Striatal plasticity in Parkinson's disease and L-dopa induced dyskinesia.

Striatal function adapts to the loss of nigrostriatal dopaminergic input in Parkinson's disease (PD) to initially maintain voluntary movement, but subsequently changes in response to drug treatment leading to the onset of motor complications, notably dyskinesia. Alterations in presynaptic dopaminergic function coupled to changes in the response of post-synaptic dopaminergic receptors causing alterations in striatal output underlie attempts at compensation and the control of movement in early PD. However, eventually compensation fails and persistent changes in striatal function ensue that involve morphological, biochemical and electrophysiological change. Key alterations occur in cholinergic and glutamatergic transmission in the striatum and there are changes in motor programming controlled by events involving LTP/LTD. Dopamine replacement therapy with L-dopa modifies altered striatal function and restores motor function but non-physiological dopamine receptor stimulation leads to altered signalling through D1 and D2 receptor systems and changes in striatal function causing abnormalities of LTP/LTD mediated through glutamatergic/nitric oxide (NO) mechanisms. These lead to the onset of dyskinesia and underlie the priming process that characterise dyskinesia and its persistence.

Continuous rotigotine administration reduces dyskinesia resulting from pulsatile treatment with rotigotine or L-DOPA in MPTP-treated common marmosets.

We previously showed that continuous infusion of rotigotine resulted in less dyskinesia than repeated pulsatile rotigotine administration or repeated oral administration of L-DOPA in MPTP-treated marmosets. Now we investigate whether continuous rotigotine delivery modifies established dyskinesia induced by prior exposure to repeated pulsatile administration of L-DOPA or rotigotine in MPTP-treated common marmosets. Repeated oral administration of L-DOPA or subcutaneous bolus administration of rotigotine over 28 days improved motor deficits but resulted in the onset of dyskinesia of moderate intensity. When these animals were switched to a 28-day continuous infusion of rotigotine, the reversal of motor disability was maintained. In those animals initially treated with L-DOPA, there was a small reduction in dyskinesia intensity but a significant reduction in the duration of dyskinesia. However, in animals initially treated with repeated bolus administration of rotigotine, dyskinesia intensity was significantly reduced. Initial treatment with a continuous infusion of rotigotine for 28 days reversed motor disability and resulted in a low incidence of dyskinesia. On switching to repeated oral administration of L-DOPA, the improvement in motor disability was maintained but the propensity of L-DOPA to provoke dyskinesia was not affected. In addition, while the continuous delivery of rotigotine prevented the expression of dyskinesia, the previously demonstrated ability of dopamine agonists to prime for dyskinesia could not be avoided. These data suggest that dyskinesia induced by pulsatile drug treatment may be improved by switching to continuous rotigotine delivery. In addition, while continuous delivery of rotigotine may prime for dyskinesia, it does not lead to its expression.

Continuous administration of rotigotine to MPTP-treated common marmosets enhances anti-parkinsonian activity and reduces dyskinesia induction.

Rotigotine is a novel, non-ergoline dopamine D(3)/D(2)/D(1)-receptor agonist for the treatment of Parkinson's disease that can be continuously delivered by the transdermal route to provide stable plasma levels. Continuous drug delivery should reduce the risk of dyskinesia induction in comparison to pulsatile dopaminergic treatment. Thus the aim of the study was to compare the reversal of motor disability and induction of dyskinesia produced by continuous compared to pulsatile rotigotine administration in MPTP-treated common marmosets. The study also investigated whether pulsatile or continuous rotigotine administration in combination with l-DOPA prevented l-DOPA-induced dyskinesia. Animals were treated for 28 days with vehicle or pulsatile (twice daily) or continuous delivery of rotigotine (via an osmotic minipump). Subsequently, l-DOPA was then co-administered for a further 28 days. Animals were assessed for locomotor activity, motor disability and dyskinesia induction. The study showed that both continuous and pulsatile administration of rotigotine improved motor deficits and normalized motor function in MPTP-treated monkeys. However, continuous rotigotine delivery reduced dyskinesia expression compared to pulsatile treatment. Both pulsatile and continuous rotigotine administration produced less dyskinesia than administration of l-DOPA alone. The addition of l-DOPA to either pulsatile or continuous rotigotine treatment resulted in the induction of marked dyskinesia similar to that produced by treatment with l-DOPA alone. These data further support the hypothesis that continuous delivery of a dopaminergic agent reduces the risk of dyskinesia induction. However, continuous rotigotine administration did not prevent l-DOPA from inducing dyskinesia suggesting that l-DOPA may induce dyskinesia by mechanisms different from dopamine agonist drugs.

Continuous delivery of ropinirole reverses motor deficits without dyskinesia induction in MPTP-treated common marmosets.

