Dynorphin mRNA Levels Research Articles

Contextual recall of fear and morphine-place conditioning modulate hippocampal kappa opioid receptor and dynorphin mRNA levels

Contextual recall of fear and morphine-place conditioning modulate hippocampal kappa opioid receptor and dynorphin mRNA levels

Short-term withdrawal from repeated exposure to cocaine during adolescence modulates dynorphin mRNA levels and BDNF signaling in the rat nucleus accumbens

BackgroundEarly-life stressful events affect the neurobiological maturation of cerebral circuitries including the endogenous opioid system and the effects elicited by adolescent cocaine exposure on this system have been poorly investigated. Here, we evaluated whether cocaine exposure during adolescence causes short- or long-term alterations in mRNAs codifying for selected elements belonging to the opioid system. Moreover, since brain-derived neurotrophic factor (BDNF) may undergo simultaneous alterations with the opioid peptide dynorphin, we also evaluated its signaling pathway as well. MethodsAdolescent male rats were exposed to cocaine (20 mg/kg/day) from post-natal day (PND) 28 to PND42, approximately corresponding to human adolescence. After short- (PND45) or long-term (PND90) abstinence, prodynorphin-κ-opioid receptor (pDYN-KOP) and pronociceptin-nociceptin receptor (pN/OFQ-NOP) gene expression were evaluated in the nucleus accumbens (NAc) and hippocampus (Hip) together with the analysis of BDNF signaling pathways. ResultsIn the NAc of PND45 rats, pDYN mRNA levels were up-regulated, an effect paralled by increased BDNF signaling. Differently from NAc, pDYN mRNA levels were down-regulated in the Hip of PND45 rats without significant changes of BDNF pathway. At variance from PND45 rats, we did not find any significant alteration of the investigated parameters either in NAc and Hip of PND90 rats. ConclusionsOur results indicate that the short-term withdrawal from adolescent cocaine exposure is characterized by a parallel pDYN mRNA and BDNF signaling increase in the NAc. Given the depressive-like state experienced during short abstinence in humans, we hypothesize that such changes may contribute to promote the risk of cocaine abuse escalation and relapse.

Dopamine D4 Receptor Counteracts Morphine-Induced Changes in µ Opioid Receptor Signaling in the Striosomes of the Rat Caudate Putamen

The mu opioid receptor (MOR) is critical in mediating morphine analgesia. However, prolonged exposure to morphine induces adaptive changes in this receptor leading to the development of tolerance and addiction. In the present work we have studied whether the continuous administration of morphine induces changes in MOR protein levels, its pharmacological profile, and MOR-mediated G-protein activation in the striosomal compartment of the rat CPu, by using immunohistochemistry and receptor and DAMGO-stimulated [35S]GTPγS autoradiography. MOR immunoreactivity, agonist binding density and its coupling to G proteins are up-regulated in the striosomes by continuous morphine treatment in the absence of changes in enkephalin and dynorphin mRNA levels. In addition, co-treatment of morphine with the dopamine D4 receptor (D4R) agonist PD168,077 fully counteracts these adaptive changes in MOR, in spite of the fact that continuous PD168,077 treatment increases the [3H]DAMGO Bmax values to the same degree as seen after continuous morphine treatment. Thus, in spite of the fact that both receptors can be coupled to Gi/0 protein, the present results give support for the existence of antagonistic functional D4R-MOR receptor-receptor interactions in the adaptive changes occurring in MOR of striosomes on continuous administration of morphine.

