Expression and function of dopamine receptors in the developing medial frontal cortex and striatum of the rat.

The timeline of dopamine (DA) system maturation and the signaling properties of DA receptors (DRs) during rat brain development are not fully characterized. We used in situ hybridization and quantitative PCR to map DR mRNA transcripts in the medial frontal cortex (mFC) and striatum (STR) of the rat from embryonic day (E) 15 to E21. The developmental trajectory of DR mRNAs revealed distinct patterns of DA receptors 1 and 2 (DRD1, DRD2) in these brain regions. Whereas the mFC had a steeper increase in DRD1 mRNA, the STR had a steeper increase in DRD2 mRNA. Both DR mRNAs were expressed at a higher level in the STR compared with the mFC. To identify the functional properties of DRs during embryonic development, the phosphorylation states of cyclic AMP response element binding protein, extracellular signal-regulated kinase 1/2, and glycogen synthase kinase 3 beta were examined after DR stimulation in primary neuronal cultures obtained from E15 and E18 embryos and cultured for 3 days to ensure a stable baseline level. DR-mediated signaling cascades were functional in E15 cultures in both brain regions. Because DA fibers do not reach the mFC by E15, and DA was not present in cultures, these data indicate that DRs can become functional in the absence of DA innervation. Because activation of DR signal transduction pathways can affect network organization of the developing brain, maternal exposure to drugs that affect DR activity may be liable to interfere with fetal brain development.

Isolation rearing impairs wound healing and is associated with increased locomotion and decreased immediate early gene expression in the medial prefrontal cortex of juvenile rats.

In addition to its maladaptive effects on psychiatric function, psychosocial deprivation impairs recovery from physical illness. Previously, we found that psychosocial deprivation, modeled by isolation rearing, depressed immediate early gene (IEG) expression in the medial prefrontal cortex (mPFC) and increased locomotion in the open field test [Levine JB, Youngs RM, et al. (2007) Isolation rearing and hyperlocomotion are associated with reduced immediate early gene expression levels in the medial prefrontal cortex. Neuroscience 145(1):42-55]. In the present study, we examined whether similar changes in behavior and gene expression are associated with the maladaptive effects of psychosocial deprivation on physical injury healing. After weaning, anesthetized rats were subjected to a 20% total body surface area third degree burn injury and were subsequently either group or isolation reared. After 4 weeks of either isolation or group rearing (a period that encompasses post-wearing and early adolescence), rats were killed, and their healing and gene expression in the mPFC were assessed. Locomotion in the open field test was examined at 3 weeks post-burn injury. We found that: 1) gross wound healing was significantly impaired in isolation-reared rats compared with group-reared rats, 2) locomotion was increased and IEG expression was suppressed for isolation-reared rats during burn injury healing, 3) the decreased activity in the open field and increased IEG expression was greater for burn injury healing group-reared rats than for uninjured group-reared rats, 4) the degree of hyperactivity and IEG suppression was relatively similar between isolation-reared rats during burn injury compared with uninjured isolation-reared rats. Thus, in the mPFC, behavioral hyperactivity to novelty (the open field test) along with IEG suppression may constitute a detectable biomarker of isolation rearing during traumatic physical injury. Implications of the findings for understanding, assessing, and treating the maladaptive effects of psychosocial deprivation on physical healing during childhood are discussed.

Isolation rearing and hyperlocomotion are associated with reduced immediate early gene expression levels in the medial prefrontal cortex.

Environmental deprivation contributes in important ways to the development of a wide range of psychiatric disorders. Isolation rearing of rodents, a model for environmental deprivation in humans, consistently produces hyperlocomotion, which provides a measurable parameter to study the underlying mechanisms of early adverse psychosocial stressors. Male Sprague-Dawley rat pups were separated from dams at postnatal (PN) day 20 and reared either in groups of three or in isolation. On PN 38, locomotion was assessed in the open field. On PN 46, rats were killed and gene expression patterns examined in the medial prefrontal cortex (mPFC). Isolation-reared rats displayed increased locomotor activity and decreased resting time in the open field. Specific gene expression patterns in the mPFC were associated with both isolation rearing and hyperlocomotive behavior in the open field. Genes involved in these expression patterns included immediate early genes (IEGs) and genes that regulate cell differentiation and apoptosis. The study of these genes could provide important insights into how abnormal early psychosocial events affect brain function and behavior.

