Co-modulation of acute ethanol-induced motor impairment by mouse cerebellar adenosinergic A1 and GABA(A) receptor systems

We have previously demonstrated that cerebellar adenosine modulates ethanol ataxia. Using Rotorod method, we investigated the role of cerebellar GABA(A) receptors in the adenosinergic modulation of ethanol ataxia in mice. Direct cerebellar microinfusion of GABA(A) agonist, muscimol (2.5, 5 and 10 ng) and antagonist, bicuculline (50, 100 and 200 ng), via permanently implanted guide cannulas, produced a marked and dose-dependent accentuation and attenuation, respectively, of ethanol (2g/kg; IP) ataxia. The accentuation of ethanol ataxia by intracerebellar muscimol was through GABA(A) receptor because intracerebellar pretreatment with bicuculline virtually abolished muscimol effect. Intracerebellar microinfusion of adenosine A(1) agonist, N(6)-cyclohexyladenosine (CHA: 4 ng), and antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX: 100 ng) markedly accentuated and attenuated, respectively, ethanol ataxia consistent with our previously published data. Intracerebellar microinfusion of CHA (4 ng) or DPCPX (100 ng) markedly enhanced and reduced, respectively, muscimol (10 ng)-induced accentuation of ethanol ataxia suggesting co-modulation of ethanol ataxia by cerebellar adenosinergic A(1) and GABA(A) receptors. Similarly, intracerebellar bicuculline (200 ng) pretreatment not only prevented CHA-induced accentuation of ethanol ataxia, but caused further decrease in ethanol ataxia. No change in the normal coordination was observed when microinfusion of the highest dose of muscimol, bicuculline, DPCPX or CHA alone or in combination was followed by saline injection instead of ethanol. The results of the present study suggest a functional similarity between GABA(A) and adenosine A(1) receptors even though both receptor types are known to couple to different signaling system and their location is on the opposite ends of the cerebellar granule cells, axons and axonal terminals (i.e., GABA(A) at the granule cells and adenosine A(1) on axons and axonal terminals of the granule cells) and act as co-modulators of ethanol ataxia.     

Role of mouse cerebellar nicotinic acetylcholine receptor (nAChR) α(4)β(2)- and α(7) subtypes in the behavioral cross-tolerance between nicotine and ethanol-induced ataxia

We have demonstrated that nicotine attenuated ethanol-induced ataxia via nicotinic-acetylcholine-receptor (nAChR) subtypes α(4)β(2) and α(7). In the present study, ethanol (2g/kg; i.p.)-induced ataxia was assessed by Rotorod performance following repeated intracerebellar infusion of α(4)β(2)- and α(7)-selective agonists. Localization of α(4)β(2) and α(7) nAChRs was confirmed immunohistochemically. Cerebellar NO(x) (nitrite+nitrate) was determined flurometrically. Repeated intracerebellar microinfusion of the α(4)β(2)-selective agonist, RJR-2403 (for 1, 2, 3, 5 or 7 days) or the α(7)-selective agonist, PNU-282987 (1, 2, 3 or 5 days), dose-dependently attenuated ethanol-induced ataxia. These results suggest the development of cross-tolerance between ethanol-induced ataxia and α(4)β(2) and α(7) nAChR agonists. With RJR-2403, the cross-tolerance was maximal after a 5-day treatment and lasted 48h. Cross-tolerance was maximal after a 1-day treatment with PNU-282987 and lasted 72h. Pretreatment with α(4)β(2)- and α(7)-selective antagonists, dihydro-β-erythroidine and methyllycaconitine, respectively, prevented the development of cross-tolerance confirming α(4)β(2) and α(7) involvement. Repeated agonist infusions elevated cerebellar NO(x) 16h after the last treatment while acute ethanol exposure decreased it. Pretreatment with repeated RJR-2403 or PNU-282987 reversed ethanol-induced decrease in NOx. The NO(x) data suggests the involvement of the nitric oxide (NO)-cGMP signaling pathway in the cross-tolerance that develops between α(4)β(2)- and α(7)-selective agonists and ethanol ataxia. Both α(4)β(2) and α(7) subtypes exhibited high immunoreactivity in Purkinje but sparse expression in molecular and granular cell layers. Our results support a role for α(4)β(2) and α(7) nAChR subtypes in the development of cross-tolerance between nicotine and ethanol with the NO signaling pathway as a potential mechanism.     