Early modulation by the dopamine D4 receptor of morphine‐induced changes in the opioid peptide systems in the rat caudate putamen

The peptides dynorphin and enkephalin modulate many physiological processes, such as motor activity and the control of mood and motivation. Their expression in the caudate putamen (CPu) is regulated by dopamine and opioid receptors. The current work was designed to explore the early effects of the acute activation of D4 and/or μ opioid receptors by the agonists PD168,077 and morphine, respectively, on the regulation of the expression of these opioid peptides in the rat CPu, on transcription factors linked to them, and on the expression of μ opioid receptors. In situ hybridization experiments showed that acute treatment with morphine (10 mg/kg) decreased both enkephalin and dynorphin mRNA levels in the CPu after 30 min, but PD168,077 (1 mg/kg) did not modify their expression. Coadministration of the two agonists demonstrated that PD168,077 counteracted the morphine-induced changes and even increased enkephalin mRNA levels. The immunohistochemistry studies showed that morphine administration also increased striatal μ opioid receptor immunoreactivity but reduced P-CREB expression, effects that were blocked by the PD168,077-induced activation of D4 receptors. The current results present evidence of functional D4 -μ opioid receptor interactions, with consequences for the opioid peptide mRNA levels in the rat CPu, contributing to the integration of DA and opioid peptide signaling.

PPAR‐gamma‐mediated neuroprotection in a chronic mouse model of Parkinson’s disease

Rosiglitazone is a commonly prescribed insulin-sensitizing drug with a selective agonistic activity on the peroxisome proliferator-activated receptor-gamma (PPAR-gamma). PPAR-gamma can modulate inflammatory responses in the brain, and agonists might be beneficial in neurodegenerative diseases. In the present study we used a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine plus probenecid (MPTPp) mouse model of progressive Parkinson's disease (PD) to assess the therapeutic efficacy of rosiglitazone on behavioural impairment, neurodegeneration and inflammation. Mice chronically treated with MPTPp displayed typical features of PD, including impairment of motor and olfactory functions associated with partial loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNc), decrease of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) content and dynorphin (Dyn) mRNA levels in the caudate-putamen (CPu), intense microglial and astroglial response in the SNc and CPu. Chronic rosiglitazone, administered in association with MPTPp, completely prevented motor and olfactory dysfunctions and loss of TH-positive cells in the SNc. In the CPu, loss of striatal DA was partially prevented, whereas decreases in DOPAC content and Dyn were fully counteracted. Moreover, rosiglitazone completely inhibited microglia reactivity in SNc and CPu, as measured by CD11b immunostaining, and partially inhibited astroglial response assessed by glial fibrillary acidic protein immunoreactivity. Measurement of striatal MPP+ levels 2, 4, 6 h and 3 days after chronic treatment indicated that MPTP metabolism was not altered by rosiglitazone. The results support the use of PPAR-gamma agonists as a putative anti-inflammatory therapy aimed at arresting PD progression, and suggest that assessment in PD clinical trials is warranted.

Behavioral and biochemical correlates of the dyskinetic potential of dopaminergic agonists in the 6‐OHDA lesioned rat

Prolonged treatment with L-DOPA induces highly disabling dyskinesia in Parkinson's disease (PD) patients. In contrast, dopaminergic agonists display variably dyskinetic outcome, depending on pharmacokinetic/pharmacodynamic profile. The present study was aimed at assessing behavioral and biochemical correlates of intense or mild dyskinesia displayed by the different dopamine (DA) receptors stimulation in a rat model of PD. The effect of subchronic stimulation of the D(1) receptor by SKF38393, and the D(2)/D(3) receptor by ropinirole was evaluated in unilaterally 6-hydroxyDA-lesioned rats. Sensitization of contralateral turning (SCT) behavior and abnormal involuntary movements (AIMs) were assessed as behavioral correlates of dyskinetic responses. Opioid peptides mRNA in the dorsolateral striatum (dlStr) and glutamic acid decarboxylase (GAD67) mRNA content in globus pallidus (GP), were evaluated as an index of neuroadaptive changes occurring in the direct and indirect basal ganglia pathways. Subchronic SKF38393 caused AIMs and SCT whereas ropinirole elicited SCT only, indicating that both drugs induced some dyskinetic response, albeit of different type. Peptides mRNA evaluation in dlStr, showed that SKF38393 subchronic treatment was associated to an overexpression of both dynorphin (DYN) and enkephalin (ENK) mRNAs, in the direct and indirect striatal pathway respectively. In contrast, a decrease in DYN mRNA levels only was observed after treatment with ropinirole. Analysis of GAD67 mRNA levels in the GP showed an increase after both D(1) and D(2)/D(3) agonist treatments. Results suggest that presence of SCT alone or SCT plus AIMs might represent correlates of the differential severity of dyskinetic movements induced by treatment with low (ropinirole) or high (SKF38393) dyskinetic potential. Neuroadaptive increases in opioid peptide expression in both direct and indirect striatal pathways were associated to the appearance of AIMs alone. In contrast, increase of GAD67 mRNA in the GP was associated to both behavioral responses (SCT and AIMs), suggesting that neuroadaptive changes in this area were unrelated to the difference in dyskinetic potential of drugs.

