Developmental alterations in locomotor and anxiety-like behavior as a function of D1 and D2 mRNA expression.

The majority of smokers start smoking in adolescence, beginning a potentially lifelong struggle with nicotine use and abuse. In rodent models of the effects of nicotine, the drug has been shown to elicit both locomotor and anxiety-like behavioral effects. Research suggests that these behavioral effects may be due in part to dopamine (DA) receptors D1 and D2 in the mesolimbic system, specifically the nucleus accumbens (NAc). We examined early adolescent (P28), late adolescent (P45), and adult (P80) male Long-Evans rats in the elevated plus maze (EPM) under normal conditions and the open field (OF) post-nicotine in order to test locomotor and anxiety-like behavior. These behavioral findings were then correlated with expression of DA D1 and D2 mRNA levels as determined via in situ hybridization. Nicotine-induced locomotor behavior was found to be significantly different between age groups. After a single injection of nicotine, early adolescents exhibited increases in locomotor behavior, whereas both late adolescents and adults responded with decreases in locomotor behavior. In addition, it was found that among, early adolescents, open arm and center time in the EPM were negatively correlated with D2 mRNA expression. In contrast, among adults, distance traveled in the center and center time in the OF were negatively correlated with D2 mRNA expression. This study suggests that DA D2 receptors play a role in anxiety-like behavior and that the relationship between observed anxiety-like behaviors and D2 receptor expression changes through the lifespan.

Non-homogeneous stereological properties of the rat hippocampus from high-resolution 3D serial reconstruction of thin histological sections.

Integrating hippocampal anatomy from neuronal dendrites to whole system may help elucidate its relation to function. Toward this aim, we digitally traced the cytoarchitectonic boundaries of the dentate gyrus (DG) and areas CA3/CA1 throughout their entire longitudinal extent from high-resolution images of thin cryostatic sections of adult rat brain. The 3D computational reconstruction identified all isotropic 16 µm voxels with appropriate subregions and layers (http://krasnow1.gmu.edu/cn3/hippocampus3d). Overall, DG, CA3, and CA1 occupied comparable volumes (15.3, 12.2, and 18.8 mm(3), respectively), but displayed substantial rostrocaudal volumetric gradients: CA1 made up more than half of the posterior hippocampus, whereas CA3 and DG were more prominent in the anterior regions. The CA3/CA1 ratio increased from ∼0.4 to ∼1 septo-temporally because of a specific change in stratum radiatum volume. Next we virtually embedded 1.8 million neuronal morphologies stochastically resampled from 244 digital reconstructions, emulating the dense packing of granular and pyramidal layers, and appropriately orienting the principal dendritic axes relative to local curvature. The resulting neuropil occupancy reproduced recent electron microscopy data measured in a restricted location. Extension of this analysis across each layer and subregion over the whole hippocampus revealed highly non-homogeneous dendritic density. In CA1, dendritic occupancy was >60% higher temporally than septally (0.46 vs. 0.28, s.e.m. ∼0.05). CA3 values varied both across subfields (from 0.35 in CA3b/CA3c to 0.50 in CA3a) and layers (0.48, 0.34, and 0.27 in oriens, radiatum, and lacunosum-moleculare, respectively). Dendritic occupancy was substantially lower in DG, especially in the supra-pyramidal blade (0.18). The computed probability of dendrodendritic collision significantly correlated with expression of the membrane repulsion signal Down syndrome cell adhesion molecule (DSCAM). These heterogeneous stereological properties reflect and complement the non-uniform molecular composition, circuit connectivity, and computational function of the hippocampus across its transverse, longitudinal, and laminar organization.

Persisting changes in basolateral amygdala mRNAs after chronic ethanol consumption.

