Dose-related neuroprotective effects of chronic nicotine in 6-hydroxydopamine treated rats, and loss of neuroprotection in alpha4 nicotinic receptor subunit knockout mice.

The present study examined the effect of a range of doses of chronic nicotine (0.75, 1.5, 3.0 and 30.0 mg kg(-1) day(-1), s.c., 14 days) upon striatal dopaminergic nerve terminal survival following 6-hydroxydopamine (6-OHDA; 10 microg intrastriatal unilaterally) in rats; and the effects of acute nicotine (1 mg kg(-1), s.c.) pretreatment upon striatal neurodegeneration induced by methamphetamine (5 mg kg(-1), i.p., three doses at 2 h intervals) in wild-type and alpha4 nicotinic receptor (nAChR) subunit knockout mice. In both models of Parkinsonian-like damage, loss of striatal dopaminergic nerve terminals was assessed by [(3)H]-mazindol autoradiography. In rats, chronic nicotine infusion delivered by osmotic minipump implanted subcutaneously 7 days prior to intrastriatal 6-OHDA injection produced significant and dose-related protection against 6-OHDA-induced neurodegeneration. Low (0.75 and 1.5 mg kg(-1) day(-1)) but not high (3.0 and 30.0 mg kg(-1) day(-1)) nicotine doses significantly inhibited 6-OHDA-induced degeneration. In wild-type mice, acute nicotine treatment produced significant inhibition of methamphetamine-induced neurodegeneration. In alpha4 nAChR subunit knockout mice, acute nicotine treatment failed to inhibit methamphetamine-induced neurodegeneration. Nicotine is capable of protecting dopaminergic neurons against Parkinsonian-like neurodegeneration in vivo. In rats, this neuroprotective effect is critically dependent upon nicotine dose and is consistent with the activation of nAChRs, as high, desensitizing doses of nicotine fail to be neuroprotective. Further, neuroprotection is absent in alpha4 nAChR subunit knockout mice. The current results therefore suggest that activation of alpha4 subunit containing nAChRs constitutes a major component of the neuroprotective effect of nicotine upon Parkinsonian-like damage in vivo.

Nicotine regulates alpha7 nicotinic receptor subunit mRNA: implications for nicotine dependence.

Recent evidence suggests that alpha7 subunit-containing nicotinic receptors (nAChRs) within the ventral tegmental area (VTA) are involved in the processes underlying nicotine tolerance and withdrawal. The current study used in situ hybridization histochemistry with multiple radiolabelled probes to amplify mRNA signal, to examine the distribution of alpha7 nAChR subunit mRNA both in control brains and following chronic nicotine treatment (1.5 and 30.0 mg/kg/day). Low levels of alpha7 transcript were detected within substantia nigra pars compacta (SNpc), substantia nigra pars reticularis (SNpr) and VTA. Higher levels of alpha7 transcript were found within the cortex and hippocampus. Following chronic nicotine treatment, levels of alpha7 subunit mRNA were significantly elevated in SNpc, SNpr and VTA, but were unchanged in cortex and hippocampus.

Nicotinic receptor subunit mRNA in the thalamus of the rat: relevance to schizophrenia?

Recent evidence suggests that aberrant nicotinic receptor (nAChR) expression plays an important role in schizophrenia. The present study sought to examine the distribution of nAChRs within the thalamus and associated cholinergic structures by examining nAChR subunit mRNA expression using in situ hybridization histochemistry. Transcripts for alpha4 and beta2 subunits were found in high levels in all thalamic nuclei and at lower levels in cholinergic nuclei (PPTg and MS/VDB). Distribution of mRNA encoding for additional subunits was restricted; lower alpha3 subunit transcript levels were detected in the anterior thalamic nuclei and portions of the lateral and posterior thalamic nuclei, with alpha7 transcripts being detected in cholinergic nuclei, the IMD and very low levels in the RTN. Low levels of alpha6 and beta3 transcript were found only within the RTN.