Role of serotonin in the regulation of the dynorphinergic system by a κ-opioid agonist and cocaine treatment in rat CNS

It has been shown that chronic cocaine increases prodynorphin mRNA in the caudate putamen and decreases it in the hypothalamus. In addition, treatment with a κ-opioid receptor agonist produced the opposite effect on prodynorphin gene expression in these brain regions and also evoked a decrease in the hippocampus. It is already known that κ-opioid receptor agonists decrease the development of sensitization to some of the behavioral effects of cocaine. The serotonin system has also been shown to regulate dynorphin gene expression and a continuous infusion of fluoxetine induced prodynorphin gene expression in the same pattern as the κ-opioid agonist (+)(5a,7a,8b)-N-methyl-N-[7-(1-pyrrolidinyl)-1 oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U-69593) in the brain regions investigated. It is interesting to note that treatment with a continuous infusion of cocaine produced different effects on this parameter. To determine whether serotonin plays a role in the regulation of prodynorphin mRNA by κ-opioid agonists or cocaine, rats were treated with the serotonin depleter parachloroamphetamine (PCA). Beginning 24 h later, rats were treated with the selective κ-opioid agonist U-69593 for 5 days or continuously with cocaine for 7 days and prodynorphin mRNA was measured. Prodynorphin mRNA was decreased significantly in the hypothalamus, caudate putamen, and hippocampus of rats treated with a single injection of PCA. Subsequent to PCA administration the effects of U-69593 or cocaine on prodynorphin mRNA were differentially affected across brain regions. Prodynorphin gene expression was still increased by U-69593 treatment in the hypothalamus and decreased in the caudate putamen. Cocaine treatment still produced a decrease in this parameter in the hypothalamus and an increase in the caudate putamen. In contrast, in the hippocampus, the decrease in prodynorphin mRNA produced by U-69593 was no longer evident after PCA and cocaine, which previously had no effect, now increased it in the serotonin-depleted group. These findings suggest that serotonin is necessary to maintain normal levels of dynorphin mRNA in all of the investigated brain areas and that the regulation of prodynorphin mRNA expression by chronic treatment with a κ-opioid receptor agonist or cocaine requires serotonin in the hippocampus, but not in the hypothalamus or caudate putamen.

The corticotropin-releasing factor receptor-1 pathway mediates the negative affective states of opiate withdrawal

The negative affective symptoms of opiate withdrawal powerfully motivate drug-seeking behavior and may trigger relapse to heroin abuse. To date, no medications exist that effectively relieve the negative affective symptoms of opiate withdrawal. The corticotropin-releasing factor (CRF) system has been hypothesized to mediate the motivational effects of drug dependence. The CRF signal is transmitted by two distinct receptors named CRF receptor-1 (CRF1) and CRF2. Here we report that genetic disruption of CRF1 receptor pathways in mice eliminates the negative affective states of opiate withdrawal. In particular, neither CRF1 receptor heterozygous (CRF1+/-) nor homozygous (CRF1-/-) null mutant mice avoided environmental cues repeatedly paired with the early phase of opiate withdrawal. These results were not due to altered associative learning processes because CRF1+/- and CRF1-/- mice displayed reliable, conditioned place aversions to environmental cues paired with the kappa-opioid receptor agonist U-50,488H. We also examined the impact of CRF1 receptor-deficiency upon opiate withdrawal-induced dynorphin activity in the nucleus accumbens, a brain molecular mechanism thought to underlie the negative affective states of drug withdrawal. Consistent with the behavioral indices, we found that, during the early phase of opiate withdrawal, neither CRF1+/- nor CRF1-/- showed increased dynorphin mRNA levels in the nucleus accumbens. This study reveals a cardinal role for CRF/CRF1 receptor pathways in the negative affective states of opiate withdrawal and suggests therapeutic strategies for the treatment of opiate addiction.