Acute amygdalar activation induces an upregulation of multiple monoamine G protein coupled pathways in rat hippocampus.

A "partial" rodent model for schizophrenia has been used to characterize the regulation of hippocampal genes in response to amygdalar activation. At 96 h after the administration of picrotoxin into the basolateral nucleus, we have observed an increase in the expression of genes associated with 18 different monoamine (ie adrenergic alpha 1, alpha 2 and beta 2, serotonergic 5HT5b and 5HT6, dopamine D4 and muscarinic m1, m2 and m3) and peptide (CCK A and B, angiotensin 1A, mu and kappa opiate, FSH, TSH, LH, GNRH, and neuropeptide Y) G-protein coupled receptors (GPCRs). These latter receptors are associated with three different G protein signaling pathways (Gq, Gs, and Gi) in which significant changes in gene expression were also noted for adenylate cyclase (AC4), phosphodiesterase (PDE4D), protein kinase A (PKA), and protein kinase C (PKC). Quantitative RT-PCR was used to validate the results and demonstrated that there were predictable increases of three GPCRs selected for this analysis, including the dopamine D4, alpha 1b, and CCK-B receptors. Eight out of the nine monoamine receptors showing these changes have moderate to high affinity for the atypical antipsychotic, clozapine. Taken together, these results suggest that amygdalar activation may play a role in the pathophysiology and treatment of psychosis by regulating the activity of multiple GPCR and metabolic pathways in hippocampal cells.

Hippocampal neurons in schizophrenia.

The hippocampus is crucial for normal brain function, especially for the encoding and retrieval of multimodal sensory information. Neuropsychiatric disorders such as temporal lobe epilepsy, amnesia, and the dementias are associated with structural and functional abnormalities of specific hippocampal neurons. More recently we have also found evidence for a role of the hippocampus in the pathophysiology of schizophrenia. The most consistent finding is a subtle, yet significant volume difference in schizophrenia. Here we review the cellular and molecular basis of smaller hippocampal volume in schizophrenia. In contrast to neurodegenerative disorders, total hippocampal cell number is not markedly decreased in schizophrenia. However, the intriguing finding of a selective loss of hippocampal interneurons deserves further study. Two neurotransmitter receptors, the GABAA and AMPA/kainate glutamate receptors, appear to be abnormal, whereas changes of the NMDA glutamate receptor are less robust. The expression of several genes, including those related to the GABAergic system, neurodevelopment, and synaptic function, is decreased in schizophrenia. Taken together, recent studies of hippocampal cell number, protein expression, and gene regulation point towards an abnormality of hippocampal architecture in schizophrenia.

cAMP response element-binding protein is required for dopamine-dependent gene expression in the intact but not the dopamine-denervated striatum.

The cAMP response element-binding protein (CREB) is believed to play a pivotal role in dopamine (DA) receptor-mediated nuclear signaling and neuroplasticity. Here we demonstrate that the significance of CREB for gene expression depends on the experimental paradigm. We compared the role of CREB in two different but related models: l-DOPA administration to unilaterally 6-hydroxydopamine lesioned rats, and cocaine administration to neurologically intact animals. Antisense technology was used to produce a local knockdown of CREB in the lateral caudate-putamen, a region that mediates the dyskinetic or stereotypic manifestations associated with l-DOPA or cocaine treatment, respectively. In intact rats, CREB antisense reduced both basal and cocaine-induced expression of c-Fos, FosB/DeltaFosB, and prodynorphin mRNA. In the DA-denervated striatum, CREB was not required for l-DOPA to induce these gene products, nor did CREB contribute considerably to DNA binding activity at cAMP responsive elements (CREs) and CRE-like enhancers. DeltaFosB-related proteins and JunD were the main contributors to both CRE and AP-1 DNA-protein complexes in l-DOPA-treated animals. In behavioral studies, intrastriatal CREB knockdown caused enhanced activity scores in intact control animals and exacerbated the dyskinetic effects of acute l-DOPA treatment in 6-OHDA-lesioned animals. These data demonstrate that CREB is not required for the development of l-DOPA-induced dyskinesia in hemiparkinsonian rats. Moreover, our results reveal an unexpected alteration of nuclear signaling mechanisms in the parkinsonian striatum treated with l-DOPA, where AP-1 transcription factors appear to supersede CREB in the activation of CRE-containing genes.