Galanin messenger RNA during postnatal development of the rat brain: expression patterns in Purkinje cells differentiate anterior and posterior lobes of cerebellum.

Following our initial mapping of preprogalanin messenger RNA in adult brain and its presence in a subpopulation of cerebellar Purkinje neurons [Ryan M. C. and Gundlach A. C. (1996) Neuroscience 70, 709-728], the present study examined the ontogenic expression of preprogalanin messenger RNA in the postnatal rat brain focussing on the Purkinje cells of the cerebellar cortex. Using in situ hybridization histochemistry, preprogalanin messenger RNA was detected in the developing forebrain and hindbrain from postnatal day 4 to day 60 (adult). On postnatal day 4 very light hybridization signal (labelling) was observed in cells of a number of nuclei including the central amygdaloid nucleus, the medial preoptic area, paraventricular nucleus and dorsomedial hypothalamic nucleus of the forebrain while lightly-labelled cells were detected in neurons of the nucleus of the solitary tract and locus coeruleus of the hindbrain. Hybridization signal was not apparent in other nuclei until later, with positively-labelled neurons first apparent in the dorsal cochlear nucleus at postnatal day 21. The abundance of preprogalanin messenger RNA-positive neurons and the intensity of the hybridization signal increased, in most regions, until postnatal day 28 when labelling resembled that of the mature rat. Preprogalanin messenger RNA was first detected in the cerebellum on postnatal day 10 only in Purkinje cells of lobule 10 of the posterior vermis and increased in distribution throughout Purkinje cell layers of the entire cerebellar cortex by postnatal day 13. The intensity of hybridization signal in Purkinje cells varied between lobules, with Purkinje cells in lobule 10 displaying a moderate to heavy degree of labelling, while lobules 6-9 and the more posterior lobules of the hemisphere including crus 2 of the ansiform lobule, the paramedian lobule and the copula pyramis, displayed only light labelling. The intensity of labelling in the anterior vermis and the remaining lobules of the hemisphere including crus 1 of the ansiform lobule, the simple lobule, the paraflocculus and the flocculus, was homogeneously weak. By postnatal day 21, Purkinje cell labelling reached maximum intensity in all lobules. Regional differences were still apparent, however, with labelling in the posterior vermis and hemisphere ranging from moderate to heavy, with only light to moderate labelling detected in the anterior vermis. The intensity of labelling in the posterior vermis and most lobules of the hemisphere was similar from postnatal day 21 to adulthood, while, in the anterior vermis, crus 1 of the ansiform lobule and the simple lobule, the intensity of hybridization decreased slightly by postnatal day 28 and was completely absent in Purkinje cells of the adult rat. Differential expression of preprogalanin messenger RNA in Purkinje cells of the developing rat cerebellum and transient expression in certain lobules suggests that galanin gene products may have a role in both the developing and mature rat brain and that galanin gene expression may represent a useful marker for differentiating the anterior and posterior cerebellar lobes.

Distribution of N-type Ca2+ channel binding sites in rabbit brain following central administration of omega-conotoxin GVIA.

Central administration of the N-type Ca2+ channel blocker omega-conotoxin GVIA in conscious rabbits has previously been shown to result in a slowly developing hypotensive and sympatholytic effect, with peak changes observed after 48 h. The aim of the current study was to examine the distribution of [125I] omega-conotoxin GVIA binding in rabbit brain alone or following a prior i.c.v. injection of omega-conotoxin GVIA to determine the site(s) of action of centrally administered omega-conotoxin GVIA. Brains were removed from rabbits 2 or 48 h after central administration of vehicle or non-labelled omega-conotoxin GVIA (30 pmol/kg, i.c.v.). Brain sections were then incubated with [125I] omega-conotoxin GVIA (50 pM) and the density of specific [125I] omega-conotoxin GVIA binding measured in dpm/mm2 was determined by quantitative receptor autoradiography. In the vehicle group, highest densities of [125I] omega-conotoxin GVIA binding sites (> 20 dpm/mm2) were detected in cortex, caudate, putamen, and the stratum oriens and stratum radiatum of the hippocampus. Prior (48 h) i.c.v. injection of omega-conotoxin GVIA resulted in a decrease in specific binding of [125I] omega-conotoxin GVIA, particularly in cortex and some portions of the caudate and hippocampus. Lesser effects were observed with a prior (2 h) i.c.v. injection of omega-conotoxin GVIA. Central administration of omega-conotoxin GVIA may be acting to disrupt neurotransmission in higher brain regions which may, in turn, affect cardiovascular control mechanisms in the rabbit.