The antidepressant effect of running is associated with increased hippocampal cell proliferation

A common trait of antidepressant drugs, electroconvulsive treatment and physical exercise is that they relieve depression and up-regulate neurotrophic factors as well as cell proliferation and neurogenesis in the hippocampus. In order to identify possible biological underpinnings of depression and the antidepressant effect of running, we analysed cell proliferation, the level of the neurotrophic factor BDNF in hippocampus and dynorphin in striatum/accumbens in 'depressed' Flinders Sensitive Line rats (FSL) and Flinders Resistant Line (FRL) rats with and without access to running-wheels. The FRL strain exhibited a higher daily running activity than the FSL strain. Wheel-running had an antidepressant effect in the 'depressed' FSL rats, as indicated by the forced swim test. In the hippocampus, cell proliferation was lower in the 'depressed' rats compared to the control FRL rats but there was no difference in BDNF or dynorphin levels in striatum/accumbens. After 5 wk of running, cell proliferation increased in FSL but not in FRL rats. BDNF and dynorphin mRNA levels were increased in FRL but not to the same extent in the in FSL rats; thus, increased BDNF and dynorphin levels were correlated to the running activity but not to the antidepressant effect of running. The only parameter that was associated to basal level of 'depression' and to the antidepressant effect was cell proliferation in the hippocampus. Thus, suppression of cell proliferation in the hippocampus could constitute one of the mechanisms that underlie depression, and physical activity might be an efficient antidepressant.

Enhanced running wheel activity of both Mch1r- and Pmch-deficient mice

Mch1r-deficient ( Mch1r −/−) mice are hyperphagic, hyperactive, lean, and resistant to diet-induced obesity. To examine whether the MCH1R is involved in regulating activity-based energy expenditure, we investigated voluntary wheel running (WR) activity of wild-type (WT) and Mch1r −/− mice basally, in response to diets with different caloric density and with different feeding schedules. We also evaluated WR activity of mice with ablation of the prepro-MCH gene ( Pmch −/− mice). Dark cycle WR activity of Mch1r −/− mice fed low fat (LF) chow was increased significantly relative to WT mice. Transition to moderate high-fat (MHF) diet was associated with an increase in nocturnal WR activity in both genotypes. Both Mch1r −/− and WT mice exhibited food anticipatory activity (FAA) before the daily scheduled feeding time, indicating that MCH1R is not required for FAA. Naloxone (3 mg/kg, i.p.) suppressed WR activity of both genotypes, suggesting opioid regulation of locomotor activity. WR increased nocturnal dynorphin mRNA levels in Mch1r −/− brain. Importantly, Pmch-deficient mice had significantly enhanced WR activity relative to WT controls. These results suggest that endogenous MCH plays an inhibitory role in regulating locomotor activity. In summary, we demonstrated enhanced WR activities in mice lacking either MCH or its cognate receptor.

Altered hippocampal expression of neuropeptides in seizure-prone GALR1 knockout mice.

