M. Saeed Dar

Mouse cerebellar adenosinergic modulation of ethanol-induced motor incoordination: possible involvement of cAMP

As an extension of our previous work pertaining to brain adenosinergic modulation of ethanol-induced motor incoordination, the effect of direct intracerebellar administration of the A1-selective adenosine agonist, N6-cyclohexyladenosine (CHA) on ethanol-induced motor incoordination was evaluated. Marked accentuation of ethanol-induced motor impairment by CHA was observed. No change in the normal motor coordination was noted when CHA administration was followed by saline instead of ethanol. Intracerebellar cAMP or its analog, 8-(4-chlorophenylthio)-cAMP, significantly inhibited ethanols motor impairment in a dose-related manner as well as abolished CHAs accentuating effect on ethanol-induced motor incoordination. These observations suggested a possible involvement of cAMP in the adenosinergic modulation and in the expression of ethanol-induced motor incoordination. Further support was provided by the observation of a marked accentuation and attenuation in a dose-related manner of ethanol-induced motor impairment as well as CHAs accentuation of ethanols motor impairment by intracerebellar miconazole and forskolin, respectively. However, equimolar intracerebellar doses of miconazole and forskolin (inhibitor and stimulator of adenylyl cyclase, respectively) failed to significantly alter ethanol-induced motor incoordination probably due to their mutual functional antagonism. The expression of adenosinergic modulation and that of ethanol-induced motor impairment most likely involved Gi protein-coupled receptor(s) (such as adenosine receptors). The involvement of receptors linked to pertussis toxin-sensitive G-proteins was suggested because intracerebellar pertussis toxin pretreatment markedly inhibited ethanol-induced motor incoordination as well as CHAs accentuation of ethanols motor impairment. Finally, cAMP, unlike its antagonism to CHAs accentuation, failed to antagonize the accentuation of ethanol-induced motor impairment by intracerebellar GABA(A) agonist (+)-muscimol. This indicated selectivity of cAMP participation in G protein coupled receptor (such as adenosine)-mediated response and not in ionic channel coupled receptor (such as GABA(A))-mediated mechanism. Overall, the data suggested a possible involvement of cerebellar adenylyl cyclase-cAMP signalling pathway in the adenosinergic modulation of ethanols ataxia.

Mediation of acute ethanol-induced motor disturbances by cerebellar adenosine in rats

The possible involvement of brain adenosine in acute ethanol-induced motor incoordination (MI) and inhibition of spontaneous motor activity (SMA) was investigated in male Sprague-Dawley rats. Pretreatment with theophylline or 7-(2-chloroethyl)-theophylline, adenosine antagonists, markedly reduced ethanol-induced MI and inhibition of SMA during a 60 min test period compared with saline + ethanol group. On the contrary, pretreatment with (-)-N6(R-phenylisopropyl)adenosine (R-PIA), an adenosine agonist, or dilazep, an adenosine uptake blocker, markedly potentiated the ethanol-induced MI as well as inhibition of SMA in a 60 min test period compared with saline + ethanol group. No effect on motor coordination was seen when the drug pretreatment was not followed by ethanol. However, the adenosine agonists and antagonists did alter SMA when the pretreatment with these drugs was not followed by ethanol. Ethanol clearance was not altered by the drug pretreatment as blood ethanol levels were similar in all groups except for lower ethanol levels in the R-PIA-treated group. Adenosine A1 binding studies, using 3H-R-PIA as the radioligand and crude membrane preparation from cerebellar cortex, revealed an increase in Bmax with no significant change in Kd in ethanol-treated animals vs. saline control. Theophylline pretreatment prevented the increase in Bmax elicited by ethanol. Collectively, the data suggest that endogenous cerebellar adenosine may be a participating factor in ethanol-induced motor dysfunctions.