Roles for nitric oxide as an intra- and interneuronal messenger at NMDA release-regulating receptors: evidence from studies of the NMDA-evoked release of [3H]noradrenaline and D-[3H]aspartate from rat hippocampal slices.

N-Methyl-D-aspartate (NMDA) receptors regulating the release of [3H]noradrenaline ([3H]NA) and D-[3H]aspartate (D-[3H]Asp) were investigated in superfused slices of rat hippocampus in the presence and absence of nitrergic drugs to examine a possible role for nitric oxide (NO) in the release process. In Mg(2+)-free Krebs-Henseleit buffer, the NMDA-evoked release of [3H]NA and D-[3H]Asp was Ca2+ dependent and inhibited by the NMDA antagonist (+/-)-3-(2-carboxypiperazin-4-yl)propenyl-1-phosphonic acid. NMDA-stimulated release of [3H]NA was tetrodotoxin (TTX; 0.1-2 microM) sensitive, whereas that for D-[3H]Asp was TTX insensitive, indicating that the NMDA receptors involved are differentially localized; those for D-[3H]Asp appear to be presynaptic, whereas those for [3H]NA are extrasynaptic in location. L-Arginine (100 microM), the natural precursor of NO synthesis, enhanced NMDA-evoked release of [3H]NA (100%) and D-[3H]Asp (700%). Exogenous NO donors--sodium nitroprusside, 3-morpholinosyndnomine, and S-nitroso-N-acetylpenicillamine (all 100 microM)--stimulated the NMDA-evoked release. An exception was the inhibition by nitroprusside of NMDA-evoked release of [3H]NA, where the presence of antioxidants may influence channel activity. Inhibitors of NO synthase (NG-nitro-, NG-methyl-, and NG-amino-L-arginine, all 100 microM) attenuated (50-80%) the NMDA-stimulated release of [3H]NA and D-[3H]Asp, as did KN-62 (10 microM), a specific inhibitor of calmodulin kinase II. Our data support roles for the NO transducing system subsequent to the activation of NMDA release-regulating receptors as both an intraneuronal (presynaptically) and an extraneuronal messenger.

Diverse roles for nitric oxide in synaptic signalling after activation of NMDA release-regulating receptors.

NMDA receptors regulating transmitter release were studied in three model systems to investigate whether their activation involves the NO transduction system. In superfused slices of rat brain, the release of [3H]D-aspartate, [3H]noradrenaline and [3H]GABA evoked by NMDA could be modulated by nitrergic drugs. Tetrodotoxin (0.1 microM) exerted differential effects in the three systems indicative of the NMDA receptors (and hence sites of NO generation) being pre- or extra-synaptic, or a combination of both types of localization. L-Arginine (100 microM) enhanced NMDA-evoked release of [3H]GABA (110%), [3H]NA (120%) and [3H]D-ASP (700%). Exogenous NO donors could increase NMDA-induced release of [3H]NA and [3H]D-ASP from hippocampal slices, although differential effects were noted, whilst inhibitors of NO synthase (NG-nitro- and NG-amino-L-arginine, both 100 microM) attenuated (60-85%) the release. NMDA-evoked release of [3H]GABA from striatal slices were insensitive to exogenous NO donors, but NG-nitro- and NG-amino-L-arginine produced 100% increases. In all cases, the NMDA receptors regulating release are linked to a NO system, although the link to the receptors modulating release of [3H]GABA appeared different. The actions of the nitrergic drugs may depend upon the redox state and/or cellular milieu of the individual NMDA receptors involved.