Mice carrying a deletion of the GALR1 galanin receptor have recently showed spontaneous seizure phenotype with 25% penetrance. To better understand the role of neuropeptides, which are known to undergo complex plasticity changes with development of epileptic seizures, we characterized their expression in the hippocampal formation in GALR1- knockout (-KO) mice with or without seizures and in wild-type (WT) mice. Immunohistochemistry and in situ hybridization were used to study expression of galanin, neuropeptide Y (NPY), substance P, enkephalin, dynorphin, and cholecystokinin (CCK). In GALR1-KO mice that had been displaying seizures, a strong upregulation of galanin immunoreactivity (ir) and messenger RNA (mRNA) was found in the polymorph layer of the dentate gyrus; galanin-ir also appeared in a dense fiber network in the supragranular layer. A strong upregulation of enkephalin was found in the granule cells/mossy fibers, whereas dynorphin mRNA levels were modestly decreased. NPY was strongly expressed in the granule cells/mossy fibers, and an increase of NPY mRNA levels in the polymorph cells was paralleled by an increase of NPY-ir in the molecular layer. An upregulation of substance P-ir was confined to the fibers in the granule and molecular layers, whereas substance P mRNA was increased in the cells of the polymorph layer. Both CCK-ir and mRNA were strongly downregulated in the granule cell/mossy fiber system, but CCK-ir appeared increased in the supragranular and molecular layers. No changes in neuropeptide-ir were found in GALR1-KO mice not displaying seizures. Complex changes in neuropeptide expression in some principal hippocampal neurons and interneurons appear as a characteristic feature of the spontaneous-seizure phenotype in GALR1-KO mice. However, to what extent causal relations exist between this "epilepsia peptidergic profile" and development of seizures requires further clarification.

Involvement of Mesolimbic Structures in Short-Term Sodium Depletion: In situ Hybridization and Ligand-Binding Analyses

Acute treatment with the diuretic furosemide (Lasix) produces a reduction in plasma Na⁺ and volume as well as increased thirst and salt appetite. The resulting hypovolemia stimulates the well-known counter-regulatory physiological response from the renin-angiotensin-aldosterone system. However, the neurochemical players underpinning the behavioral responses of thirst and salt appetite are less clear. Previously, we have reported that salt-replete deoxycorticosterone (DOCA) treatment activates mesolimbic structures associated with reward and goal-seeking behavior. The present study was designed to test whether the same brain regions are affected in a salt-depleted state. In experiment 1, two groups of adult male Sprague-Dawley (SD) rats were injected with Lasix (10 mg/rat, s.c.) and 18 h later were allowed access either to 2% NaCl solution (‘Lasix+salt’) or only to tap water (‘Lasixnosalt’) for 2 h. For comparison purposes, a third group received an isotonic saline injection instead of Lasix and was allowed access to the 2% salt solution (Vehicle). All groups were permitted 24 h access to tap water. We found no differences in dynorphin-mRNA levels in any striatal and accumbal regions among any of the treatment groups. However, as found previously in DOCA-treated rats, there were increased enkephalin (ENK)-mRNA and decreased dopamine transporter (DAT) binding levels throughout the striatum in Lasix+salt and decreased ENK-mRNA in Lasixnosalt rats versus Vehicle. In experiment 2, the involvement of the ENKergic and/or dopaminergic system was tested in rats divided into the same three groups described in experiment 1. However, before access to salt or water, the Lasix+salt and the vehicle groups were administered either a δ-opioid, naltrindole or a dopamine D₂ antagonist, raclopride. Only the naltrindole-treated rats showed a blunted intake of salt solution. Thus, these findings along with our neurochemical results suggest that mesolimbic enkephalin might impact salt intake through dopaminergic systems.

Differential regulation of GAD67, enkephalin and dynorphin mRNAs by chronic-intermittent L-dopa and A2A receptor blockade plus L-dopa in dopamine-denervated rats.

