed nucleus, and piriform cortex. The most robust increase in tyrosine kinase-B expression occurred in dorsal aspects of the striatum, which also showed elevated levels after a single amphetamine injection. These findings indicate that changes in striatal tyrosine kinase-B signaling could play a role in neuroadaptations and behavioral changes induced by amphetamine treatment.

Quantitative proteomic analysis of mitochondrial proteins: relevance to Lewy body formation and Parkinson's disease.

The mechanisms underlying Parkinson's disease (PD) and Lewy body (LB) formation, a pathological hallmark of PD, are incompletely understood; however, mitochondrial dysfunction is likely to be at least partially responsible. To study the processes that might be related to nigral neurodegeneration and LB formation, we employed nonbiased quantitative proteomics with isotope-coded affinity tag (ICAT) to compare the mitochondrial protein profiles in the substantia nigra (SN) between controls and mice treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a potent mitochondrial toxicant, and an adjuvant, probenecid (prob), for 5 weeks, which produced selective nigrostriatal neurodegeneration with formation of LB-like cytoplasmic inclusions in the remaining nigral neurons. This method identified a total of more than 300 proteins; of these proteins, more than 100 displayed significant changes in relative abundance in the MPTP/prob-treated mice compared to the controls. We validated one of these proteins, DJ-1, whose mutation has been implicated in familial PD, with Western blot analysis, followed by immunohistochemical studies of its distribution in the SN in relation to cytoplasmic inclusions in mice, as well as in classical LBs in PD patients. The results demonstrated that DJ-1 was not only colocalized with alpha-synuclein in dopaminergic neurons but also to cytoplasmic inclusions in mice treated with MPTP/prob. In addition, DJ-1 was present in the halo but not in the core of classical LBs in patients with PD. Our findings suggested that DJ-1 might play an important role in mitochondrial dysfunction, as well as LB formation in PD.

Elevated neuronal nitric oxide synthase expression in chronic haloperidol-treated rats.

Long-term administration of neuroleptic drugs, such as haloperidol, in the management of psychiatric disorders may adversely cause an irreversible neurological syndrome of tardive dyskinesia, which is associated with dopamine (DA(2)) receptor supersensitivity in the basal ganglia. Recent studies also indirectly suggest an involvement of nitric oxide synthase (NOS) in dopaminergic supersensitivity; however, chronic neuroleptic effects on neuronal NOS (nNOS) expression in the basal ganglia have not been reported. In this investigation, we treated rats with saline or haloperidol (1 mg/kg, s.c.) daily for 21 days. Five days later, we detected a significant increase of NOS activity in the striatum of haloperidol-treated rats when compared to saline controls. This effect was associated with elevated levels of nNOS mRNA and protein expression in the striatum, but not in the nucleus accumbens, as evidenced by the use of in situ hybridization, Western blot and immunohistochemical techniques. The involvement of the nNOS system after chronic haloperidol treatment coincides with increases of striatal DA(2) receptor sites, calmodulin kinase II activity, animal locomotor and stereotypy behaviors. This study suggests an integral role between nNOS, DA(2) receptor and calmodulin system in the development of dopaminergic behavioral supersensitivity resulting from chronic neuroleptic drug treatment. Furthermore, the toxic effect of chronic haloperidol on NOS system selectivity takes place in the neostriatum.

Relationship of orofacial movements to behavioural repertoire as assessed topographically over the course of 6-month haloperidol treatment followed by 4-month withdrawal.

RATIONALE: Late-onset vacuous chewing movements (VCMs) arise in a significant proportion of rats treated chronically with conventional antipsychotic drugs. Given their common action to block dopamine D2-like receptors, VCMs may be related to changes in dopaminergic function; if so, other typical dopamine-mediated behaviours might be altered also. OBJECTIVE: To examine this hypothesis, behavioural repertoire was studied topographically over the course of chronic treatment and withdrawal. METHODS: Animals were injected with haloperidol decanoate 28 mg/kg IM, or vehicle, every 3 weeks for 27 weeks, and then maintained without treatment for a further 18 weeks. Immediately before each injection and during withdrawal, VCMs and other topographies of behaviour were assessed. RESULTS: In both control and haloperidol-treated rats, exploratory behaviours declined over the study, indicating habituation effects. Conversely, VCMs emerged after 6 weeks of treatment with haloperidol and persisted after withdrawal; VCM and locomotion were not related, indicating that in treated rats, increased VCMs are not an artifact of reduced locomotion. Treated animals with VCMs evidenced increases in buccal tremor and grooming behaviour relative to those without VCMs, although no clear relationship to the emergence of VCMs was established; there were no material differences in any other topographies of behaviour. CONCLUSION: The effect of long-term treatment with haloperidol to induce VCMs is not reflected in fundamental changes in dopamine-mediated behavioural topography but, rather, appears to affect neural mechanisms involved in orofacial movement preferentially.