Adolescent alcohol use is common and evidence suggests that early use may lead to an increased risk of later dependence. Persisting neuroadaptions in the amygdala as a result of chronic alcohol use have been associated with negative emotional states that may lead to increased alcohol intake. This study assessed the long-term impact of ethanol consumption on levels of several basolateral amygdala mRNAs in rats that consumed ethanol in adolescence or adulthood. Male Long-Evans rats were allowed restricted access to ethanol or water during adolescence (P28, n=11, controls=11) or adulthood (P80, n=8, controls=10) for 18 days. After a sixty day abstinent period, the brain was removed and sections containing the basolateral amygdala were taken. In situ hybridization was performed for GABA(A) alpha(1), glutamic acid decarboxylase (GAD(67)), corticotropin releasing factor (CRF), and N-methyl-D-aspartate (NMDA) NR2A mRNAs. A significant decrease was observed in GABA(A) alpha(1), GAD(67), and CRF, but not NR2A, mRNAs in adult rats that consumed ethanol in comparison to controls. No significant changes were seen in adolescent consumers of ethanol for any of the probes tested. A separate analysis for each probe in the piriform cortex ascertained that the changes after ethanol consumption were specific to the basolateral amygdala. These results indicate that chronic ethanol consumption induces age-dependent alterations in basolateral amygdala neurochemistry.

Reduced GAP-43 mRNA in dorsolateral prefrontal cortex of patients with schizophrenia.

Schizophrenia has been associated with anatomical and functional abnormalities of the dorsolateral prefrontal cortex (DLPFC), which may reflect abnormal connections of DLPFC neurons. We measured mRNA levels of growth-associated protein (GAP-43), a peptide linked to the modifiability of neuronal connections, in post-mortem brain tissue from two cohorts of patients with schizophrenia and controls. Using the RNase protection assay (RPA), we found a significant reduction in GAP-43 mRNA in the DLPFC, but not in the hippocampus, of patients with schizophrenia. With in situ hybridization histo- chemistry (ISHH), performed on a separate cohort, we confirmed the reduction of GAP-43 mRNA in the DLPFC of patients with schizophrenia. We detected reduced GAP-43 mRNA per neuron in layers III, V and VI of patients with schizophrenia compared with normal controls and patients with bipolar disorder. Thus, glutamate neurons in DLPFC of schizophrenic patients may synthesize less GAP-43, which could reflect fewer and/or less modifiable connections than those in normal human brain, and which may be consistent with the deficits of prefrontal cortical function that characterize schizophrenia.

Mammal-like striatal functions in Anolis. II. Distribution of dopamine D(1) and D(2) receptors, and a laminar pattern of basal ganglia sub-systems.

We used in situ autoradiographic ligand binding methods to determine the occurrence and distribution of dopamine D(1) and D(2) receptor sub-types in the anole lizard, Anolis carolinensis. Both were present and exhibited pharmacological specificity characteristics similar to those described for mammals. However, unlike in mammals where in the neostriatum [outside the nucleus accumbens/olfactory tubercle complex (NA/OT)] these receptors exhibit only slight dorsolateral (D(2) high, D(1) low) to ventromedial (D(1 )high, D(2) low) gradients that co mingle extensively, in the anole striatum outside the NA/OT there was a striking laminar pattern, with little if any overlap between D(2) (high in a dorsal band) and D(1) (high ventral to the D(2) band) distributions. As D(1) receptors are related to the direct and D(2) to the indirect basal ganglia (BG) subsystems in mammals, we also determined anole striatal distributions of pre-proenkephalin mRNA, a marker for striatal efferents to the indirect BG subsystem in mammals. Here, too, there was a striking laminar pattern, with pre-proenkephalin mRNA in a band similar to that seen for D(2) receptors. The crisp neuroanatomical separation between these classic BG subsystem markers in Anolis striatum make this species attractive for the study of such systems' functions during behavior.

Inbred mouse strains differ in their sensitivity to an antiseizure effect of MK-801.