Adenosine A2A receptor antagonists have been proposed as an effective therapy in the treatment of Parkinson's disease. In the present study, we compared the modifications on striatal glutamate decarboxylase (GAD67), enkephalin, and dynorphin mRNA levels produced by a chronic-intermittent administration of L-3,4-dihydroxyphenyl-alanine (L-dopa) (6 mg/kg) with those produced by the adenosine A2A receptor antagonist SCH 58261 (5 mg/kg) plus L-dopa (3 mg/kg) in unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. As previously reported, L-dopa (6 mg/kg) and SCH 58261 (5 mg/kg) plus L-dopa (3 mg/kg) produced the same degree of turning behavior after the first administration. However, while L-dopa (6 mg/kg) induced a sensitized turning behavior response during the course of the treatment, which indicated a dyskinetic potential, SCH 58261 (5 mg/kg) plus L-dopa (3 mg/kg) produced a stable turning behavior response, which was predictive of absence of dyskinetic side effects. Unilateral 6-OHDA lesion produced an elevation in striatal GAD67 and enkephalin mRNA levels and to a decrease in dynorphin mRNA levels. Chronic-intermittent L-dopa (6 mg/kg) treatment increased the striatal levels of GAD67, dynorphin, and enkephalin mRNA in the lesioned side as compared to the vehicle treatment. Chronic-intermittent SCH 58261 (5 mg/kg) plus L-dopa (3 mg/kg) as well as L-dopa (3 mg/kg) or SCH 58261 (5 mg/kg) alone did not produce any significant modification in GAD67, dynorphin, or enkephalin mRNA levels in the lesioned striatum as compared to the striatum of vehicle-treated rats. The results show that combined SCH 58261 plus L-dopa did not produce long-term changes in markers of striatal efferent neurons activity and suggest that the lack of modifications in GAD67 and dynorphin mRNA after SCH 58261 plus L-dopa might correlate with the lack of turning behavior sensitization which predicts drug dyskinetic potential.

MK-801 alters the effects of priming with L-DOPA on dopamine D1 receptor-induced changes in neuropeptide mRNA levels in the rat striatal output neurons.

In a previous study, we have shown in unilaterally dopamine-depleted rats that increased behavioral responsiveness to the dopamine D1-receptor agonist SKF-38393, which was induced by pretreatment with L-DOPA, is paralleled by specific alterations in striatal neuropeptide mRNA levels. The behavioral 'priming' effect of L-DOPA is prevented if L-DOPA is preceded by the NMDA-receptor antagonist MK-801. In the present study, the question is addressed whether blockade of the increased behavioral responsiveness with MK-801 also prevents the observed changes in striatal neuropeptide mRNA levels. After a challenge with SKF-38393 (3 mg/kg, s.c.), the striatal levels of preprodynorphin, preprotachykinin, and preproenkephalin mRNA were compared between unilaterally dopamine-depleted rats that were either primed with a single administration of L-DOPA (50 mg/kg, i.p.) or with L-DOPA preceded by MK-801 (0.1 mg/kg, i.p.). Priming with L-DOPA enhanced the increase in dynorphin mRNA levels in the dorsolateral part of the dopamine-depleted striatum that occurred after SKF-38393. On the other hand, it had no significant effect on substance P or enkephalin mRNA levels. MK-801 prior to L-DOPA prevented the increased responsiveness of dynorphin regulation. However, it induced a decreased response to dopamine D1-receptor stimulation in the substance P mRNA levels in dorsal regions of the dopamine-depleted striatum. The levels of enkephalin mRNA after challenge with SKF-38393 were not affected by the MK-801 administration. These results demonstrate that the increased behavioral responsiveness to the D1-receptor agonist SKF-38393 after priming with L-DOPA is primarily related to the upregulation of dynorphin mRNA levels in the dopamine-depleted striatum.

Running and cocaine both upregulate dynorphin mRNA in medial caudate putamen

Physical activities such as long-distance running can be habit forming and associated with a sense of well-being to a degree that justifies comparison with drug-induced addictive behaviours. To understand molecular similarities and dissimilarities controlling these behaviours in humans we compared the effects of running in running wheels to the effects of chronic cocaine or morphine administration on mRNA levels in brain reward pathways in the inbred Fischer and Lewis rat strains. These strains are both inbred from the Sprague-Dawley strain; Lewis rats display a higher preference towards addictive drugs and running than do Fischer rats. After chronic cocaine or running a similar increase of dynorphin mRNA in medial caudate putamen was found in the Lewis rat, suggesting common neuronal adaptations in this brain region to both cocaine and running. Fischer and Lewis rats both responded to cocaine with increased dynorphin mRNA levels in medial caudate putamen. However, only Lewis rats increased dynorphin mRNA after running, possibly reflecting the much higher degree of running by the Lewis strain as compared to the Fischer strain. Moreover, the running-induced upregulation of dynorphin mRNA was blocked by the opioid receptor antagonist naloxone. We suggest that running increases dynorphin mRNA by a mechanism that involves endogenous opioids. The voluntary wheel-running model in rats might be used to study natural reward and compulsive behaviours and possibly also to screen candidate drugs for treatment of compulsive disorders.

