Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands.

Cannabinoids, endocannabinoids and marijuana activate two well-characterized cannabinoid receptors (CB-Rs), CB1-Rs and CB2-Rs. The expression of CB1-Rs in the brain and periphery has been well studied, but neuronal CB2-Rs have received much less attention than CB1-Rs. Many studies have now identified and characterized functional glial and neuronal CB2-Rs in the central nervous system. However, many features of CB2-R gene structure, regulation and variation remain poorly characterized in comparison with the CB1-R. In this study, we report on the discovery of a novel human CB2 gene promoter transcribing testis (CB2A) isoform with starting exon located ca 45 kb upstream from the previously identified promoter transcribing the spleen isoform (CB2B). The 5' exons of both CB2 isoforms are untranslated 5'UTRs and alternatively spliced to the major protein coding exon of the CB2 gene. CB2A is expressed higher in testis and brain than CB2B that is expressed higher in other peripheral tissues than CB2A. Species comparison found that the CB2 gene of human, rat and mouse genomes deviated in their gene structures and isoform expression patterns. mCB2A expression was increased significantly in the cerebellum of mice treated with the CB-R mixed agonist, WIN55212-2. These results provide much improved information about CB2 gene structure and its human and rodent variants that should be considered in developing CB2-R-based therapeutic agents.

A single high dose of methamphetamine increases cocaine self-administration by depletion of striatal dopamine in rats.

Psychostimulant addicts often take high doses of drugs, and high doses of psychostimulants such as methamphetamine (METH) are neurotoxic to striatal dopamine (DA) terminals. Yet, the effects of high doses of METH on drug-seeking and drug-taking behavior have not been examined. In the present study, we found that single high doses of METH in rats (10-20 mg/kg) dose-dependently increased cocaine self-administration under fixed-ratio 2 (FR2) reinforcement conditions, while higher doses (40 mg/kgx1 or 10 mg/kg/2 hx4) caused high mortality among rats maintained on daily cocaine self-administration. The increased cocaine self-administration appeared to be a compensatory response to reduced cocaine reward after METH, because the same doses of METH caused a dose-dependent reduction both in "break-point" levels for cocaine self-administration under progressive-ratio reinforcement and in nucleus accumbens DA response to acute cocaine. Further, METH (10-20 mg/kg) produced large DA release (4000%-6000% over baseline), followed by a significant reduction in striatal DA and 3,4-dihydroxyphenylacetic acid (DOPAC) contents, but without significant changes in striatal DA transporter levels. These findings suggest that the present high doses of METH caused striatal DA depletion or hypofunction without severe damage in DA terminals, which may contribute to the increased cocaine-taking behavior observed in the present study. Provided that the present doses of METH may mimic METH overdose incidents in humans, the present findings suggest that METH-induced DA depletion or neurotoxicity may lead to an increase in subsequent drug-taking and drug-seeking behavior.

Increased mesolimbic GABA concentration blocks heroin self-administration in the rat.

Opiate reinforcement has been hypothesized to be mediated by an inhibition of mesolimbic gamma-aminobutyric acid (GABA) release that subsequently disinhibits ventral tegmental area (VTA) dopamine neurons. In support of this hypothesis, this study demonstrates that when administered directly into the lateral ventricle, the VTA, or the ventral pallidum, but not the nucleus accumbens, gamma-vinyl-GABA (GVG, an irreversible GABA-transaminase inhibitor, 20-50 microg) dose dependently blocked heroin (0.06 mg/kg) self-administration (SA), as assessed by an increase in heroin SA at low doses of GVG and an initial increase followed 1 to 2 h later by a blockade of heroin SA at higher GVG doses. This effect lasted 3 to 5 days. In drug-naïve rats, intra-VTA GVG pretreatment also prevented or delayed acquisition of heroin SA for 2 days. This GVG effect was prevented or reversed by systemic or intra-VTA pretreatment with the GABA(B) antagonist 2-hydroxysaclofen, but not the GABA(A) antagonist bicuculline. Similarly, coadministration of heroin with aminooxy-acetic acid (1-4 mg/kg) or ethanolamine-O-sulfate (50-100 mg/kg), two reversible GABA transaminase inhibitors, dose dependently reduced heroin reinforcement. Coadministration of (+/-)-nipecotic acid (0.1-5 mg/kg) with heroin, or intra-VTA or -ventral pallidum pretreatment with (+/-)-nipecotic acid (10 microg) or NO-711 (2 microg), two GABA uptake inhibitors, significantly increased heroin SA behavior, an effect also blocked by systemic 2-hydroxysaclofen, but not bicuculline. Taken together, these experiments, for the first time, demonstrate that pharmacological elevation of mesolimbic GABA concentration blocks heroin reinforcement by activating GABA(B) receptors, supporting the GABAergic hypothesis of opiate reinforcement and the incorporation of GABA agents in opiate abuse treatment.

