John Q. Wang

D1 and D2 receptor regulation of preproenkephalin and preprodynorphin mRNA in rat striatum following acute injection of amphetamine or methamphetamine

Our previous work has demonstrated a dose‐dependent induction of striatal preprodynorphin (PPD) in response to a single injection of the psychostimulants amphetamine (AMPH) or methamphetamine (METH). In the present study, dose‐response effects of acute administration of these stimulants on preproenkephalin (PPE) mRNA expression in the rat striatum were investigated with quantitative in situ hybridization histochemistry 3 h after injection. Acute AMPH or METH at equimolar doses (3.75, 7.5, 15, and 30 μmol/kg) significantly increased PPE mRNA expression in dorsal (caudoputamen), but not ventral (nucleus accumbens), striatum in a dose‐dependent fashion. In addition, the role of D1 and D2 dopamine receptors in mediating AMPH‐ and METH‐stimulated PPE and PPD expression was also evaluated by using subtype‐specific antagonists. Pretreatment of rats with SCH 23390 (0.1 mg/kg, i.p.), a selective D1 receptor antagonist, completely blocked acute AMPH (21 μmol/kg, i.p.)‐ or METH (21 μmol/kg, i.p.)‐induced PPE as well as PPD mRNA expression in the caudoputamen. Pretreatment with eticlopride (0.5 mg/kg, i.p.), a selective D2 receptor antagonist, also blocked PPD induction by the two stimulants, but PPE induction was unaffected. Furthermore, SCH 23390 decreased, and eticlopride elevated, constitutive PPE mRNA levels in the caudoputamen. Neither antagonist had a significant effect on the basal level of PPE or PPD mRNA in the nucleus accumbens. These results demonstrate a clear dose‐related responsiveness of PPE gene expression in striatal neurons in response to acute administration of amphetamines, although the intensity of the response is far less than that for striatal PPD. Furthermore, both D1 and D2 subtypes of dopamine receptors mediate AMPH‐ and METH‐stimulated striatal PPD mRNA expression, whereas D1 receptor activation alone mediates amphetamine‐stimulated PPE mRNA expression in the rat striatum.

Alterations in striatal zif/268, preprodynorphin and preproenkephalin mRNA expression induced by repeated amphetamine administration in rats

Quantitative in situ hybridization histochemistry was used to compare the effects of AMPH administration on mRNAs coding for zif/268, a member of the zinc finger family of immediate early genes, and the opioid peptides, preprodynorphin (PPD) and preproenkephalin (PPE), in rat striatum after 3 dosing schedules: (1) acute; (2) once a day for 5 days, and (3) once a day for 5 days followed 10 days later by a challenge dose. Behavioral ratings indicated that the activity of rats was significantly higher after 5 daily AMPH (5 mg/kg, i.p.) injections than after a single 5 mg/kg injection and that the activity of AMPH-pretreated rats was significantly higher than that of saline-pretreated rats after a 1 mg/kg challenge dose 10 days later. Three hours after acute administration of AMPH to naive rats, PPD mRNA expression in the dorsal (caudatoputamen) and ventral (nucleus accumbens, NAc) striatum as well as PPE and zif/268 mRNA expression in the dorsal, but not ventral, striatum were increased as compared to saline-treated rats. Five daily treatments with AMPH augmented acute AMPH-induced increases in PPD mRNA expression in the caudatoputamen but not in the NAc. The increase in striatal zif/268 mRNA expression induced by acute AMPH was, in contrast, reduced after 5 daily treatments with AMPH. AMPH induced PPE expression to the same extent in rats treated with one or 5 daily injections. Hybridization performed 3 h after AMPH challenge in AMPH-pretreated rats failed to demonstrate augmentation of AMPH-stimulated zif/268, PPD, or PPE mRNA expression in the striatum as compared to saline-pretreated rats. These results indicate that an augmented response of PPD and a decreased response of zif/268 mRNA expression in striatal neurons are closely associated with the initiation of behavioral sensitization induced by repeated, intermittent AMPH treatments. In contrast, a clear dissociation exists between the expression of sensitized behaviors and unsensitized zif/268, PPD and PPE mRNA in the striatum in response to a challenge dose of AMPH. Possible functional implications of these alterations in the initiation and expression of AMPH-induced behavioral sensitization are discussed.