Hippocampal lesions induced by microinjection of the nitric oxide donor nitroprusside.

Unilateral intrahippocampal microinjection of the nitric oxide (NO) donor, sodium nitroprusside (33 nmol in 1 microliter), in the rat resulted in a marked degeneration of the ipsilateral hippocampal formation characterised by a virtual absence of pyramidal cells and dentate granule cells. Damage was not observed contralateral to the injection site. Quinolinic acid (100 nmol in 1 microliter) also produced neuronal damage within the ipsilateral hippocampus although the lesion was considerably smaller and more discrete than that caused by sodium nitroprusside. Injection of equimolar amounts of potassium ferricyanide failed to mimic the neurotoxic effects of sodium nitroprusside suggesting that NO is responsible specifically for the neuronal damage observed.

Effect of nicotine and tacrine on acetylcholine release from rat cerebral cortical slices.

The effect of nicotine (1-10 microM) and tacrine (9-amino-1,2,3,4-tetrahydroacridine; THA) on stimulation evoked release of [3H]acetylcholine from the rat brain slice preparation preincubated with [3H]choline was investigated. In these preparations, nicotine enhanced while tacrine inhibited evoked [3H]acetylcholine release. These effects were blocked by (+)tubocurarine (1 microM) and atropine (0.1 microM) respectively. In the presence of idazoxan (0.3 microM) plus atropine (0.1 microM), nicotine (3 microM) continued to enhance evoked [3H]acetylcholine release while the inhibitory effect of tacrine (1 microM) on evoked [3H]acetylcholine release was reversed to an enhancement. Under these circumstances the effects of both nicotine and tacrine were blocked by (+)tubocurarine (1 microM). These findings demonstrate that tacrine can both inhibit or enhance [3H]acetylcholine release, most likely through its activity as a cholinesterase inhibitor. Under normal circumstances following tacrine the predominant effect of the elevated levels of acetylcholine will be activation of inhibitory presynaptic muscarine receptors on cholinergic nerves and an inhibition of evoked [3H]acetylcholine release. Under conditions where both presynaptic inhibitory muscarine and alpha 2-adrenoceptors are blocked, the elevated levels of acetylcholine produced by tacrine will lead to the activation of facilitatory presynaptic nicotine cholinoceptors on cholinergic nerves and an enhancement of evoked [3H]acetylcholine release.

Increased vascular reactivity induced by essential fatty acid deficiency in rat autoperfused hindquarters.

The effects of essential fatty acid deficiency (EFAD) on vascular reactivity to vasoconstrictor stimuli were studied in rat autoperfused hindquarters. Weanling male Sprague-Dawley rats (aged 21 days) were fed diets containing 8% (weight/weight) of stearax plus 2% safflower oil (control diet) or 10% stearax (EFAD diet) for 8 weeks. There was no difference in systemic blood pressure or body weight between the two groups. Basal production of immunoreactive 6-keto-PGF1 alpha by aortic segments was much less in EFAD aortae than in control aortae. In contrast, immunoreactive 6-keto-PGF1 alpha produced by incubating aortic segments with exogenous arachidonic acid (12 mumol/l) was much greater in EFAD aortae than in control aortae. Moreover, conversion of [14C]-arachidonate to [14C]-6-keto-PGF1 alpha was more pronounced in EFAD aortae than in control aortae. Vasoconstrictor responses to noradrenaline (0.01-1.0 mumol/l) and angiotensin II (0.001-1.0 mumol/l) infused into the blood perfused hindquarters were then examined. The rats on the EFAD diet were more sensitive to both noradrenaline and angiotensin II than rats on the control diet (P less than 0.05, two-way ANOVA). Thus, a deficiency of essential fatty acids can lead to increased vascular sensitivity to vasoconstrictor stimuli. Deficiency of arachidonic acid in phospholipid stores is also accompanied by augmented cyclo-oxygenase activity in the vessel wall, similar to that observed previously in spontaneously hypertensive rats (SHR) and rats with one kidney renovascular hypertension.