Antipsychotic drugs and neuroplasticity: insights into the treatment and neurobiology of schizophrenia.

This paper reviews the evidence that antipsychotic drugs induce neuroplasticity. We outline how the synaptic changes induced by the antipsychotic drug haloperidol may help our understanding of the mechanism of action of antipsychotic drugs in general, and how they may help to elucidate the neurobiology of schizophrenia. Studies have provided compelling evidence that haloperidol induces anatomical and molecular changes in the striatum. Anatomical changes have been documented at the level of regional brain volume, synapse morphology, and synapse number. At the molecular level, haloperidol has been shown to cause phosphorylation of proteins and to induce gene expression. The molecular responses to conventional antipsychotic drugs are predominantly observed in the striatum and nucleus accumbens, whereas atypical antipsychotic drugs have a subtler and more widespread impact. We conclude that the ability of antipsychotic drugs to induce anatomical and molecular changes in the brain may be relevant for their antipsychotic properties. The delayed therapeutic action of antipsychotic drugs, together with their promotion of neuroplasticity suggests that modification of synaptic connections by antipsychotic drugs is important for their mode of action. The concept of schizophrenia as a disorder of synaptic organization will benefit from a better understanding of the synaptic changes induced by antipsychotic drugs.

Altered responsiveness to cocaine and increased immobility in the forced swim test associated with elevated cAMP response element-binding protein expression in nucleus accumbens.

Drugs of abuse regulate the transcription factor cAMP response element-binding protein (CREB) in striatal regions, including the nucleus accumbens (NAc). To explore how regulation of CREB in the NAc affects behavior, we used herpes simplex virus (HSV) vectors to elevate CREB expression in this region or to overexpress a dominant-negative mutant CREB (mCREB) that blocks CREB function. Rats treated with HSV-mCREB in place conditioning studies spent more time in environments associated with cocaine, indicating increased cocaine reward. Conversely, rats treated with HSV-CREB spent less time in cocaine-associated environments, indicating increased cocaine aversion. Studies in which drug-environment pairings were varied to coincide with either the early or late effects of cocaine suggest that CREB-associated place aversions reflect increased cocaine withdrawal. Because cocaine withdrawal can be accompanied by symptoms of depression, we examined how altered CREB function in the NAc affects behavior in the forced swim test (FST). Elevated CREB expression increased immobility in the FST, an effect that is opposite to that caused by standard antidepressants and is consistent with a link between CREB and dysphoria. Conversely, overexpression of mCREB decreased immobility, an effect similar to that caused by antidepressants. Moreover, the kappa opioid receptor antagonist nor-Binaltorphimine decreased immobility in HSV-CREB- and HSV-mCREB-treated rats, suggesting that CREB-mediated induction of dynorphin (an endogenous kappa receptor ligand) contributes to immobility behavior in the FST. Exposure to the FST itself dramatically increased CREB function in the NAc. These findings raise the possibility that CREB-mediated transcription within the NAc regulates dysphoric states.

Potassium chloride depolarization mediates CREB phosphorylation in striatal neurons in an NMDA receptor-dependent manner.

Potassium chloride (KCl)-depolarization has been used to study the properties of L-type Ca2+ channel-mediated signal transduction in hippocampal neurons. Calcium influx through L-type Ca2+ channels stimulates a second messenger pathway that transactivates genes under the regulatory control of the Ca2+-and cyclic AMP-responsive element (CRE). Here, we show that in striatal neurons, but not in hippocampal neurons, CRE binding protein (CREB) phosphorylation and CRE-mediated gene expression after KCl-depolarization depends on functional NMDA receptors. This difference in NMDA receptor dependence is not due to different properties of L-type Ca2+ channels in either neuronal type, but rather to different neuron-intrinsic properties. Despite this variation, the second messenger pathway activated by KCl requires Ca2+/calmodulin (CaM) kinase for CREB phosphorylation in both neuronal types. We conclude that depolarization by KCl works differently in striatal and hippocampal neurons.

Intracellular modulation of NMDA receptor function by antipsychotic drugs.