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 ethanol's motor impairment in a dose-related manner as well as abolished CHA's 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 CHA's accentuation of ethanol's 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 G_i 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 CHA's accentuation of ethanol's motor impairment. Finally, cAMP, unlike its antagonism to CHA's 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 ethanol's ataxia.     

Effects of anti-glutamic acid decarboxylase antibodies associated with neurological diseases

OBJECTIVE: Glutamic acid decarboxylase (GAD) catalyzes the conversion of glutamic acid into GABA. GAD autoantibodies (GAD-Ab) have been described in diabetes mellitus and in diseases involving the central nervous system such as stiff-person syndrome and cerebellar ataxia. However, the pathogenic role of GAD-Ab in neurological diseases remains a matter of debate. METHODS: Using neurophysiological and neurochemical methods, we analyzed the effects of intracerebellar and paraspinal administration of GAD-Ab in rats. RESULTS: Intracerebellar administration of IgG from patients with GAD-Ab and neurological involvement (IgG-GAD) blocked the potentiation of the corticomotor response normally associated with trains of repetitive peripheral nerve stimulation. When injected in the lumbar paraspinal region, IgG-GAD induced continuous motor activity with repetitive discharges, abnormal exteroceptive reflexes, and increased excitability of anterior horn neurons, as assessed by F/M ratios. Furthermore, IgG-GAD significantly reduced the N-methyl-D-aspartate-mediated production of nitric oxide in cerebellar nuclei and impaired the synaptic regulation of glutamate after N-methyl-D-aspartate administration. These effects were not observed after administration of IgG from the following groups: (1) patients with GAD-Ab, diabetes mellitus, and without neurological complications; and (2) control patients. INTERPRETATION: These results indicate that stiff-person syndrome and cerebellar ataxia are the direct consequence of antibody-mediated neuronal dysfunction.     

Cerebellar CB(1) receptor mediation of Delta(9)-THC-induced motor incoordination and its potentiation by ethanol and modulation by the cerebellar adenosinergic A(1) 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.     

Mouse cerebellar GABAB participation in the expression of acute ethanol-induced ataxia and in its modulation by the cerebellar adenosinergic A1 system

The possible modulation and of co-modulation by the cerebellar GABA_B and adenosine A₁ receptors of ethanol-induced motor impairment were investigated in the mice using rotorod performance as the test response. Direct cerebellar microinfusion of GABA_B agonist, baclofen, and antagonist, phaclofen, into the permanently cannulated mice, produced a dose-dependent accentuation and attenuation, respectively, of ethanol-induced motor impairment. The baclofen and phaclofen exhibited accentuation and attenuation, respectively, via GABA_B receptors linked to pertussis toxin-sensitive G protein. A co-modulation by the cerebellar adenosine A₁ receptors was also observed because intracerebellar microinfusion of adenosine agonists N⁶-cyclohexyladenosine (CHA), 5′-N-ethylcarboxamidoadenosine (NECA), and 2-p-(2-carboxyethyl)-phenyl-ethylamino-5′-N-ethylcarbox-amidoadenosine (CGS-21680), and antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), also accentuated and attenuated, respectively, ethanol-induced motor impairment. The accentuation of ethanol-induced motor impairment by baclofen was further enhanced after the intracerebellar microinfusion of CHA, suggesting a co-modulation by the co-localized adenosine A₁ receptors. A similar response was observed after the intracerebellar microinfusion of adenosine A₁ = A₂ agonist NECA and the several-fold higher dose of adenosine A₂-selective agonist CGS-21680. Ethanol-induced motor impairment was markedly blocked by intracerebellar A₁-selective antagonist, DPCPX, as well as by the intracerebellar pertussis toxin pretreatment suggesting again a co-modulation by the adenosine A₁ receptors and the involvement of pertussis toxin-sensitive G protein, respectively. The almost 25-fold higher dose of CGS-21680 to accentuate and DPCPX to attenuate, respectively, ethanol-induced motor impairment together with the reported cerebellar localization of adenosine A₁ subtype only, suggested A₁ receptor activation by NECA and CGS-21680. The functional similarity between GABA_B and adenosine A₁, receptors associated with their anatomical co-localization on the cerebellar granule cells, mainly axons and axonal terminals, may suggest a possible common adenylate cyclase catalytic unit as the basis of modulation of ethanol's motor impairment by these two receptor mechanisms.     