The ability of MK-801 to antagonize the electrical precipitation of tonic hindlimb extension was studied in four inbred mouse strains (BALB/c, C57BL/6, AKR, and DBA/2) and the outbred NIH Swiss strain, using an incremental electroconvulsive shock (IECS) procedure. Strains differed in the threshold voltages required for the electrical precipitation of tonic hindlimb extension. They also differed in their sensitivity to antagonism of electrically precipitated tonic hindlimb extension by MK-801. The BALB/c, C57BL, and DBA strains required a significant elevation of the threshold voltage for the elicitation of tonic hindlimb extension in response to 0.18 mg/kg of MK-801, the lowest dose tested; whereas the AKR and NIH Swiss strains did not respond to this dose with an increase of threshold voltage for the elicitation of tonic hindlimb extension. Moreover, a higher percentage of BALB/c mice were maximally protected against seizure elicitation by MK-801 over a dosage range of 0.18 to 0.56 mg/kg, compared to the other strains. The demonstration of strain differences suggests that genetic factors influence the anticonvulsant properties of MK-801.

Effects of prefrontal cortical lesions on neuropeptide and dopamine receptor gene expression in the striatum-accumbens complex.

In the rat, neurochemical, behavioral, and anatomical investigations suggest that medial prefrontal cortical input modulates the activity of the basal ganglia. To understand how prefrontal dysfunction might alter striatal-accumbens function, in situ hybridization histochemistry with S35-labeled oligonucleotide probes was used to assess changes in striatal-accumbens gene expression following bilateral excitotoxic ibotenic acid (IA) lesions of the rat medial prefrontal cortex. Quantitative densitometry was used to measure changes in mRNA levels for preproenkephalin A (ENK), D1 dopamine receptor, protachykinin (SubP), glutamic acid decarboxylase (GAD65), and D2 dopamine receptor. No differences were found between sham and lesion groups for ENK, D1, SubP, or GAD65 mRNA levels in the striatum or nucleus accumbens (NAC). D2 receptor mRNA levels were, however, significantly higher in the dorsomedial striatum and in the core area of the NAC of the lesioned rats. Although the functional significance of increased D2 mRNA is unclear, these findings demonstrate that glutamate mPFC projections modulate gene expression in relatively regionally-localized subcortical neuronal populations.

VASE-containing N-CAM isoforms are increased in the hippocampus in bipolar disorder but not schizophrenia.

The neural cell adhesion molecule (N-CAM) is a cell recognition molecule that is involved in cellular migration, synaptic plasticity, and CNS development. In schizophrenia, a 105- to 115-kDa N-CAM protein is increased in CSF and in the hippocampus and prefrontal cortex. The variable alternatively spliced exon (VASE) of N-CAM is developmentally regulated and can be spliced into any of the major 120-, 140-, and 180-kDa N-CAM isoforms. We determined that the variable alternative spliced exon of N-CAM (VASE) also is increased in bipolar disorder by quantitative Western immunoblot. VASE immunoreactive proteins (triplet bands around 140 kDa and a single band around 145 kDa) were identified in soluble and membrane brain extracts and quantified in the hippocampus. Soluble VASE 140 kDa was increased in the hippocampus of patients with bipolar disorder as compared to controls, patients with schizophrenia, and suicide cases. Membrane-extracted VASE 140 and 145 kDa were unchanged in the same groups. Multiple 145-kDa VASE-immunoreactive proteins that also reacted to an N-CAM antibody were separated by isoelectric focusing and electrophoresis followed by western immunoblotting; however, the VASE 140-kDa proteins were only weakly N-CAM immunoreactive. By immunohistochemistry, VASE colocalized with GFAP-positive astrocytes in the hippocampus. VASE immunostaining was also observed in the cytoplasm of CA4 pyramidal neurons that were positive for phosphorylated high molecular weight neurofilament and synaptophysin terminals. Thus no differences in VASE were found in patients with schizophrenia, but there was a marked increase of VASE immunoreactive proteins in bipolar disorder. It is possible that abnormal regulation of N-CAM proteins results in differing patterns of abnormal expression in neuropsychiatric disorders.

Abnormal cholecystokinin mRNA levels in entorhinal cortex of schizophrenics.