Cerebellar CB1 receptor mediation of Δ9-THC-induced motor incoordination and its potentiation by ethanol and modulation by the cerebellar adenosinergic A1 receptor in the mouse

The effect of intracerebellar microinfusion of antisense oligodeoxynucleotide to Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and other naturally occurring cannabinoid receptor (CB(1)) mRNA on Delta(9)-THC-induced motor impairment was investigated in mice. Delta(9)-THC (15-30 microgram/microliter intracerebellar) resulted in a significant motor impairment in a dose-related manner. The intracerebellar pretreatment with antisense oligodeoxynucleotide (3.0 microgram/100 nl/12 h; six administrations/mouse) virtually abolished Delta(9)-THC (15 and 25 microgram/1 microliter intracerebellar)-induced motor impairment. However, intracerebellar pretreatment with the mismatched oligodeoxynucleotide in exactly the same manner as the antisense was completely ineffective in altering the Delta(9)-THC-induced motor impairment. These results strongly suggest the involvement of CB(1) receptor in the expression of Delta(9)-THC-induced motor impairment. The intracerebellar microinfusion of adenosine A(1)-selective agonist, N(6)-cyclohexyladenosine (CHA) (4 ng/100 nl) significantly enhanced Delta(9)-THC-induced motor impairment, suggesting a cerebellar A(1) adenosinergic modulation of motor impairment. A pretreatment with the antisense and the mismatched oligodeoxynucleotide also markedly attenuated and did not alter, respectively, the cerebellar A(1) adenosinergic modulation (enhancement) of Delta(9)-THC-induced motor impairment. There was no change in the normal motor coordination due to intracerebellar pretreatment with antisense and its mismatch, in the presence as well as absence of intracerebellar CHA indicating the selectivity of interactions with Delta(9)-THC. The Delta(9)-THC-induced motor incoordination was also significantly enhanced dose-dependently by systemic (i.p.) ethanol administration suggesting behavioral synergism between the two psychoactive drugs. Pretreatment (intracerebellar) with pertussis toxin (PTX) markedly attenuated Delta(9)-THC- and Delta(9)-THC+CHA-induced motor incoordination suggesting coupling of CB(1) receptor to PTX-sensitive G-protein (G(i)/G(o)). These data suggested co-modulation by cerebellar cannabinoid and adenosine system of Delta(9)-THC-induced motor impairment. Conversely, the results in the present study also suggested co-modulation by cerebellar adenosine A(1) and CB(1) receptors of ethanol-induced motor impairment, thereby indicating a possible common signal transduction pathway in the expression of motor impairment produced by Delta(9)-THC as well as ethanol.

Effect of Acute Ethanol on Release of Endogenous Adenosine from Rat Cerebellar Synaptosomes

Abstract: The effects of pharmacologically relevant concentrations of ethanol on the release of endogenous adenosine from rat cerebellar synaptosomes were investigated. Release was conducted for 5, 10, 30, or 60 s after which time the incubation medium (containing the released adenosine) was rapidly separated from the synaptosomal membranes by vacuum filtration. The adenosine content of the filtrate was measured by HPLC–fluorescence detection. Both basal and KCl‐stimulated adenosine release consisted of an initial rapid phase, for the first 10 s, that was followed by a relatively slower phase. Basal endogenous adenosine release was estimated as 199 ± 14 pmol/mg protein/5 s. Potassium (chloride) increased adenosine release from the basal level to 433 ± 83 pmol/mg protein/5 s. Ethanol caused a dose‐dependent increase of adenosine release. The interaction between dilazep and ethanol indicates that ethanol‐stimulated release does not involve the dilazep‐sensitive transport system. The results support previous findings that indicate that cerebellar adenosine is involved in the mediation of ethanol‐induced motor disturbances in the rat.

Release of endogenous glutamate from rat cerebellar synaptosomes interactions with adenosine and ethanol

The effects of ethanol and adenosine receptor agonist R-PIA and antagonist theophylline on release of endogenous glutamate were tested in rat cerebellar synaptosomal preparation. Release was carried out for 5 to 60 sec after which time the released glutamate was separated from the synaptosomal membranes by rapid filtration. The amount of released glutamate in the filtrate was measured by an enzyme-linked fluorometric assay. Basal endogenous glutamate release was estimated as 3.7 +/- 0.3 nmol/mg protein/5 sec and was stimulated by high K+. Glutamate release consisted of an initial rapid phase for the first 10 sec that was followed by a relatively slower phase. Both Ca2+-dependent and Ca2+-independent glutamate release were observed which suggested the involvement of both neuronal and glial constituents of the synaptosomal preparation, respectively. Pharmacologically relevant concentrations of ethanol (25-100 mM) caused a trend toward a dose-dependent inhibition of glutamate release. R-PIA and theophylline inhibited and stimulated, respectively, basal release of glutamate and R-PIA-inhibited release was blocked by theophylline. Ethanol (25 mM) blocked the stimulatory effect of theophylline and the results of experiments following the inclusion of adenosine deaminase suggested the involvement of adenosine in this effect of ethanol. The results support our previous findings that suggest an involvement of cerebellar adenosine in the motor disturbing effects of acute ethanol and extend those findings by indicating that ethanol inhibits glutamate release from granule cells of the cerebellar cortex through an adenosine-sensitive mechanism.