Effects of arachidonate deficiency on endothelium-dependent vascular reactions.

1. The release of endothelium-derived relaxing factor (EDRF), which appears to be impaired in vessels chronically exposed to hypertension, may involve mobilization of arachidonate from phospholipids. In this study the effects of arachidonate deficiency on endothelium-dependent responses were examined in rat isolated aorta. 2. Weanling rats were fed an essential fatty acid-deficient (EFAD) diet for 8 weeks which reduced plasma and aortic phospholipid arachidonate content from 17 to 1.8% and from 21 to 8%, respectively. After this time the rats were killed and the reactivity of aortic rings was studied in organ baths. 3. In aortic rings from control rats the concentration-response curves for the contractile action of phenylephrine were shifted to the left 3.5-fold by removal of the endothelium, and the maximum was not altered. 4. In contrast, in EFAD rings with endothelium, the maximal vasoconstriction to phenylephrine was less than in control rings, and removal of the endothelium increased the maximum (from 1.9 +/- 0.2 to 3.2 +/- 0.1 g, P less than 0.05) and reduced the EC50 7-fold. 5. In EFAD rings precontracted with phenylephrine (0.3 mumol/l) the relaxations produced by the endothelium-dependent dilator acetylcholine were not significantly different from those produced in control rings. The dilator actions of sodium nitroprusside were also similar in EFAD and control rings. 6. Thus, endothelium-dependent dilatation in the aorta is not impaired by partial depletion of phospholipid arachidonate. However, contractile responses to alpha-adrenoceptor agonists are depressed by spontaneously released EDRF in rat aorta, so that the results suggest that depletion of phospholipid arachidonate either augments spontaneous release of EDRF, or impairs EDRF inactivating mechanisms.

Modulation of neuroeffector transmission.

Local mechanisms that regulate transmitter release at autonomic neuroeffector junctions may be classified into four main types: (a) Automodulation, involving a feedback effect of the transmitter on receptors associated with the prejunctional terminals resulting in a restraint on the facilitation of release that occurs when a train of nerve impulses invades the terminals. Changes in the composition of the transmitter, such as the presence of adrenaline as a cotransmitter together with noradrenaline, can result in increased facilitation of transmission. (b) Transneuronal modulation involving an effect of the transmitter released from terminals of one type on adjacent terminals of another type; thus, noradrenaline release may be inhibited by acetylcholine released from cholinergic nerve terminals adjacent to the noradrenergic terminals. (c) Transjunctional modulation involving a feedback effect on the prejunctional nerve terminals of one or more factors released from effector cells. Such substances include adenyl compounds (adenosine and/or ATP) and metabolites of arachidonic acid. (d) Hormonal modulation involving the action of blood-borne hormones or locally generated hormone-like substances on prejunctional terminals. Some of the substances involved in modulation may act in more than one way; thus, opioids may function as cotransmitters or as hormones, and adenyl compounds may be cotransmitters or be released from effector cells. The effects of exogenous drugs on the substances involved in the modulation of transmission and on the prejunctional receptors for these substances account for many anomalous actions of drugs used or proposed for use in therapeutics.

Effect of alpha-adrenoceptor agonists and antagonists on cholinergic transmission in guinea-pig isolated atria.