Limbic cortical regions, including anterior cingulate cortex (ACC), prefrontal cortex (PFC) and entorhinal cortex (ERC), have been implicated in the neuropathology of schizophrenia. Glutamate projection neurons connect these limbic cortical regions to each other, as well as to the terminal fields of the striatal/accumbens dopamine neurons. Subsets of these glutamate projection neurons, and of the GABA interneurons in cortex, contain the neuropeptide cholecystokinin (CCK). In an effort to study the limbic cortical glutamate projection neurons and GABA interneurons in schizophrenia, we have measured CCK mRNA with in situ hybridization histochemistry in postmortem samples of dorsolateral (DL)PFC, ACC and ERC of seven schizophrenics, nine non-psychotic suicides and seven normal controls. CCK mRNA is decreased in ERC (especially layers iii vi) and subiculum in schizophrenics relative to controls. Cellular analysis indicates that there is a decrease in density of CCK mRNA in labelled neurons. In so far as ERC CCK mRNA is not reduced in rats treated chronically with haloperidol, this decrease in schizophrenics does not appear to be related to neuroleptic treatment. In contrast, in DLPFC, where schizophrenics do not differ from normals, the suicide victims have elevated CCK mRNA (especially in layers v and vi), and increased cellular density of CCK mRNA, relative to both normals and schizophrenics. These results lend further support for the involvement of ERC and hippocampus in schizophrenia, suggesting that neurons that utilize CCK may be particularly important. Similarly, an increase in CCK mRNA levels in the PFC of suicides adds to a growing body of evidence implicating this structure in this pathological state. In so far as CCK is co-localized with GABA or glutamate in cortical neurons, both of these neuronal populations need to be studied further in schizophrenia and suicide.

Neuropathology of suicide. A review and an approach.

Neuropathology is one approach to the effort to elucidate the pathophysiology of suicide. Initial neurochemical studies focusing on the roles of serotonin (5-HT) and noradrenaline (NE) abnormalities in brains of suicide victims have been somewhat inconsistent. More recently developed methodologies, including quantitative receptor autoradiography, immunoblotting, immunohistochemistry, cell morphometry, in situ hybridization, Northern analysis, solution hybridization/RNase protection assay, reverse transcriptase polymerase chain reaction, and genotyping, which have already been applied successfully in studies of other disorders of brain structure or function, are now increasingly being adopted for postmortem studies of suicide. These new strategies are adding convergent evidence for brain 5-HT and NE dysfunction in the etiology of suicide susceptibility, refining the neuroanatomical localization of this dysfunction, and in addition, implicating heretofore unsuspected candidate neurotransmitter systems in the neuropathological substrates of suicide susceptibility. It is argued here that the confluence of the availability of suitable postmortem samples and this augmentation of our armamentarium of techniques promises the attainment of important new insights into the biological underpinnings of suicide from postmortem research. It is to be hoped that this new knowledge might inspire novel pharmacotherapeutic strategies for the prevention of suicide.

Pharmacological and neurochemical differences between acute and tardive vacuous chewing movements induced by haloperidol.

Late onset vacuous chewing movements (VCMs) from chronic neuroleptic treatment have been used as a rat model of tardive dyskinesia (TD). Early onset VCMs have also been observed, raising questions about the validity of this model. To assess the relationship between these two types of VCMs, pharmacological and neurochemical properties of early and late onset VCMs were compared. "Acute" VCMs were induced by daily intraperitoneal injections for 1-21 days. "Tardive" VCMs were induced by intramuscular injections of haloperidol decanoate every 3 weeks for 30 weeks followed by a 24-week withdrawal period. Suppression was attempted for both types of VCMs using several doses of intraperitoneal haloperidol. Striatonigral activation was assessed by measuring mRNA expression levels of the neuropeptides dynorphin and substance P using in situ hybridization histochemistry. Enkephalin mRNA was also measured as an index of striatopallidal activation. The results indicate that acute VCMs cannot be suppressed with increased doses of haloperidol and are associated with reduced dynorphin and substance P. This profile is similar to that seen with an animal model of parkinsonism. Tardive VCMs, in contrast, were markedly suppressed by haloperidol. They have previously been shown to be associated with increased striatonigral activation as indicated by increased dynorphin mRNA. Enkephalin mRNA was elevated following both short and long term treatment. Although superficially similar, acute and tardive VCMs appear to have different pharmacological and neurochemical profiles, suggesting they are related to acute extrapyramidal side effects and tardive dyskinesia, respectively.