Differential effects of D1 and D2 dopamine receptor antagonists on acute amphetamine- or methamphetamine- induced up-regulation of zif/268 mRNA expression in rat forebrain

Abstract: This study investigated the hypothesis that D1 and D2 dopamine receptors interact to regulate the expression of zif/268 mRNA in rat forebrain after an acute injection of amphetamine or methamphetamine. Forty‐five minutes and 3 h after a single injection of amphetamine (4 mg/kg i.p.) or methamphetamine (4 mg/kg i.p.), the mRNA expression of zif/268 in dorsal striatum and sensorimotor cortex was increased, as revealed by quantitative in situ hybridization histochemistry. Induction was more intense in striatal patches at 45 min than at 3 h, when a more homogeneous pattern of zif/268 mRNA induction was observed. SCH 23390, a selective D1 receptor antagonist, suppressed, and eticlopride, a D2 receptor antagonist, elevated, constitutive zif/268 mRNA levels in the striatum, but neither antagonist had a significant effect on the constitutive expression of zif/268 in sensorimotor cortex. Pretreatment with SCH 23390 completely blocked the stimulant‐induced zif/268 expression in striatum and partially blocked the stimulant‐induced zif/268 expression in cortex. Pretreatment with eticlopride augmented zif/268 mRNA expression in patch and matrix compartments of dorsal and ventral striatum 45 min after amphetamine or methamphetamine injection. However, at 3 h, eticlopride completely blocked amphetamine‐ and methamphetamine‐stimulated zif/268 mRNA in dorsomedial, but not dorsolateral, striatum. In addition, eticlopride partially blocked cortical zif/268 induction by both amphetamines. Both antagonists prevented stimulant‐induced hyperlocomotion and stereotypies. These results demonstrate that D1 and D2 receptors in mesolimbic/mesostriatal pathways both regulate amphetamine‐and methamphetamine‐stimulated behaviors and zif/268 mRNA expression. Furthermore, the effect of D2 receptor blockade on zif/268 expression was found to be contingent on the time interval investigated after psychostimulant administration.

Intrastriatal injection of a muscarinic receptor agonist and antagonist regulates striatal neuropeptide mRNA expression in normal and amphetamine-treated rats.

Systemic administration of the muscarinic receptor antagonist, scopolamine, augments, whereas the muscarinic receptor agonist, oxotremorine, attenuates behaviors (locomotion and stereotypies) and preprodynorphin (PPD) and substance P (SP) gene expression in striatonigral neurons induced by the indirect dopamine receptor agonist, amphetamine (AMPH). In contrast, systemic scopolamine blocks, whereas oxotremorine augments, AMPH-stimulated preproenkephalin (PPE) gene expression in striatopallidal neurons. This study investigated the site of action of these effects by administering scopolamine and oxotremorine directly into the striatum and assessing the expression of neuropeptide mRNAs with quantitative in situ hybridization. Unilateral injection of scopolamine into the dorsal striatum augmented, and oxotremorine attenuated, AMPH (2.5 mg/kg, i.p.)-stimulated behaviors. Intrastriatal scopolamine at a concentration of 62 mM, but not 6.2 mM, increased basal levels of PPD and SP mRNAs in the dorsal striatum. In addition, both 6.2 and 62 mM scopolamine significantly augmented AMPH-stimulated PPD and SP mRNA levels. Intrastriatal infusion of 1.6 or 8.1 mM oxotremorine did not alter basal levels of striatal PPD and SP mRNAs. However, both concentrations of oxotremorine completely blocked AMPH-stimulated SP mRNA and oxotremorine at 8.1 mM blocked AMPH-stimulated PPD mRNA. In contrast, PPE induction by AMPH was blocked by 62, but not 6.2, mM scopolamine. Both concentrations of oxotremorine tended to augment basal and AMPH-stimulated PPE mRNA in the dorsal striatum but the trend was not significant. These data demonstrate an inhibition of striatonigral, and facilitation of striatopallidal, gene expression through activation of local striatal muscarinic receptors, which is consistent with the changes seen after systemic administration of muscarinic agents. Therefore, muscarinic cholinergic regulation of basal and stimulated expression of neuropeptide mRNA is processed within the striatum.