Heroin-induced neuronal activation in rat brain assessed by functional MRI.

The present study demonstrates the application of fMRI technology to neuropharmacology and the interaction of drug/receptor in the rat brain. Specifically, we have observed two different types of fMRI signal changes induced by acute i.v. heroin administration in rat brains under conditions of spontaneous and artificial respiration. Under spontaneous respiration, a global decrease in fMRI signal was observed; under artificial respiration, a region-specific increase in fMRI signal was identified and the activation sites are consistent with the distribution of opiate mu-receptors in rat brain as previously reported by autoradiography. Both heroin-induced fMRI signal changes were suppressed by pretreatment of naloxone, an opiate mu-receptor antagonist, and reversed by injection of naloxone following heroin infusion. These results suggest that fMRI has specific advantages in spatial and temporal resolution for studies of neuropharmacology and drugs of abuse.

Baclofen inhibits heroin self-administration behavior and mesolimbic dopamine release.

An emerging hypothesis to explain the mechanism of heroin-induced positive reinforcement states that opiates inhibit gamma-aminobutyric acid (GABA)-ergic interneurons within the mesocorticolimbic dopamine (DA) system to disinhibit DA neurons. In support of this hypothesis, we report that the development of heroin self-administration (SA) behavior in drug-naive rats and the maintenance of SA behavior in heroin-trained rats were both suppressed when the GABA(B) receptor agonist baclofen was coadministered with heroin. Microinjections of baclofen into the ventral tegmental area (VTA), but not the nucleus accumbens, decreased heroin reinforcement as indicated by a compensatory increase in SA behavior. Additionally, baclofen administered alone or along with heroin dose-dependently reduced heroin-induced DA release. This effect was blocked partially by intra-VTA infusion of the GABA(B) antagonist 2-hydroxysaclofen, suggesting an additional, perhaps GABA(A) receptor-mediated, disinhibitory effect. Taken together, these experiments, for the first time, demonstrate that heroin-reinforced SA behavior and nucleus accumbens DA release are mediated predominantly by GABA(B) receptors in the VTA and suggest that baclofen may be an effective agent in the treatment of opiate abuse.

Nucleus accumbens dopamine release modulation by mesolimbic GABAA receptors-an in vivo electrochemical study.

The role of GABA receptors in regulating the mesolimbic dopamine (DA) system and drug reinforced behaviors has not been well characterized. Using fast-cyclic voltammetry, the effects of specific GABA receptor modulation on DA release in the nucleus accumbens (NAcc) and heroin self-administration (SA) behavior was investigated. The GABAA agonist muscimol, administered either intravenously or directly into the ventral tegmental area (VTA), significantly increased DA release in the NAcc in 7 of the 10 rats tested. DA release decreased in the remaining three rats; both effects were blocked by pretreatment with the GABAA receptor antagonist bicuculline. In contrast, the GABAB agonist baclofen decreased, while 2-OH-saclofen (a GABAB antagonist) increased DA release in the NAcc. However, when VTA GABAB receptors were previously activated or inactivated by microinjections of baclofen or 2-OH-saclofen, systemic injections of muscimol caused an inhibition of NAcc DA release. These results suggest that GABAA receptors may be co-localized on both DA neurons and non-DA (GABAergic) interneurons in the VTA, with the effects of GABAA determined by the net effect of both direct inhibition and indirect disinhibition of DA neurons. Finally, although a DA releaser, muscimol was neither self-administered in drug naive rats, nor did it substitute for heroin in rats previously trained to self-administer heroin, suggesting that GABAA receptors appear to play a complex role in mediating drug reinforcement, depending upon the dynamic functional state of GABAA receptors on both tegmental DA and non-DA neurons.