Dopamine D3 receptor antisense reduces neuropeptide mRNA levels in rat nucleus accumbens.

We have investigated the effects of a dopamine D3 receptor antisense oligodeoxynucleotide (ODN), on neurotensin and dynorphin mRNA levels in rat nucleus accumbens. Intracerebroventricular injections of ODNs were made into the lateral ventricle (5 and 10 microg h(-1), for 5 days). Receptor autoradiography of dopamine D2 and D3 receptor subtypes was performed. Dynorphin and neurotensin mRNA levels were evaluated by in situ hybridization. Dopamine D3 receptor levels were significantly decreased in nucleus accumbens shell (NAS) of rats that received the D3 antisense ODN. Dynorphin and neurotensin mRNA levels were also significantly decreased in the NAS after D3 antisense ODN treatments. Our results show that D3 receptors may regulate neuropeptide gene expression and demonstrate that an antisense strategy could be useful to identify molecular targets under control of specific dopamine receptor subtypes.

Maternal Cocaine Treatment Alters Dynorphin and Enkephalin mRNA Expression in Brains of Fetal Rhesus Macaques

Cocaine exposure in utero is known to cause a variety of behavioral and motor deficits that may be attributable to alterations in the dopamine neurocircuitry. To ascertain cocaine effects in the fetus, we developed a nonhuman primate model in which pregnant monkeys were administered cocaine from day 20 through day 60 or 70 of gestation. Fetuses from these pregnancies develop a repertoire of neural deficiencies, including decreased mRNA expression of tyrosine hydroxylase in the midbrain and increased mRNA expression of dopamine receptor subtypes in the rostral forebrain. Presently, we studied the effects of maternal cocaine treatment on the mRNA expression of the endogenous opioids preprodynorphin (PPD) and preproenkephalin (PPE) in fetal monkey brains. Fetuses exposed to saline (0.9%) or cocaine (3 mg/kg) were delivered by Caesarean section, the fetal brains were dissected, and tissue RNA was extracted and quantified using ribonuclease protection assay analysis. The opioid peptides PPD and PPE were expressed in the fetal monkey brain by day 60, and even higher levels were found in day 70 fetuses. Maternal exposure to cocaine increased gene expression of PPD and PPE in the fetus at both day 60 and day 70 of gestation. Dynorphin mRNA levels were significantly elevated in the striatum, whereas enkephalin mRNA was elevated in both the frontal cortex and the striatal area of fetuses whose mothers received cocaine. Changes in the expression of these opioid peptides in presumed dopamine target neurons, which mediate motivation and reward, as well as motor control, provide further evidence for profound consequences of in utero cocaine exposure on the developing dopamine neurocircuitry.

Delayed kindling development after rapidly recurring seizures: relation to mossy fiber sprouting and neurotrophin, GAP-43 and dynorphin gene expression

Development of kindling and mossy fiber sprouting, and changes of gene expression were studied after 40 seizures produced during about 3 h by electrical stimulation every 5 min in the ventral hippocampus. As assessed by 5 test stimulations, enhanced responsiveness was present already after 6–24 h but from 1 week post-seizure increased gradually up to 4 weeks without additional stimuli. Sprouting of mossy fibers in the dentate gyrus was demonstrated only at 4 weeks with Timm's staining. In situ hybridization showed a transient increase (maximum at 2 h) of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), TrkB and TrkC mRNA levels and reduction (maximum at 12–24 h) of neurotrophin-3 (NT-3) mRNA expression in dentate granule cells after the seizures. In addition, BDNF mRNA levels were elevated in CAI and CA3 regions, amygdala and piriform cortex. Marked increases of mRNA for growth-associated protein (GAP-43), with maximum expression at 12–24 h, were observed in dentate granule cells and in amygdala-piri-form cortex. Dynorphin mRNA levels showed biphasic changes in dentate granule cells with an increase at 2 h followed by a decrease at 24 h. No long-term alterations of gene expression were observed. These findings indicate that increased responsiveness develops rapidly after recurring seizures but that the kindled state is reached gradually in about 4 weeks. Mossy fiber sprouting occurs in parallel to epileptogenesis and may play a causative role. Short-term changes of neurotrophin and Trk, GAP-43 and dynorphin mRNA levels and the assumed alterations of the corresponding proteins could trigger structural rearrangements underlying kindling but might also contribute to the initial increase of seizure susceptibility.