Nicotine-induced NO-mediated increase in cortical cerebral blood flow is blocked by beta2-adrenoceptor antagonists in the anesthetized rats

Involvement of nitric oxide (NO) and beta-adrenoceptors in an increase in the cortical cerebral blood flow (CBF) following an intravenous (i.v.) injection of a small dose of nicotine which did not affect the systemic blood pressure in the rats was investigated. I.v. injection of nicotine (30 microg/kg) for 1 min produced a significant increase in CBF lasting for more than 20 min without a significant effect on the systemic blood pressure. I.v. injection of L-N(G)-nitroarginine methylester (30 mg/kg) significantly attenuated nicotine-induced increase in the cortical CBF. The attenuation was reversed by i.v. injection of L-arginine (300 mg/kg), suggesting an intimate role of nitric oxide (NO) in nicotine-induced increase in the cortical CBF. The nicotine-induced increase in the cortical CBF was significantly attenuated by propranolol (10 mg/kg, i.v.) and ICI 118,551 (a beta2-adrenoceptor antagonist, 10 mg/kg, i.v.) but not by metoprolol (a beta1-adrenoceptor antagonist, 10 mg/kg, i.v.). Beta2-adrenoceptors on presynaptic nitrergic nerves may be involved in nicotine-induced NO-mediated increase in the cortical CBF.     

Cerebellar CB1 receptor mediation of Δ-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 Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and other naturally occurring cannabinoid receptor (CB₁) mRNA on Δ⁹-THC-induced motor impairment was investigated in mice. Δ⁹-THC (15–30 μg/μl; intracerebellar) resulted in a significant motor impairment in a dose-related manner. The intracerebellar pretreatment with antisense oligodeoxynucleotide (3.0 μg/100 nl/12 h; six administrations/mouse) virtually abolished Δ⁹-THC (15 and 25 μg/1 μl; 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 Δ⁹-THC-induced motor impairment. These results strongly suggest the involvement of CB₁ receptor in the expression of Δ⁹-THC-induced motor impairment. The intracerebellar microinfusion of adenosine A₁-selective agonist, N⁶-cyclohexyladenosine (CHA) (4 ng/100 nl) significantly enhanced Δ⁹-THC-induced motor impairment, suggesting a cerebellar A₁ adenosinergic modulation of motor impairment. A pretreatment with the antisense and the mismatched oligodeoxynucleotide also markedly attenuated and did not alter, respectively, the cerebellar A₁ adenosinergic modulation (enhancement) of Δ⁹-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 Δ⁹-THC. The Δ⁹-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 Δ⁹-THC- and Δ⁹-THC+CHA-induced motor incoordination suggesting coupling of CB₁ receptor to PTX-sensitive G-protein (G_i/G_o). These data suggested co-modulation by cerebellar cannabinoid and adenosine system of Δ⁹-THC-induced motor impairment. Conversely, the results in the present study also suggested co-modulation by cerebellar adenosine A₁ and CB₁ receptors of ethanol-induced motor impairment, thereby indicating a possible common signal transduction pathway in the expression of motor impairment produced by Δ⁹-THC as well as ethanol.     

Impairment of the nitric oxide/cyclic GMP pathway in cerebellar slices prepared from thehph-1 mouse

In this study, the effect of tetrahydrobiopterin deficiency on the nitric oxide/cGMP pathway has been investigated in cerebellar slices derived from thehph-1 mouse. This animal displays a partial deficiency of tetrahydrobiopterin. Basal levels of cGMP were significantly reduced (−29.5%) in thehph-1 mouse cerebellum compared to controls. Following kainate stimulation (500 μM) cGMP levels increased in both control andhph-1 preparations but were again significantly lower (−29.1 %) in thehph-1 mouse. Exposure of slices to the nitric oxide donors,S-nitroso-N-acetylpenicillamine andS-nitroso-glutathione, revealed no difference in cGMP accumulation between the two groups. These findings suggest that the cerebellar nitric oxide/cGMP pathway may be impaired in partial tetrahydrobiopterin deficiency states due to diminished nitric oxide formation.     

Intercellular action of nitric oxide increases cGMP in cerebellar Purkinje cells

cGMP is thought to play a role in cerebellar signalling yet its production within Purkinje cells has never been detected. In the present study, the hydrolysis of a fluorescent substrate analogue, 2'-O-anthranyloyl cyclic GMP, by type 5 phosphodiesterase was monitored within Purkinje cells in slices and in culture. Nitric oxide, either endogenously released from adjacent neurons or pharmacologically applied, accelerated the rate of hydrolysis in a manner that was dependent on soluble guanylyl cyclase, demonstrating that nitric oxide triggers cyclic GMP production in Purkinje cells, which in turn activates type 5 phosphodiesterase. We conclude that NO acts as an intercellular messenger in the cerebellar cortex and that parallel fibre terminals are a probable source of nitric oxide.     