Dopamine release in the nucleus accumbens during heroin self-administration is modulated by kappa opioid receptors: an in vivo fast-cyclic voltammetry study.

Mu and kappa opioid agonists are known to produce different, and sometimes opposite, effects on several pharmacological and behavioral measures. However, whether kappa agonists can be used to antagonize the reinforcing and putative dopamine (DA)-releasing properties of a mu agonist such as heroin is unclear. With the use of the high temporal and spatial resolution of in vivo fast-cyclic voltammetry to measure changes in extracellular DA in the nucleus accumbens (NAcc), we observed (1) dose-dependent increases in DA in the NAcc during heroin self-administration (SA), (2) that coadministration of the kappa agonist U50,488H with heroin or intracerebroventricular dynorphin A pretreatment significantly depressed the heroin-stimulated DA release during SA, where U50,488H alone inhibited the basal DA release in the NAcc, (3) that coadministration of low-dose U50,488H or dynorphin A significantly increased heroin SA behavior, whereas high-dose U50,488H, which alone did not support SA behavior, reduced or completely blocked heroin SA and (4) that nor-binaltorphimine dihydrochloride (a selective kappa receptor antagonist) potentiated DA release in the NAcc and modestly decreased heroin SA. Taken together, these data suggest that endogenous kappa receptor activation can inhibit mu agonist-induced activation of the mesolimbic DA pathway, which may in turn depress heroin-induced reinforcement.

Baclofen reduces GABAA receptor responses in acutely dissociated neurons of bullfrog dorsal root ganglia.

The effect of baclofen on the function of the gamma-aminobutyric acidA (GABAA) receptor was examined in acutely dissociated neurons of bullfrog dorsal root ganaglia (DRG) by using the whole-cell voltage-clamp method. Baclofen (0.1-100 microM) depressed the inward currents produced by GABA (100 microM) and muscimol (100 microM). Baclofen shifted the concentration-response curve for GABA (1 microM-1 mM) downward. Baclofen decreased the maximum response (Vmax) to GABA without changing the apparent dissociation constant (Kd), suggesting a noncompetitive antagonism. The effect of baclofen on the GABA current was blocked by antagonists for the GABAB receptor; the rank order of potency was P-[3-Aminopropyl]-P-diethoxymethylphosphinic acid (CGP 55845A) > > 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P- benzyl-phosphinic acid (CGP 35348) > saclofen > > phaclofen. Baclofen produced an irreversible depression of the GABA current in neurons dialyzed with an internal solution containing guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S, 100 microM). Intracellular guanosine 5'-O-(2-thiodiphosphate) (GDP beta S, 100 microM) blocked the inhibitory effect of baclofen on the GABA current. Forskolin (10 microM) and dibutyryl N6, 2'-O-dibutyryladenosine 3':5'-cyclic monophophate (db-cyclic AMP) (200 microM) depressed the GABA current. N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9, 40 microM) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 50 microM), protein kinase A (PKA) inhibitors, reduced the depressant effect of baclofen on the GABA current. The baclofen-induced depression of the GABA current was blocked by PKI(5-24), a specific PKA inhibitor, but not by PKC(19-36), a specific protein kinase C (PKC) inhibitor. We suggest that GABAB receptors regulate the GABAA receptor function through a G-protein linked to the adenylyl cyclase-PKA pathway in bullfrog DRG neurons.

Opioid peptides modulate GABA(A) receptor responses in neurons of bullfrog dorsal root ganglia.