Methamphetamine-induced changes in AP-1 binding and dynorphin in the striatum: correlated, not causally related events?

Activation of D1 dopamine (DA) receptors in the striatum increases the expression of the opioid neuropeptide, dynorphin (DYN). While cAMP is generally accepted as a second messenger in this signal transduction pathway, the role of Fos/FRA proteins and AP-1 binding in mediating D1 receptor-induced changes in DYN expression remains uncertain. In this study, DYN peptide and mRNA levels, as well as Fos/FRA proteins and AP-1 DNA binding activity, were measured in individual animals following acute challenge with the psychostimulant drug methamphetamine (METH). METH caused an initial decrease in striatal levels of DYN A, reflecting increased peptide release. Six hours postinjection, DYN mRNA levels were significantly elevated by METH as compared to vehicle-injected controls. At the same time, these drugs increased the expression of Fos/FRA-ir proteins, in particular the 35- and 40-kDa molecular weight species, and increased binding to the AP-1 DNA element. Analyses of the time course of METH's effects revealed that DYN mRNA levels, Fos/FRA proteins and AP-1 binding activity showed variable increases by 3 h but all were significantly elevated above control levels by 6 h posttreatment. The D1-specific antagonist, SCH 23390, completely blocked the METH-induced changes in DYN peptide and mRNA levels while a D2 receptor antagonist (sulpiride) had little or no effect. These data indicate that stimulant drugs such as METH increase the expression of DYN and AP-1 factors in the striatum via a D1 receptor-mediated mechanism. However, the finding that AP-1 binding merely paralleled, but did not precede, the increase in DYN expression, as would be expected if it were mediating increased gene transcription, suggests these may be correlative, not causally related events.

Effects of adrenal steroid manipulations and repeated restraint stress on dynorphin mRNA levels and excitatory amino acid receptor binding in hippocampus

Adrenal steroid and stress effects were determined in hippocampus on levels of dynorphin (DYN) mRNA, expressed in dentate gyrus, and excitatory amino acid receptors, measured in Ammon's horn and dentate gyrus. Adrenalectomy (ADX) decreased DYN mRNA levels in dentate gyrus and replacement with aldosterone (ALDO), a specific type I adrenal steroid receptor agonist, prevented the decrease. Ru28362, a specific type II receptor agonist, had no effect. Likewise, kainate receptor binding to the stratum lucidum and hilus region of dorsal hippocampus was decreased after ADX and this decrease was prevented by ALDO but not by Ru28362 treatment. Similar though smaller effects were found for CNQX binding to AMPA receptors but only in the dentate gyrus molecular or infra- and supragranular layers. Although corticosterone (CORT) treatment of intact rats (40 mg/kg for 3 weeks) elevated DYN mRNA levels in dentate gyrus, up to 14 days of daily restraint stress (1 or 6 h/day) had no significant effect. Neither CORT treatment nor repeated restraint stress altered NMDA and non-NMDA glutamate receptors in hippocampus. The results of this study showing ADX-induced decreases of DYN mRNA and CNQX binding in dentate gyrus and decreased kainate binding in mossy fiber terminal regions are consistent with morphological evidence showing that adrenal steroids maintain normal integrity and structure of dentate gyrus neurons and do so via type I adrenal steroid receptors. These same parameters are apparently not sensitive to chronic restraint stress although the effects of other stressors must be examined.