L-DOPA treatment of Parkinson's disease induces a high incidence of motor complications, notably dyskinesia. Longer acting dopamine agonists, e.g. ropinirole, are thought to produce more continuous dopaminergic stimulation and less severe dyskinesia. However, standard oral administration of dopamine agonists does not result in constant plasma drug levels, therefore, more continuous drug delivery may result in both prolonged reversal of motor deficits and reduced levels of dyskinesia. Therefore, we compared the effects of repeated oral administration of ropinirole to constant subcutaneous infusion in MPTP-treated common marmosets. Animals received oral administration (0.4 mg/kg, BID) or continuous infusion of ropinirole (0.8 mg/kg/day) via osmotic minipumps for 14 days (Phase I). The treatments were then switched and continued for a further 14 days (Phase II). In Phase I, locomotor activity was similar between treatment groups but reversal of motor disability was more pronounced in animals receiving continuous infusion. Dyskinesia intensity was low in both groups however there was a trend suggestive of less marked dyskinesia in those animals receiving continuous infusion. In Phase II, increased locomotor activity was maintained but animals switched from oral to continuous treatment showing an initial period of enhanced locomotor activity. The reversal of motor disability was maintained in both groups, however, motor disability tended towards greater improvement following continuous infusion. Importantly, dyskinesia remained low in both groups suggesting that constant delivery of ropinirole neither leads to priming nor expression of dyskinesia. These results suggest that a once-daily controlled-release formulation may provide improvements over existing benefits with standard oral ropinirole in Parkinson's disease patients.

MPTP treatment of common marmosets impairs proteasomal enzyme activity and decreases expression of structural and regulatory elements of the 26S proteasome.

Dysfunction of the ubiquitin-proteasome system occurs in the substantia nigra (SN) in Parkinson's disease (PD). However, it is unknown whether this is a primary cause or a secondary consequence of other components of the pathogenic process. We have investigated in nonhuman primates whether initiating cell death through mitochondrial complex I inhibition using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) altered proteasomal activity or the proteasomal components in the SN. Chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolase (PGPH) activating of 20S proteasome were decreased in SN homogenates of MPTP-treated marmosets compared to naïve animals. Western blotting revealed a marked decrease in the expression of 20S-alpha subunits, but no change in 20S-beta subunits in the SN of MPTP-treated marmoset compared to naïve animals. There was a marked decrease in the expression of the proteasome activator 700 (PA700) and proteasome activator 28 (PA28) regulatory complexes. The 20S-alpha4 subunit immunoreactivity was decreased in the nucleus of colocalized tyrosine hydroxylase (TH)-positive cells of MPTP-treated animals compared to naïve animals but no difference in the intensity of 20S-beta1i subunit staining. Immunoreactivity for PA700-Rpt5 and PA28-alpha subunits within surviving TH-positive cells of MPTP-treated marmoset was reduced compared to naïve controls. Overall, the changes in proteasomal function and structure occurring follow MPTP-induced destruction of the SN in common marmosets were very similar to those found in PD. This suggests that altered proteasomal function in PD could be a consequence of other pathogenic processes occurring in SN as opposed to initiating cell death as previously suggested.

3-Nitrotyrosine-dependent dopaminergic neurotoxicity following direct nigral administration of a peroxynitrite but not a nitric oxide donor.

The presence of 3-nitrotyrosine (3-NT) adducts in Lewy bodies in Parkinson's disease suggests a role for nitrative stress in dopaminergic cell death. Whether this is a direct effect of increased nitric oxide (NO) formation or requires its reaction with superoxide to form peroxynitrite is not clear. In the present study, we show that direct nigral administration of a NO donor, SNOG, in the rat produced only local toxicity to dopaminergic neurones pre-labeled with fluorogold with no 3-NT formation. However, administration of a peroxynitrite donor, SIN-1, caused widespread damage to dopaminergic neurones and marked expression of 3-NT immunoreactivity. Importantly, dopaminergic cell loss and the expression of 3-NT were completely prevented when SIN-1 was co-administered with the NO/peroxynitrite scavenger, carboxy-PTIO. The results suggest that increased NO formation is not inherently toxic to dopaminergic neurons, but when both oxidative and nitrative stress combine to cause peroxynitrite formation, neurotoxicity occurs.

Sonic hedgehog delivered by an adeno-associated virus protects dopaminergic neurones against 6-OHDA toxicity in the rat.