Effects of enkephalin and selective opioid-receptor agonists on GABA-induced current were examined in dissociated neurons of bullfrog dorsal root ganglia (DRG) by using whole-cell patch-clamp method. Leucine- (Leu)-enkephalin and methionine- (Met)-enkephalin depressed GABA(A) receptor-mediated currents. DPDPE, DAMGO and dynorphin-A (Dyn-A) also depressed the inward current produced by GABA; the order of agonist potency was DPDPE > DAMGO > Dyn-A. Naloxone blocked the inhibitory effects of enkephalins and other opioid agonists on the GABA current. Naltrindole (NTI), a delta-receptor antagonist, prevented the DPDPE-induced depression of the GABA current. beta-Funaltrexamine (beta-FNA), a mu-receptor antagonist, reduced the DAMGO-induced depression of GABA currents. Nor-binaltorphimine (nor-BNI), a kappa-receptor antagonist, reduced the effects of Dyn-A in depressing the GABA current. The results suggest that enkephalin down-regulates GABA(A) receptor function through mainly delta- and mu-opioid receptors in bullfrog DRG neurons.

[Renin-angiotension system--stress hormone response system].

Angiotensin II (A II) was significantly increased in both acutely and chronically stressed animals as found in brain, heart, blood vessels and adrenal glands. After running a race, the plasma A II contents of sportsmen similar to cortisol increased markedly too. In the chronical stress-hypertensive animals, the contents of circulating A II and tissue A II were increased quite lastingly. It was also found that the high concentration of A II may stimulate the adrenal gland secreting glucosteroid, and increase of tissue A II may be faciliated by the activation of beta adrenergic receptors. On basis of the above finding, we tend to assume that A II is a stress hormone and the renin angiotension system (RAS) is a stress hormone responses system. Morever there exists a close relation between RAS and two classic stress hormone response systems, i.e. the hypothalamus-pituitary-adrenal cortex system and the sympathetic-adrenal medulla system.

N-methyl-D-aspartate depresses GABAA receptor-mediated currents in neurons of bullfrog dorsal root ganglia.

The effect of N-methyl-D-aspartate (NMDA) on the function of GABAA receptors was examined in acutely dissociated neurons of bullfrog dorsal root ganglia (DRG) by using whole-cell patch clamp methods. The application of NMDA (100 microM) to DRG neurons suppressed the inward currents produced by GABA (100 microM) and muscimol (100 microM). DL-2-Amino-5-phosphonovaleric acid (APV; 100 microM) antagonized the NMDA-induced depression of the GABA current in a competitive manner. Kainic acid (100 microM) did not depress the GABA current. NMDA induced no depression of the GABA current in a nominally Ca(2+)-free solution containing 5 mM Mg2+. These data indicate that the activation of the NMDA receptor inhibits the GABAA receptor-mediated current via a Ca(2+)-dependent process.

Presynaptic GABAA receptors in vertebrate synapses.

The presynaptic gamma-aminobutyric acidA (GABAA) receptor has long been considered as the site for 'presynaptic inhibition' of synaptic transmission in the central nervous system (CNS). However, recent reports have indicated that the activation of GABAA receptors depolarizes primary afferent neurons and actually facilitates the release of GABAA, noradrenaline, adenosine, and luteinizing hormone (LH) in central and peripheral tissues. Isoguvacine, a GABAA receptor agonist, enhances substance P (SP) release in the spinal cord. GABAB receptor agonists, bu not GABAA receptor agonists, produce behavioral antinociception in the spinal cord. Baclofen does not directly depolarize the postsynaptic membrane, but presynaptically inhibits the activity of dorsal horn neurons. The excitatory postsynaptic potential (EPSP) evoked by stimulation of dorsal root C-fibers is inhibited by baclofen. Baclofen and GABA inhibit SP release from the primary afferent terminals by activating GABAB receptors. The activation of GABAB receptors inhibits calcium currents in neurons of dorsal roots ganglia (DRG). It is likely that the GABAA receptors act as a site for 'presynaptic facilitation' of transmitter release in the CNS.