Evidence was sought for the existence on cholinergic nerve terminals in guinea-pig atria of alpha-adrenoceptors subserving inhibition of acetylcholine release. The experiments were performed with atria which had been incubated with 3H-choline and transmitter release was deduced from the efflux of radioactivity elicited by field stimulation. In preparations which had been incubated with 3H-choline, field stimulation (60 pulses, 2 Hz) evoked release and radioactivity which was inhibited by 1.0 mumol/l noradrenaline, in the presence of propranolol (1.0 mumol/l), but was unaltered by clonidine (1.0 and 10.0 mumol/l). The inhibitory effect noradrenaline on the stimulation-induced efflux of radioactivity was blocked by idazoxan (0.3 mumol/l), and phentolamine (1.0 mumol/l) but not by prazosin (0.3 mumol/l). In the presence of propranolol (1.0 mumol/l), neither phentolamine (1.0 mumol/l), idazoxan (0.3 mumol/l) nor prazosin (0.3 mumol/l) had any effect on stimulation-induced efflux of radioactivity. Stimulation of the extrinsic vagus nerve of atrial preparations with trains of pulses at frequencies of 2, 4, 8, and 16 Hz produced graded decreases in the rate of atrial beating. The negative chronotropic responses to vagus stimulation were unaffected by noradrenaline (1.0 mumol/l) in the presence of propranolol (1.0 mumol/l). These findings indicate that the release of acetylcholine from the cholinergic terminals in guinea-pig atria can be inhibited by a mechanism apparently involving prejunctional alpha 2-adrenoceptors. However, under the experimental conditions used here the chronotropic responses of atria to stimulation of the extrinsic vagus nerve was not affected by activation of the prejunctional alpha 2-adrenoceptors associated with the cholinergic terminals.

Interaction between the inhibitory action of acetylcholine and the alpha-adrenoceptor autoinhibitory feedback system on release of [3H]-noradrenaline from rat atria and rabbit ear artery.

Stimulation-induced increases in the efflux of radioactivity (S-I efflux) were measured in the bathing medium following labelling of the noradrenergic transmitter pools of rat atria and rabbit artery preparations with [3H]-noradrenaline. In atria stimulated with trains of 16 or 60 pulses at 2 Hz, phentolamine enhanced, whereas acetylcholine inhibited S-I efflux. With trains of 16 pulses phentolamine had a smaller enhancing effect than with trains of 60 pulses, whereas the inhibitory effect of acetylcholine was more pronounced with 16 pulses of stimulation. The inhibitory effect of acetylcholine was markedly enhanced by phentolamine when stimulation was with 60 pulses. With 16 pulses of stimulation the effect of acetylcholine was unaltered by phentolamine and abolished by the alpha 2-adrenoceptor agonist 3,4-dihydroxyphenylimino-2-imidazolidine (DPI). Phentolamine had no effect on the negative inotropic effect of acetylcholine on driven left atrial preparations. In arterial preparations stimulated with trains of 30 pulses at 1 Hz, both acetylcholine and clonidine inhibited S-I efflux, whereas yohimbine and idazoxan enhanced S-I efflux. Combining acetylcholine with clonidine did not alter the inhibitory effect of clonidine but the combination of acetylcholine with yohimbine or idazoxan abolished the marked enhancing effects of yohimbine or idazoxan on S-I efflux. These findings indicate that there may be a reciprocal interaction between prejunctional alpha-adrenoceptors and prejunctional muscarinic cholinoceptors.

Acetylcholine-induced inhibition of responses to field stimulation in rabbit pulmonary artery is unaffected by endothelium removal.

Acetylcholine (0.01-10 mumol litre-1) relaxed normal rings (endothelium-retained) of rabbit pulmonary artery precontracted with clonidine (10 mumol litre-1) while preparations with the endothelium removed responded with contraction only. Removal of the endothelium had no effect on contractions of the preparation to clonidine or field stimulation of the adventitial nerves (2 Hz, 10 s). Furthermore, the inhibitory effect of acetylcholine (0.3 and 1.0 mumol litre-1) on contractions induced by field stimulation was not influenced by the vascular endothelium.