The Nitric Oxide/Cyclic GMP Messenger System in Olfactory Pathways of the Locust Brain

Nitric oxide is generated by a Ca²⁺/calmodulin-stimulated nitric oxide synthase and activates soluble guanylyl cyclase. Using NADPH diaphorase (NADPHd) staining as a marker for the enzyme nitric oxide synthase and an antiserum against cGMP, we investigated the cellular organization of nitric oxide donor and target cells in olfactory pathways of the brain of the locust ( Schistocerca gregaria ). A small subset of neuronal and glial cells expressed cGMP immunoreactivity after incubation of tissue in a nitric oxide donor. Nitric oxide-induced increases in cGMP immunoreactivity were quantified in a tissue preparation of the antennal lobe and in primary mushroom body cell cultures. The mushroom body neuropil is a potential target of a transcellular nitric oxide/ cGMP messenger system since it is innervated by extrinsic NADPHd-positive neurons. The mushroom body-intrinsic Kenyon cells do not stain for NADPHd but can be induced to express cGMP immunoreactivity. The colocalization of NADPHd and cGMP immunoreactivity in a cluster of interneurons of the antennal lobe, the principal olfactory neuropil of the insect brain, suggests a role of the nitric oxide/cGMP system in olfactory sensory processing. Colocalization of NADPHd staining and cGMP immunoreactivity was also found in certain glial cells. The cellular organization of the nitric oxide/cGMP system in neurons and glia raises the possibility that nitric oxide acts not only as an intercellular but also as an intracellular messenger molecule in the insect brain.     

Cat carotid body chemosensory responses to non-hypoxic stimuli are inhibited by sodium nitroprusside in situ and in vitro

We studied the effects of sodium nitroprusside, a nitric oxide donor, on the chemosensory responses to cyanide and nicotine in the cat carotid body. In situ, sodium nitroprusside infusion reduced the cyanide-evoked responses in a dose-dependent manner. In vitro, Tyrode containing nitroprusside reversibly reduced the cyanide- (by 59%) and nicotine-induced (by 45%) chemosensory responses. The present results suggest that chemosensory responses induced by cyanide and nicotine are reduced by increased nitric oxide content, similarly to the hypoxic chemosensory responses.     

A review of in vivo modulation of cerebellar cGMP levels by excitatory amino acid receptors: role of NMDA, quisqualate and kainate subtypes.

1. Agonists of all three EAA receptor subtypes augment cerebellar cGMP levels in vivo. 2. Agonists of the NMDA-associated glycine receptor also increase cerebellar cGMP levels but with lesser efficacy than EAA agonists. 3. Pharmacological agents, such as harmaline and pentylenetetrazol, which enhance endogenous EAA transmission also increase cerebellar cGMP levels in vivo. 4. Increases in cerebellar cGMP elicited by EAA receptor agonists are blocked by inhibition of nitric oxide synthase with N-monomethyl-L-arginine. 5. Basal cerebellar cGMP levels are decreased in a dose-dependent manner by competitive and non-competitive NMDA receptor antagonists but not by blockade of the NMDA-associated glycine receptor. 6. Selective alpha-1 blockade also antagonizes the actions of NMDA-dependent increases in cerebellar cGMP, suggesting NMDA receptor modulation of NE release from noradrenergic mossy fibers. 7. Quisqualate-dependent increases in cerebellar cGMP were blocked by the nonselective EAA antagonist, DNQX, but not by glycine antagonists or noncompetitive NMDA antagonists. 8. The sigma ligands, ifenprodil and BMY 14802, which did not alter or increased basal cerebellar cGMP levels, respectively, antagonized NMDA-dependent increases in cGMP levels. 9. The polyamines, spermine and spermidine, also did not change basal cGMP levels but nonselectively antagonized EAA-mediated increases in cGMP. 10. In summary, all 3 major EAA subtypes appear to modulate cerebellar cGMP levels in vivo. These actions also involve the intermediate generation of nitric oxide. The NMDA receptor population also appears to reside mainly on noradrenergic nerve endings in the cerebellum.     