Cannabinoid-induced motor incoordination through the cerebellar CB1 receptor in mice

Cannabinoids are known to impair motor function in humans and laboratory animals. We have observed dose-dependent motor incoordination in mice evaluated by rotorod following direct intracerebellar (i.c.b.) microinjection of synthetic cannabinoid agonists CP55,940 (5-25 microg) and HU-210 (1.56-6.25 microg), through permanently implanted stainless steel guide cannulas. The motor incoordination was marked at 15, 35 and 55 min post-microinjection. The motor incoordination elicited by HU-210 (6.25 microg) and CP55,940 (20 microg) was significantly blocked by the CB(1) receptor-selective antagonist SR141716A (25 microg i.c.b.), indicating mediation by a cerebellar CB(1) receptor. Further direct evidence of CB(1) mediation was obtained through a CB(1) receptor antisense/mismatch oligodeoxynucleotide approach (3 microg/12 h; total of six doses). Mice treated with intracerebellar antisense had a significantly diminished motor incoordination response to intracerebellar CP55,940 15 microg compared to mice that received intracerebellar mismatch or no prior treatment. Also, the response to intracerebellar CP55,940 in the CB(1) mismatch-treated mice did not differ from the mice that received only CP55,940. A separate study using a cerebellar tissue punching technique, following intracerebellar [3H]-CP55,940 microinjection, confirmed that cannabinoid drug dispersion following microinjections was exclusively confined to the cerebellum. Microinjection of CP55,940 (20 microg) into the hippocampus, an area with a large density of CB(1) receptors, did not impair motor coordination. Taken together, these results indicate that cannabinoid-induced motor impairment occurs by activation of a CB(1) receptor in the cerebellum. The participation of other brain motor areas in cannabinoid-induced motor incoordination will require future study.

The effect of acute ethanol on dopamine metabolism and other neurotransmitters in the hypothalamus and the corpus striatum of mice

The effect of acute ethanol on the levels of NE, DA and its metabolites DOPAC and HVA, as well as on the levels of GABA, in the corpus striatum and hypothalamus were investigated in mice in the first two hours after acute ethanol administration. There was a marked increase in the concentration of DOPAC and HVA in the corpus striatum from 30 to 120 minutes after a dose of 3.5 g/kg of ethanol even though the concentration of DA was only elevated at 60 minutes after ethanol. A dose of 1.75 g/kg of ethanol did not increase DA levels 60 minutes after administration although it did increase the concentration of DOPAC and HVA at this time. In the hypothalamus a dose of 3.5 g/kg of ethanol did not change the concentration of NE or DA but did produce a marked increase in the levels of DOPAC and HVA at 60 and 120 minutes post ethanol. A lower dose of ethanol, 1.75 g/kg, produced the same effect 60 minutes after ethanol. Ethanol caused a dose-dependent accumulation of DOPA in the corpus striatum after inhibition of DOPA-decarboxylase suggesting an increased synthesis of DA. These data suggest that the increased concentrations of DA metabolites after ethanol is secondary to enhanced DA synthesis and turnover. The concentration of NE and GABA in the hypothalamus and the corpus striatum was unchanged at any time period after ethanol.