The present study deals with the functional interaction of antipsychotic drugs and NMDA receptors. We show that both the conventional antipsychotic drug haloperidol and the atypical antipsychotic drug clozapine mediate gene expression via intracellular regulation of NMDA receptors, albeit to different extents. Data obtained in primary striatal culture demonstrate that the intraneuronal signal transduction pathway activated by haloperidol, the cAMP pathway, leads to phosphorylation of the NR1 subtype of the NMDA receptor at (897)Ser. Haloperidol treatment is likewise shown to increase (897)Ser-NR1 phosphorylation in rats in vivo. Mutation of (896)Ser and (897)Ser to alanine, which prevents phosphorylation at both sites, inhibits cAMP-mediated gene expression. We conclude that antipsychotic drugs have the ability to modulate NMDA receptor function by an intraneuronal signal transduction mechanism. This facilitation of NMDA activity is necessary for antipsychotic drug-mediated gene expression and may contribute to the therapeutic benefits as well as side effects of antipsychotic drug treatment.

Anatomic and molecular principles of psychopharmacology. A primer for psychiatrists.

Psychopharmacology uses chemicals to modulate human brain function. Three basic principles of neurotransmission may help to understand the current practice of clinical psychopharmacology. First, the anatomic organization of neurotransmitter systems determines their behavioral affiliation. Second, neurotransmitter receptors modulate the electrical properties (via ion channels) or the biochemical properties (via second-messenger systems) of neurons. Third, the intracellular integration of receptor-mediated responses leads to immediate or delayed effects on neuronal function.

Glutamate carboxypeptidase II is expressed by astrocytes in the adult rat nervous system.

The enzyme glutamate carboxypeptidase II (GCP II) has been cloned from rat brain and human prostate. This enzyme, which catabolizes the neuropeptide N-acetylaspartylglutamate, has also been known as N-acetylated alpha-linked acidic dipeptidase (NAALADase), and is identical to the prostate-specific membrane antigen and to the jejunal folylpoly-gamma-glutamate carboxypeptidase. The goals of the present study were to elucidate the cell specificity and regional pattern of GCP II expression in the rat nervous system by using Northern blots and enzymatic assays of brain and subfractionated primary neuronal and glial cultures together with in situ hybridization histochemistry (ISHH) in sections of adult rat tissue. GCP II activity was assayed in astrocyte cultures (4.4 pmol/mg protein per minute), neuronal-glial cocultures (2.5 pmol/mg protein per minute) and neuron-enriched cultures (0.38 pmol/mg protein per minute), with the activity in each preparation correlating to its astrocytic content (r = 0.99). No activity was detected in cultured oligodendrocytes or microglia. Northern blots probed with a GCP II cDNA detected mRNAs exclusively in activity-positive cell preparations. ISHH results show that GCP II is expressed by virtually all astrocytes, by Bergmann glial cells in cerebellum, by Müller cells in retina and by the satellite cells in dorsal root ganglia. Astrocytes in select groups of nuclei (e.g., habenula, supraoptic nucleus, pontine nucleus) contained pronounced levels of GCP II message. The data of the present study suggest that GCP II is expressed in the adult rat nervous system exclusively in astrocytic glial cells.

L-Type Ca(2+) channels are essential for glutamate-mediated CREB phosphorylation and c-fos gene expression in striatal neurons.

The second messenger pathways linking receptor activation at the membrane to changes in the nucleus are just beginning to be unraveled in neurons. The work presented here attempts to identify in striatal neurons the pathways that mediate cAMP response element-binding protein (CREB) phosphorylation and gene expression in response to NMDA receptor activation. We investigated the phosphorylation of the transcription factor CREB, the expression of the immediate early gene c-fos, and the induction of a transfected reporter gene under the transcriptional control of CREB after stimulation of ionotropic glutamate receptors. We found that neither AMPA/kainate receptors nor NMDA receptors were able to stimulate independently a second messenger pathway that led to CREB phosphorylation or c-fos gene expression. Instead, we saw a consecutive pathway from AMPA/kainate receptors to NMDA receptors and from NMDA receptors to L-type Ca(2+) channels. AMPA/kainate receptors were involved in relieving the Mg(2+) block of NMDA receptors, and NMDA receptors triggered the opening of L-type Ca(2+) channels. The second messenger pathway that activates CREB phosphorylation and c-fos gene expression is likely activated by Ca(2+) entry through L-type Ca(2+) channels. We conclude that in primary striatal neurons glutamate-mediated signal transduction is dependent on functional L-type Ca(2+) channels.