Cytochemical Evidence for Nitric Oxide/Cyclic GMP Signal Transmission in the Visual System of the Locust

Nitric oxide is a membrane-permeant messenger molecule which activates soluble guanylyl cyclase. Using NADPH diaphorase staining as a marker for the enzyme nitric oxide synthase and an antiserum against cyclic GMP (cGMP), we investigated the possible sites of nitric oxide and cGMP synthesis in the retina and lamina of Schisfocerca gregaria. The photoreceptor cells did not express NADPH diaphorase staining but monopolar cells of the lamina were strongly stained. After inhibition of phosphodiesterase activity and incubation of tissue in a nitric oxide donor, the photoreceptor cells showed cGMP immunoreactivity. In contrast to the photoreceptors, the monopolar cells of the lamina were not stained. Since the presynaptic photoreceptors were cGMP-immunoreactive and the postsynaptic targets of the monopolar cells did not express immunoreactivity, it is conceivable that nitric oxide released by monopolar cells may play a role as a retrograde messenger in visual information processing.     

Protective effects of nicotine on ethanol-induced toxicity in cultured cerebellar granule cells

Alcoholism is associated with a higher incidence of smoking. In addition to the stimulatory effects of both ethanol and nicotine on the mesolimbic reward pathway, nicotine's ability to counteract some of the adverse effects of ethanol (e.g. ataxia) may be a powerful incentive for alcohol consumers to increase their tobacco (nicotine) intake. The cerebellum is believed to play an important role in ethanol-induced ataxia. In this study, we sought to test the hypothesis that nicotine would protect against toxic effects of ethanol in primary cultures of cerebellar granule cells. Moreover, it was postulated that the effects of nicotine would be mediated through nicotinic receptors. Primary cultures of cerebellar granule cells were prepared from 20-day embryos obtained from timed-pregnant Sprague Dawley rats. Cells were cultured for 10 days and were then exposed for 3 days to various concentrations of ethanol with and without pretreatment with nicotine and nicotinic antagonists. Cellular toxicity was evaluated by measuring the lactate dehydrogenase level. Administration of ethanol (10–100 mM) resulted in a dose-dependent toxicity. Pretreatment with nicotine 1–20 μM resulted in a dose-dependent protection against ethanol-induced toxicity. The effects of nicotine were blocked by pretreatment with nicotinic antagonists such as mecamylamine 1–20 μM, dihydro-β-erythroidine 1.0 nM–1.0 μM and methyllycaconitine 5 nM-5 μM in a dose-dependent manner. Thus, ethanol-induced cytotoxicity in primary cultures of cerebellar granule cells is blocked by pretreatment with nicotine. The effects of nicotine, in turn, may be blocked by nicotinic antagonists, implicating both high and low affinity nicotinic receptors in mediating the actions of nicotine. The exact mechanism of ethanol-induced toxicity and/or neuroprotection through activation of nicotinic receptors in this paradigm remains to be elucidated. The neuroprotective effect of nicotine against ethanol-induced toxicity in cerebellar neurons may be a contributing factor to the high incidence of smoking among alcoholics.     

Nitric oxide synthase,cGMP, and NO-mediated cGMP production in the olfactory bulb of the rat

High levels of nitric oxide synthase and cyclic 3′,5′-guanosine monophosphate (cGMP) in the olfactory bulb suggest that nitric oxide, acting as a diffusible intercellular messenger molecule inducing increased synthesis of cGMP, plays an important role in olfaction. The localization of cGMP after sodium nitroprusside stimulation of in vitro slices of rat olfactory bulb was compared with the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase, nitric oxide synthase, and glial fibrillary acidic protein. cGMP was detected immunohistochemically in cryostat sections. In the presence of the phosphodiesterase blocker isobutyl methylxanthine, cGMP was present in neurons in the glomerular layer, axons in the external and internal plexiform layers, and in a few somata and axons of the granule cell layer. This staining was blocked by N^G-nitro-L-arginine methylester hydrochloride or hemoglobin. After sodium nitroprusside stimulation, the olfactory nerve layer was intensely stained, as were the glomeruli and periglomerular cells. In the external plexiform layer, axonal staining was increased substantially, and there were occasional multipolar cGMP-positive neurons. In the internal plexiform and granule cell layers, axonal staining was greatly increased. Many granule cells were also cGMP positive after sodium nitroprusside stimulation. cGMP and nitric oxide synthase-positive neuronal elements overlapped in the glomerular and granule cell layers, but staining was not colocalized. cGMP was not found in astrocytes. The glutamatergic antagonists D-2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline caused differential inhibition of cGMP accumulation in layers of the olfactory bulb. These findings support the hypothesis that nitric oxide is an intercellular messenger in the olfactory bulb (Breer and Shepherd [1993] Trends Neurosci. 16:5–9). © 1996 Wiley-Liss, Inc.     