[Changes of angiotensin II contents in rat plasma, brain, cardiovascular system and adrenal during stress].

The changes of content of angiotensin II (A II) in plasma, anterior hypothalamus, medulla oblongata, myocardium, vasculature and adrenals during acute and chronic stress were studied in rats. Compulsive cold-water swimming and trauma by limb-breaking were used to set acute stress models while cold environment of 4-8 degrees C was used to set chronic stress model. The results showed that: (1) The A II levels in plasma were significantly increased in all three stress- model groups, reaching to 900%, 390% and 134% of the control in the swimming group, the limb-broken group and the cold environment group, respectively. It's clear that the level of angiotension II in the acute stress groups were much higher than those in the chronic stress group. (2) The A II levels of the rat brain, myocardium and blood vessel were also increased in the stress animals except the limb-broken group. Furthermore, the contents of angiotensin II of the chronic stress animals were significantly higher than those of the acute stress animals in the brain, myocardium and blood vessels, but not in the adrenals. (3) The adrenal A II content was significantly higher than that of the control in all stressed animals. (4) The plasma corticosterone was also significantly increased over the control level in both the acute and chronic stress groups. These results suggest that circulating angiotensin II and tissue angiotensin II may play a role during the development of acute and chronic stress, respectively.

Changes in circulating and tissue angiotensin II during acute and chronic stress.

Changes of angiotensin II and cAMP in plasma, brain tissue, adrenal gland and cardiovascular tissue during the acute and chronic stress were studied in rats. The acute stress group was subjected to compulsive cold water swimming for 20 min, while the chronic stress group was exposed to an ambient temperature of 4-8 degrees C for 5 days. The results indicated that plasma angiotensin II levels were significantly increased in both stress groups, reaching up to 900% and 134% of the control in the acute and chronic groups, respectively. Angiotensin II contents in the anterior hypothalamus, medulla oblongata, myocardium, vasculature and adrenals were also elevated in both groups. With the exception of the adrenals, the contents of tissue angiotensin II in the chronic stress animals were significantly higher than those of the acute stress animals. In contrast, cAMP levels in plasma and tissue (hypothalamus and adrenals) and corticosterone levels in plasma in the acute stress group were all higher than those in the chronic stress animals, although the levels of the latter group were also increased compared with the control group. These results suggest that circulating and tissue angiotensin II may play an important role in the acute and chronic stress responses and that angiotensin II should be classified as a stress hormone.

[Morphine decreased the content of cyclic AMP in the rat spinal cord].

It has been reported that morphine or opiate-like substances (OLS) can affect the contents of cyclic AMP and/or cyclic GMP in mammalian brain, but very little is known whether similar effects also occur in spinal cord. Using RIA method, we showed that morphine significantly decreased the content of cyclic AMP in the rat spinal cord in vivo and in vitro and this effect could be completely blocked by naloxone, while the concentration of cyclic GMP in rat spinal cord is unchanged. In view of the present experiment it is suggested that the changes of cyclic AMP in the central nervous system may be partly mediated by the action of morphine.

Study on the genome of cauliflower mosaic virus (Xinjiang isolate). Restriction mapping of virion and cloned viral DNA.

The genomic DNA of CaMV (Xinjiang isolate) was mapped on the virion DNA with a number of restriction endonucleases by double digestions and partial digestions of one-end 32P-labelled fragments. It contained the unique sites of BglI, SalI and XhoI. BamHI and HhaI each cut it at two sites, EcoRI at five sites (three of which were located), HpaII at seven sites, BglII at eight sites, and HaeIII at ten sites. The sites of all the enzymes used above, as well as HindIII which cut at ten sites were also mapped on the cloned CaMV DNA. Virion DNA and cloned viral DNA gave the same results in the number and location of cleavage sites of the restriction enzymes tested on both DNAs. Like most of the other isolates, CaMV-Xinjiang isolate has three single-stranded discontinuities. From the digestion patterns of SalI, XhoI, EcoRI and HhaI, it is concluded that the restriction map of CaMV-XJ genome is closely related to that of BKMV isolate from East Germany.