Role of kainate/AMPA receptors in induction of striatal zif/268 and preprodynorphin mRNA by a single injection of amphetamine

The role of kainate/AMPA excitatory amino acid receptors in D-amphetamine (AMPH)-induced behavioral changes and the induction of immediate early gene and preprodynorphin (PPD) mRNA in various regions of rat forebrain was investigated with quantitative in situ hybridization histochemistry. Three hours after a single injection of AMPH (5 mg/kg, i.p.), PPD mRNA and mRNA of the transcription factor zif/268, but not c-fos, was increased in dorsal striatum (caudate). Zif/268 mRNA was also increased in the sensorimotor cortex. Pretreatment of rats with DNQX, a kainate/AMPA receptor antagonist, did not affect the behaviors elicited by AMPH. However, the AMPH-stimulated increase in PPD and zif/268 mRNA levels in striatum, but not zif/268 mRNA in cortex, was blocked by DNQX pretreatment. In contrast, DNQX alone attenuated basal (constitutive) levels of zif/268 mRNA expression in sensorimotor cortical, but not in striatal, neurons. These studies indicate that kainate/AMPA receptors mediate the induction of zif/268 and PPD mRNA expression in the caudate nucleus induced by a single injection of AMPH. The fact that DNQX blocked genomic, but not behavioral, responses to acute AMPH suggests that kainate/AMPA receptor mechanisms may be involved in the long-term (possibly sensitizing) effects, rather than the acute effects, of the drug. In addition, tonic kainate/AMPA receptor stimulation may play a key role in maintaining constitutive expression of the zif/268 gene in cortical neurons.

Acute methamphetamine-induced zif/268, preprodynorphin, and preproenkephalin mRNA expression in rat striatum depends on activation of NMDA and kainate/AMPA receptors

This study tested the role of N-methyl-D-aspartate and kainate/AMPA receptors in mediating mRNA expression of the immediate early gene zif/268 and the opioid peptide genes preprodynorphin and preproenkephalin in rat forebrain following a single injection of methamphetamine. At 3 h after acute methamphetamine [4 mg/kg, intraperitoneally (IP)], quantitative in situ hybridization histochemistry revealed that zif/268 mRNA expression was increased in the dorsal striatum (caudoputamen) and in the sensory cortex. Preprodynorphin was increased in both dorsal and ventral striatum (nucleus accumbens) and preproenkephalin was increased in the dorsal striatum. Pretreatment with (+ or -)-3-(2-carboxypiperazin-4-yl)-propyl-1 -phosphonic acid (CPP) (10 mg/kg, IP), an N-methyl-D-aspartate receptor antagonist, blocked the methamphetamine-induced zif/268 mRNA expression in the striatum and in the region of sensory cortex representing the upper limb and nose. 6,7-Dinitro-quinoxaline-2,3-dione (DNQX) (100 mg/kg, IP), a kainate/AMPA receptor antagonist, did not reduce the ability of methamphetamine to induce zif/268 mRNA in striatal and cortical neurons. Furthermore, both antagonists caused a parallel blockade of methamphetamine-stimulated preprodynorphin mRNA expression in the dorsal and ventral striatum but did not significantly affect methamphetamine-stimulated preproenkephalin mRNA expression. CPP and DNQX reduced basal levels of zif/268 mRNA in cortical and striatal neurons but did not affect the constitutive expression of the two opioid mRNAs in the striatum. Neither antagonist had a significant effect on methamphetamine-induced hyperlocomotion and stereotypies. These results demonstrate that both N-methyl-D-aspartate and kainate/AMPA receptor-mediated glutamatergic transmission is linked to modulation of the methamphetamine-stimulated oploid peptide gene expression in rat forebrain. Furthermore, N-methyl-D-aspartate receptors participate in methamphetamine-stimulated zif/268 expression.

Intrastriatal injection of the metabotropic glutamate receptor antagonist MCPG attenuates acute amphetamine-stimulated neuropeptide mRNA expression in rat striatum

In chronically cannulated rats, microinjection of a competitive metabotropic glutamate receptor (mGluR) antagonist, (+)-alpha-methyl-4-carboxyphenylglycine (MCPG), into the dorsal striatum at doses of 0.4, 2 and 10 micrograms/1 microliter did not affect basal levels of preprodynorphin, substance P and preproenkephalin mRNAs in the dorsal striatum as revealed by quantitative in situ hybridization. However, intrastriatal MCPG (0.08, 0.4 and 2 micrograms/1 microliter) dose-dependently attenuated increases in the three mRNA expression induced by acute amphetamine injection (2 mg/kg, i.p.). MCPG had no significant effect on spontaneous, and amphetamine-stimulated, behavioral activities. These data indicate that activation of MCPG-sensitive mGluRs is necessary for upregulation of striatal neuropeptide mRNA expression in response to amphetamine exposure. However, the mGluR activity is not implicated in maintaining basal levels of the peptide gene expression in the striatum.