Molecular components of striatal plasticity: the various routes of cyclic AMP pathways.

Neuroplasticity serves an important role for normal striatal function and in disease states. One route to neuroplasticity involves activation of the transcription factor cyclic 3', 5'-adenosine monophosphate (cyclic AMP) response element binding protein (CREB) by phosphorylation of the amino acid 133Ser. Dopamine and glutamate, the two predominant neurotransmitters in the striatum, induce CREB phosphorylation in primary cultures of rat striatum through cyclic AMP and Ca2+ pathways. Here we present the role of N-methyl-D-aspartate receptors and Ca2+ in cyclic AMP-mediated CREB phosphorylation.

Kainic acid induces apoptosis in neurons.

Growing evidence suggests that non-N-methyl-D-aspartate receptor activation may contribute to neuronal death in both acute and chronic neurological diseases. The intracellular processes that mediate this form of neuronal death are poorly understood. We have previously characterized a model of kainic acid neurotoxicity using cerebellar granule cell neurons in vitro and we sought to determine the mechanism of kainic acid-induced neuronal degeneration. We found DNA laddering by agarose gel electrophoresis, cellular DNA fragmentation by in situ end labeling of DNA, and chromatin condensation using a fluorescent DNA intercalating dye, in cerebellar granule cells following exposure to kainic acid (100 microM). Aurintricarboxylic acid protected cerebellar granule cells from kainic acid-induced death. While the morphological and biochemical features of neuronal death induced by kainic acid resembled low K(+)-induced apoptosis in cerebellar granule cells, the time interval from the institution of the death promoting condition to neuronal death was shorter with kainic acid and did not require new protein or RNA synthesis. These results demonstrate that kainic acid receptor activation can induce transcription-independent apoptosis in neurons. This in vitro model should be useful in identifying the intracellular pathways that link kainic acid receptor activation with apoptosis.

Kainate induces apoptosis in neurons.

Growing evidence suggests that non-N-methyl-D-aspartate receptor activation may contribute to neuronal death in both acute and chronic neurological diseases. The intracellular processes that mediate this form of neuronal death are poorly understood. We have previously characterized a model of kainate neurotoxicity using cerebellar granule cell neurons in vitro and we sought to determine the mechanism of kainate-induced neurons degeneration. We found DNA, and chromatin condensation using a fluorescent DNA intercalating dye, in cerebellar granule cells following exposure to kainate (100 microM). Aurintricarboxylic acid protected cerebellar granule cells from kainate-induced death. While the morphological and biochemical features of neuronal death induced by kainate resembled low-K(+)-induced apoptosis in cerebellar granule cells; the time interval from the institution of the death-promoting condition to neuronal death was briefer with kainate and did not require new protein or RNA synthesis. These results demonstrate that kainate receptor activation can induce transcription-independent apoptosis in neurons. This in vitro model should be useful in identifying the intracellular pathways that link kainate receptor activation with apoptosis.

Amphetamine and dopamine-induced immediate early gene expression in striatal neurons depends on postsynaptic NMDA receptors and calcium.

Amphetamine and cocaine induce the expression of both immediate early genes (IEGs) and neuropeptide genes in rat striatum. Despite the demonstrated dependence of these effects on D1 dopamine receptors, which activate the cyclic AMP pathway, there are several reports that amphetamine and cocaine-induced IEG expression can be inhibited in striatum in vivo by NMDA receptor antagonists. We find that in vivo, the NMDA receptor antagonist MK-801 inhibits amphetamine induction of c-fos acutely and also prevents downregulation of IEG expression with chronic amphetamine administration. Such observations raise the question of whether dopamine/glutamate interactions occur at the level of corticostriatal and mesostriatal circuitry or within striatal neurons. Therefore, we studied dissociated striatal cultures in which midbrain and cortical presynaptic inputs are removed. In these cultures, we find that dopamine- or forskolin-mediated IEG induction requires Ca2+ entry via NMDA receptors but not via L-type Ca2+ channels. Moreover, blockade of NMDA receptors diminishes the ability of dopamine to induce phosphorylation of the cyclic AMP responsive element binding protein CREB. Although these results do not rule out a role for circuit-level dopamine/glutamate interactions, they demonstrate a requirement at the cellular level for interactions between the cyclic AMP and NMDA receptor pathways in dopamine-regulated gene expression in striatal neurons.