The ultrastructural distribution of alpha-synuclein-like protein in normal mouse brain.

The synaptic protein alpha-synuclein is found throughout the brain, although its function remains ill-defined. Abnormal accumulations of alpha-synuclein have been recognised to be associated with a number of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Nevertheless, little is known about the precise localisation of this protein within the normal brain, information which might contribute to our understanding of its role in both health and disease. We raised an antibody which recognises both human and murine alpha-synuclein and this was used to study the distribution of the protein in the normal mouse brain. We used morphological characteristics to classify the immunopositive presynaptic elements and their targets. We conclude that the protein is present in synaptic boutons of axons with different neurochemical phenotypes but that it is not present in all synaptic terminals. Furthermore, the protein is present in the terminals of neurons such as the dopaminergic neurons of the substantia nigra and the glutamatergic neurons of the hippocampus, cell types which accumulate alpha-synuclein in disease. Nevertheless alpha-synuclein is also found in terminals of neurons which have not been reported to accumulate the protein in neurodegenerative disorders.

Brain-derived neurotrophic factor expression is increased in the rat amygdala, piriform cortex and hypothalamus following repeated amphetamine administration.

The amygdala plays an important role in the regulation of motivational states, especially those associated with addiction. The amygdala also expresses high levels of brain-derived neurotrophic factor (BDNF), an activity-dependent neurotrophin that can influence the reinforcing and locomotor activating properties of psychostimulants. In the present study, we examined the effects of acute and repeated amphetamine administration on the expression and production of this factor in the forebrain of rats. Animals given a single, acute injection (5 mg/kg, i.p.) of D-amphetamine developed hyperactivity followed by stereotypical behavior but showed no change in the basal expression of BDNF mRNA or its immunocytochemical profile in any region except the piriform cortex. Repeated injections (5 days) of 5 mg/kg amphetamine were accompanied by an enhanced onset of stereotypical behavior and elevated BDNF mRNA in the basolateral amygdala, rostral piriform cortex and paraventricular nucleus of the hypothalamus. Repeated treatment also increased BDNF immunoreactivity in perikarya of these same regions. In addition, increased BDNF immunoreactivity was found in fibers of many projection targets of the basolateral amygdala--the central extended amygdala, olfactory tubercle, medial nucleus accumbens, and in small zones resembling striosomes in the dorsal medial striatum. These results suggest that the upregulation of BDNF expression and protein in the basolateral nucleus of the amygdala and its targets could be an important part of the neuroadaptive response to psychostimulants.

A critical review of the development and importance of proteinaceous aggregates in animal models of Parkinson's disease: new insights into Lewy body formation.

The pace of development of new animal models of Parkinson's disease (PD) has increased dramatically in the recent past, primarily because of the identification of the protein, alpha-synuclein, in Lewy bodies in both idiopathic and familial PD. This discovery has allowed the production of transgenic models that incorporate a form of human, mutant alpha-synuclein from rare familial cases, and has enabled the search for Lewy-body-like aggregations of this protein in toxin-induced models. Indeed, alpha-synuclein-positive inclusions, some of which bear strong resemblance to Lewy bodies, have now been recognized and their formation investigated in several different, environmentally-induced and transgenic models. Nevertheless, these data have yet to provide a uniform theory of inclusion pathogenesis for PD. The aim of this review is not only to summarize the findings to date on alpha-synuclein-immunopositive inclusion bodies, including some new information on Lewy bodies, but also provide a concise viewpoint on their origin and formation in animal models. We will provide evidence for a predicted series of intracellular events that underlie inclusion formation. Triggered by oxidative and metabolic stress, chronic, toxin-treated animals, rather than transgenic models transfected with human alpha-synuclein, eventually produce inclusion bodies that most closely resemble early stages of Lewy bodies. Elucidating the common mechanisms in animal models is a first step towards understanding the role of Lewy bodies and their formation in Parkinson's disease.

MPTP mouse models of Parkinson's disease: an update.

Among the most widely used models of Parkinson's disease (PD) are those that employ toxins, especially 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Depending on the protocol used, MPTP yields large variations in nigral cell loss, striatal dopamine loss and behavioral deficits. Motor deficits do not fully replicate those seen in PD. Nonetheless, MPTP mouse models mimic many aspects of the disease and are therefore important tools for understanding PD. In this review, we will discuss the ability of MPTP mouse models to replicate the pathophysiology of PD, the mechanisms of MPTP-induced neurotoxicity, strain differences in susceptibility to MPTP, and the models' roles in testing therapeutic approaches.