Amphetamine-induced c-fos mRNA expression is altered in rats with neonatal ventral hippocampal damage.

To further characterize the mechanisms underlying enhanced dopamine-related behaviors expressed during adulthood in rats with neonatal excitotoxic ventral hippocampal (VH) damage, we studied the expression of c-fos mRNA in these rats after a single saline or amphetamine (AMPH) (10 mg/kg, i.p.) injection using in situ hybridization. The VH of rat pups was lesioned with ibotenic acid on postnatal day 7 (PD7). At the age of 90 days, rats were challenged with AMPH or saline, and the expression of c-fos mRNA using an oligonucleotide probe was assessed 30, 90, and 180 min later. AMPH significantly increased c-fos mRNA expression in medial prefrontal cortex, piriform cortex, cingulate cortex, septal region, and dorsolateral and ventromedial striatum in control and lesioned rats. However, this response to AMPH was attenuated 30 min after AMPH injection in all of these regions in the lesioned as compared to the sham-operated rats. No significant changes were seen at other time points. These results indicate that the neonatal VH lesion alters time-dependent intracellular signal transduction mechanisms measured by AMPH-induced c-fos mRNA expression in cortical and subcortical brain regions. Changes in c-fos mRNA expression in this putative animal model of schizophrenia may have implications for long-term alterations in cellular phenotype because of altered regulation of certain target genes.

The neuropathology of schizophrenia.

Neuropharmacologic discoveries have driven much of the research on neural substances of schizophrenia since the advent of neuroleptic drugs, which appear to share blockade of dopamine receptors. as their common denominator. Yet, despite concerted efforts to identify the source of putative dopaminergic hyperactivity in the brain in schizophrenia, definitive evidence for the "dopamine hypothesis of schizophrenia" remains elusive. More recently, a "neural systems" approach, focussing on the limbic system, has yielded substantial convergent evidence, from both in vivo imaging and postmortem morphological, biochemical, and molecular biological methods, implicating limbic cortex in the neuropathology underlying schizophrenia. Moreover, these limbic cortical regions modulate dopaminergic function in the striatum and nucleus accumbens, via glutamatergic projections. Increasingly, focus is shifting to a role for glutamatergic dysfunction in schizophrenia, opening the possibility that drugs that act upon glutamate function, either directly or indirectly via co-modulators of glutamate transmission, could potentially be developed as adjunctive or primary novel pharmacotherapeutic strategies.

Co-administration of progabide inhibits haloperidol-induced oral dyskinesias in rats.

Vacuous chewing movements in rats may be an animal analogue of the human motor disorder, tardive dyskinesia. The movements are phenomenologically and pharmacologically similar to tardive dyskinesia. The pathophysiology of these involuntary oral movements, and perhaps of tardive dyskinesia, are likely to include both dopamine receptor changes, and alterations in GABA (gamma-aminobutyric acid) system function. In an attempt to test the involvement of GABA system dysfunction in these movements, we treated rats chronically with water alone, haloperidol alone, the GABA agonist progabide alone, and haloperidol plus progabide. Sprague-Dawley rats received haloperidol (1.5 mg/kg per day) in their drinking water and progabide (100 mg/kg per day) in their food for 12 months. After 12 months of treatment, haloperidol had induced vacuous chewing movements when administered alone, but the prevalence of the movements was decreased by 40% with the coadministration of progabide. Moreover, the haloperidol-progabide-treated animals did not merely demonstrate movement suppression but actual inhibition of movement onset, as determined by an additional progabide-withdrawal experiment. These data would suggest that progabide and perhaps other GABAmimetic compounds can prevent the development of tardive dyskinesia in man.