Comparative effects of intrahippocampal injection of dynorphin A(1-8), dynorphin A(1-13), dynorphin A(1-17), U-50,488H, and dynorphin B on blood pressure and heart rate in spontaneously hypertensive and normotensive Wistar-Kyoto rats

We previously demonstrated centrally mediated hypotensive and bradycardic effects of dynorphin A(1-8) (DA1-8) on microinjection into various areas of the hippocampal formation (HF) of both anesthetized and conscious male normotensive and spontaneously hypertensive rats (SHR). The purpose of the present study was to determine whether other dynorphin fragments also had this activity. We microinjected DA1-8, dynorphin A(1-13), dynorphin A(1-17), dynorphin B (DB), and the nonpeptide kappa-opioid agonist U-50,488H into HF areas previously found to react to DA1-8, at doses ranging from 0.05 to 50 nmol. The subjects were male SHR and normotensive Wistar-Kyoto (WKY) rats in which arterial pressure and heart rate were monitored. Dose-related centrally mediated hypotension and bradycardia were found in both strains with all agents used, except for DB, which had no effects. Similarly injected drug vehicle was also without effect. In general, the responses were greater in SHR than in WKY rats. Preinjection of the active HF areas with 2 nmol of nor-binaltorphimine (nor-BNI), a selective kappa-opioid receptor antagonist, which itself had no blood pressure or heart rate effects, abolished both the decrease in blood pressure and heart rate of all dynorphins and U-50,488H. The results demonstrated the equivalent abilities of all the dynorphin fragments studied, except DB, to cause HF-mediated hypotension and bradycardia. The results with U-50,488H and nor-BNI strongly implicate kappa-opiate receptor activation of the HF in these effects.

Dentate granule cells as a central cardioregulatory site in the rat

Dentate granule cells can be selectively destroyed by intrahippocampal injections of colchicine. This study evaluates the consequences of granule cell destruction on blood pressure regulation in the normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rat (SHR). Bilateral destruction of dentate granule cells at 6 weeks of age produced a significant increase in blood pressure in the WKY that lasted for approximately 3 weeks, and a biphasic effect (increase then decrease) in the SHR that resulted in a significant hypotensive period that persisted for 6 weeks. Granule cell destruction at 11 weeks produced a maximal hypertension in the SHR that preceded age-matched controls by 4 weeks, but produced only a small transient increase in WKY blood pressure. Dentate granule cells are the exclusive source of prodynorphin-derived peptides in the hippocampal formation and their synthesis is regulated by glucocorticoids. Evidence suggests glucocorticoids may be involved in the regulation of blood pressure and hypertension. We determined that chronic high levels of corticosterone significantly reduced hippocampal dynorphin B levels in normotensive Sprague-Dawley rats. In addition, we confirmed that naive SHRs also contain significantly lower levels of hippocampal dynorphin B. These results suggest (i) that dentate granule cells represent a discrete neural site that may exert a tonic inhibitory influence on blood pressure, (ii) that dentate granule cells are not required for the full expression of hypertension in the SHR, and (iii) that chronic high levels of corticosterone can reduce dynorphin B levels in the dentate granule cells of normotensive rats.

The muscarinic toxin 3 augments neuropeptide mRNA in rat striatum in vivo.

The selective M4 muscarinic receptor toxin, MT3, was used in vivo to evaluate the role of M4 receptors in cholinergic inhibition of neuropeptide mRNA expression in striatonigral neurons. Unilateral injection of the muscarinic toxin 3 (0.04-4 nmol) into the dorsal striatum of chronically-cannulated rats elevated basal levels of preprodynorphin, substance P and preproenkephalin mRNAs in the ipsilateral dorsal striatum as revealed by quantitative in situ hybridization. Pretreatment with muscarinic toxin 3 also augmented amphetamine (2.5 mg/kg, i.p.)-stimulated preprodynorphin and substance P expression in the dorsal striatum in a manner similar to that observed after the muscarinic antagonist, scopolamine. Since muscarinic toxin 3 has a much greater affinity for muscarinic M4 receptors than for other subtypes, it is possible that muscarinic toxin 3, by interacting with the muscarinic M4 subtype, regulates basal and/or dopamine-stimulated striatal neuropeptide gene expression.