Effects of ethanol on nicotine-induced conditioned place preference in C57BL/6J mice

It has been shown that small doses of ethanol (相似文献   

实验性癫痫大鼠海马结构内一氧化氮-环磷酸鸟苷途径的研究

目的研究实验性癫痫发作大鼠海马结构内一氧化氮（ＮＯ）环磷酸鸟苷（ｃＧＭＰ）信使机制及其意义。方法雄性ＳＤ大鼠４１只，随机分为对照组（５只）、红藻氨酸（ＫＡ）１０、３０、６０分钟组（每组６只）和Ｌ硝基精氨酸甲酯（ＬＮＡＭＥ）＋ＫＡ１０、３０、６０分钟组（每组６只）。用放射免疫法测定ＫＡ诱导性癫痫发作中各时点海马结构内ｃＧＭＰ含量及ＬＮＡＭＥ的干预效应。结果ＫＡ注射引起大鼠海马结构内ｃＧＭＰ浓度升高，并加重大鼠癫痫发作（湿狗样摇动提早出现和发生次数增多）；ＫＡ注射前３０分钟给予ＬＮＡＭＥ可明显抑制ＫＡ１０、３０分钟组ｃＧＭＰ浓度的升高，但ＬＮＡＭＥ对ＫＡ６０分钟组ｃＧＭＰ的抑制作用不显著。结论在ＫＡ发作早期，ｃＧＭＰ浓度升高与内源性ＮＯ有关；ＮＯ的抗发作效应可能与ｃＧＭＰ信使机制存在某种联系。     

Natriuretic Peptides Stimulate Cyclic GMP Production in an Immortalized LHRH Neuronal Cell Line

The role of cyclic 3',5'-guanosine monophosphate (cGMP) as a second messenger in LHRH neurons is not well understood. Recent studies involving nitric oxide, a direct activator of soluble guanylate cyclase (GC), have implicated cGMP in the regulation of LHRH secretion both in vivo and in vitro . Evidence for the membrane-bound form of GC in LHRH neurons has thus far not been reported. In polymerase chain reaction screening of various cell lines for the natriuretic peptide receptors—which represent GCs—we identified both GC-A and GC-B cDNAs by southern blot hybridization in reverse transcribed and amplified extracts of the GT1-7 cell line, an immortalized LHRH neuronal cell line. Subsequent experiments demonstrated that all of the natriuretic peptides elevated cGMP production with a rank order of potency: CNP > ANP > BNP. Time course studies revealed a rapid intracellular accumulation of cGMP following exposure to CNP with a peak at 2.5 min. CNP was some 200-fold more potent than the NO donor, sodium nitroprusside, in stimulating cGMP accumulation in these cells. These data show for the first time the presence of functional mGCs on LHRH cells, and suggest that the natriuretic peptides may also participate in the regulation of LHRH activity.     

Fructose-1,6-bisphosphate preserves intracellular glutathione and protects cortical neurons against oxidative stress

The use of alcohol and nicotine are highly correlated, suggesting an underlying biochemical interaction. Chronic nicotine exposure results in a deactivation and subsequent upregulation of the expression of nicotinic acetylcholine receptors (nAChRs). Upregulation is thought to represent certain aspects of physical dependence on nicotine. If alcohol also alters nAChR expression or modulates the nicotine-induced upregulation, it could partially explain the high rate of co-abuse of these two drugs. We examined the effects of ethanol on the expression and nicotine-induced upregulation of nAChRs in two cell lines expressing different receptor subtypes. As measured by ligand binding, ethanol initially decreased nAChR expression in M10 cells but increased expression with a more chronic exposure. In the presence of nicotine, the effect of ethanol was similar; initially acting to blunt the upregulation of receptor expression caused by nicotine but enhancing the upregulation with 96 h of exposure. The upregulation of nAChRs was long lasting, remaining above control levels for as long as 7 days following removal of nicotine and ethanol. In PC12 cells, ethanol increased expression at all time points examined. A protein phosphatase inhibitor reduced nicotine-induced upregulation and a PKC inhibitor blocked the ethanol-induced decrease in nAChR expression. These data suggest that ethanol and nicotine interact at the level of the PKC pathway to regulate expression of nAChRs.