Antagonism by intracerebellar Ro15–4513 of acute ethanol-induced motor incoordination in mice

The possible antiethanol effect of intracerebellarly microinjected Ro15-4513 was investigated using motor incoordination as the test response. The results of this study further confirmed reports from this and other laboratories that this partially negative ligand of benzodiazepine selectively attenuated and nearly reversed the motor impairment of acute ethanol. The attenuation observed after microinjections of doses of 0.05, 0.1, and 0.5 ng was significant and dose related. There was no effect on normal coordination when the highest dose, 0.5 ng, was administered followed by saline instead of a test dose of ethanol. When 0.5 ng of Ro15-4513 alone was microinjected into the cerebellum, no significant change in the locomotor activity was observed. Even a 10-fold higher intracerebellar dose (5 ng) of Ro15-4513 administered alone produced no significant changes in locomotor activity. This suggests that attenuation of ethanol-induced motor incoordination was most likely due to the selective antiethanol effect of Ro15-4513 at the dose range used in the present investigation. The antiethanol effect of intracerebellar Ro15-4513 also reaffirmed the well-known belief that the cerebellum is an important brain region for ethanols motor-impairing effect. The results also indirectly suggest the inhibition of GABAA-gated chloride ion channel activity as the most likely basis of Ro15-4513s antiethanol effect.

Effect of chronically administered methylxanthines on ethanol-induced motor incoordination in mice.

The effect of chronic (10 days) administration of methylxanthines, caffeine, IBMX and theophylline on acute ethanol-induced motor incoordination has been investigated in the mice. In animals that received caffeine, 45 and 90 mg/kg/24 h, ethanol, 1.5 g/kg, produced motor incoordination significantly greater compared to that in the control groups. Significantly greater ethanol-induced motor incoordination was seen in animals fed IBMX, 30 and 60 mg/kg/24 h, compared to controls. Ethanol-induced increased motor incoordination in caffeine and IBMX-fed animals was also associated with significantly greater 3H-R-PIA binding in whole brains compared to tap water controls indicating an increase in brain adenosine binding sites. However neither motor incoordination nor 3H-R-PIA binding was altered in theophylline 75 and 150 mg/kg/24 h, fed animals. The increased motor incoordination associated with increased adenosine binding sites in the brains of caffeine and IBMX-fed animals suggests an involvement of central adenosine mechanisms in the motor incoordinating effect of ethanol and further supports our earlier suggestion for the role of adenosine in some of the central effects of ethanol.

Antagonism of ethanol ataxia by intracerebellar nicotine: Possible modulation by mouse cerebellar nitric oxide and cGMP

We have reported previously that intracerebellar nicotine attenuates ethanol ataxia via nicotinic-cholinergic receptors. We report now that attenuation of ethanol ataxia by intracerebellar nicotine is modulated by cerebellar nitric oxide-guanylyl cyclase (GC) messenger system. Intracerebellar microinfusion of SNP (sodium nitroprusside, a nitric oxide donor; 15, 30, and 60 pg) and SMT (S-methylisothiourea; 70, 140, and 280 fg; an inhibitor of inducible nitric oxide synthase), significantly enhanced and reduced, respectively, intracerebellar nicotine-induced attenuation of ethanol ataxia in a dose-related manner. Similarly, intracerebellar isoliquiritigenin (an activator of GC; 1, 2, and 4 pg) and ODQ (1H [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, an inhibitor of GC; 375, 750, and 1500 fg), significantly enhanced and reduced, respectively, intracerebellar nicotine-induced attenuation of ethanol ataxia in a dose-related fashion. These results suggest that the functional interaction between nicotine and ethanol may involve modulation by cerebellar nitric oxide and cGMP. Intracerebellar microinfusion of isoliquiritigenin (4, 8, and 16 pg) in the absence of nicotine significantly attenuated ethanol ataxia dose-dependently indicating a tonic involvement of cGMP in ethanol ataxia. Finally, intracerebellar nicotine (5 ng) significantly increased and ethanol 2 g/kg i.p. decreased levels of total cerebellar nitrite+nitrate (NOx) which were functionally correlated with ethanol ataxia and its attenuation by intracerebellar nicotine. The ethanol-induced decrease in NOx was significantly antagonized by intracerebellar nicotine. The NOx data further supported an involvement of nitric oxide in the behavioral interaction between nicotine and ethanol. Overall, the results of the present investigation demonstrate a functional correlation between cerebellar nitric oxide messenger system and the behavioral interaction between nicotine and ethanol.