Oral movements induced by interference with nigral GABA neurotransmission: relationship to tardive dyskinesias.

Previous studies have shown that the emergence of spontaneous dyskinetic behaviors, such as vacuous chewing movements, following several months of neuroleptic treatment in the rat, is correlated with depletion of nigral GABA. To explore the specificity of this relationship, we acutely interfered with nigral GABA transmission pharmacologically, by microinfusing either the GABA receptor antagonist, bicuculline, or the GABA-depleting agent, isoniazid, bilaterally into substantia nigra. We found that both acute treatments induced vacuous chewing movements in rats. Moreover, the time to onset of action of each of these drugs corresponded to the onsets of their respective effects on GABA transmission. In addition, we found that the application of muscimol into the target field of the nigrotegmental projection, which has been shown to block gnawing elicited by nigral GABA receptor stimulation, completely abolished elicitation of vacuous chewing movements by intranigral isoniazid. In contrast, bilateral microinfusions of muscimol into the nigrocollicular target region, in the deep layers of superior colliculus, blocked elicitation of gnawing by intranigral muscimol, but completely spared elicitation of vacuous chewing movements by intranigral isoniazid. We conclude that qualitatively different dyskinetic syndromes can be produced by bidirectional perturbations of nigral GABA function and are differentially mediated by nigrotegmental and nigrotectal projections. These syndromes may represent animal models of distinct components of extrapyramidal side effects of chronic neuroleptic administration.

Differential effects of chronic treatment with chlorpromazine versus cocaine on behavioral responsiveness to nigral GABA receptor stimulation.

We examined whether any locus postsynaptic to the GABAergic striatonigral projection might be involved in the enhancement of behavioral response to dopaminergic stimulants induced by chronic treatment with either neuroleptic drugs or stimulants. Rats exposed chronically to either chlorpromazine or cocaine were tested for behavioral responses to bilateral intranigral microinfusions of muscimol (2.5 ng bilaterally). Chronic chlorpromazine-treated rats responded with more intense stereotypy to nigral muscimol than controls, but chronic cocaine-treated rats were less responsive than controls to intranigral muscimol. It therefore appears that changes in the responsiveness of neural outputs from, or distal to, substantia nigra may contribute to the enhanced behavioral effects of dopaminergic stimulants caused by chronic exposure to dopaminergic antagonists, but that the critical neural changes responsible for cocaine sensitization probably do not occur at, or distal to, the nigral outflow from the basal ganglia. At least at the nigral level, different neural mechanisms evidently mediate the increased stereotypy response to dopaminergic stimulation induced by chronic administration of either neuroleptic or stimulant drugs.

Muscimol microinfused into the nigrotegmental target area blocks selected components of behavior elicited by amphetamine or cocaine.

We examined whether behaviors elicited by systemic administration of the stimulants amphetamine and cocaine involve the relay of outflow from basal ganglia to the target region of the GABAergic nigrotegmental pathway (nigrotegmental target area: NTT), in the region of the pedunculopontine nucleus. Bilateral microinfusions of the GABAA agonist muscimol (25 ng in each side) were administered into the NTT in stimulant-treated rats. Amphetamine- and cocaine-induced stereotyped sniffing and repetitive head movements were totally abolished by muscimol infusions. In contrast, stimulant-induced locomotion and snout contact fixation were spared or enhanced. These observations, which extend those of Childs and Gale (1983b) with apomorphine-induced gnawing, further implicate the GABAergic nigrotegmental pathway in the mediation of basal ganglia-related motor dysfunction. On the other hand, cocaine- and amphetamine-induced locomotion and snout contact fixation do not appear to depend upon mediation through GABA-receptive neurons in the NTT. Thus, the NTT may be selective for the processing of outflow from the basal ganglia.