Direct intracerebral administration of sonic hedgehog (SHH) reduces 6-OHDA and MPTP toxicity to nigral dopaminergic cells in rats and primates. To determine whether transfection of the DNA sequence for SHH using viral vectors also protects against 6-OHDA toxicity, a type 2 adeno- associated virus (AAV) incorporating 600 base pairs of N-terminal SHH DNA was generated to induce SHH expression in rat striatum.AAV-SHH was injected into the striatum, 3 weeks prior to the initiation of an unilateral partial 6-OHDA nigro-striatal lesion. Animals receiving 4x10(7) viral particles of AAV-SHH showed a reduction in (+)-amphetamine induced ipsilateral turning over 4 weeks, when compared to animals receiving vehicle or a LacZ encoding vector. Following vehicle or AAV-LacZ administration, 6-OHDA caused a marked loss of striatal dopamine content and nigral tyrosine hydroxylase (TH) immunopositive cells. Following treatment with 4x10(7) viral particles of AAV-SHH the loss of striatal dopamine content was reduced and there was marked preservation of nigral dopaminergic cells. However, administration of 4x10(8) particles of AAV-SHH did not cause a significant change in (+)-amphetamine-induced rotation, striatal dopamine levels or the number of nigral TH immunoreactive cells following 6-OHDA lesioning compared to vehicle or AAV-LacZ treated animals. The results show that SHH delivered via a viral vector can protect dopaminergic neurons against 6-OHDA toxicity and suggest that this could be developed into a novel treatment for PD. However, the effects maybe dose limited due to uncoupling of hedgehog receptor signalling at higher levels of SHH expression.

Pramipexole protects against apoptotic cell death by non-dopaminergic mechanisms.

We have investigated the ability of pramipexole, a dopamine agonist used in the symptomatic treatment of Parkinson's disease (PD), to protect against cell death induced by 1-methyl-4-phenylpyridinium (MPP+) and rotenone in dopaminergic and non-dopaminergic cells. Pre-incubation with either the active (-)- or inactive (+)-enantiomer forms of pramipexole (10 microm) decreased cell death in response to MPP+ and rotenone in dopaminergic SHSY-5Y cells and in non-dopaminergic JK cells. The protective effect was not prevented by dopamine receptor blockade using sulpiride or clozapine. Protection occurred at concentrations at which pramipexole did not demonstrate antioxidant activity, as shown by the failure to maintain aconitase activity. However, pramipexole reduced caspase-3 activation, decreased the release of cytochrome c and prevented the fall in the mitochondrial membrane potential induced by MPP+ and rotenone. This suggests that pramipexole has anti-apoptotic actions. The results extend the evidence for the neuroprotective effects of pramipexole and indicate that this is not dependent on dopamine receptor occupation or antioxidant activity. Further evaluation is required to determine whether the neuroprotective action of pramipexole is translated to a disease-modifying effect in PD patients.

Behavioural and immunohistochemical changes following supranigral administration of sonic hedgehog in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated common marmosets.

Sonic hedgehog (SHH) has trophic actions on dopaminergic cell cultures and protects them from MPP(+) toxicity but its in vivo actions have not been explored. We now investigate the effects of unilateral supranigral administration of SHH on nigro-striatal function in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated common marmosets. SHH (0.1 or 1.0 microg) or vehicle was stereotaxically injected into the region of the right substantia nigra twice with an interval of 5 weeks between administrations. The first or second administration of low dose SHH (0.1 microg) did not significantly improve motor disability or locomotor activity compared to time-matched vehicle-treated animals. There was, however, an approximately 30% improvement in both motor disability and locomotor activity following the first administration of high dose SHH (1.0 microg). No further improvements occurred following the second high dose SHH treatment. Acute oral administration of L-3,4-dihydroxyphenylalanine (L-DOPA) produced a smaller increase in locomotor activity and greater reversal of motor disability in animals treated with SHH than occurred in vehicle-treated common marmosets. In the substantia nigra pars compacta, ipsilateral to SHH administration, the number of tyrosine hydroxylase-positive neurones was increased by 21% (P > 0.05) and 57% (P < 0.05) in low and high dose SHH groups respectively compared to the untreated contralateral hemisphere. There was no difference in the number of glial fibrillary acidic protein-positive cells. SHH may improve nigro-striatal function by restoring tyrosine hydroxylase positivity. This is reflected by an improvement in basal disability and a reduction in the lesion-induced response to L-DOPA.

Involvement of inducible nitric oxide synthase in inflammation-induced dopaminergic neurodegeneration.

The loss of dopaminergic neurones in the substantia nigra with Parkinson's disease may result from inflammation-induced proliferation of microglia and reactive macrophages expressing inducible nitric oxide synthase (iNOS). We have investigated the effects of the supranigral administration of lipopolysaccharide on iNOS-immunoreactivity, 3-nitrotyrosine formation and tyrosine hydroxylase-immunoreactive neuronal number, and retrogradely labelled fluorogold-positive neurones in the ventral mesencephalon in male Wistar rats. Following supranigral lipopolysaccharide injection, 16-18 h previously, there was intense expression of NADPH-diaphorase and iNOS-immunoreactivity in non-neuronal, macrophage-like cells. This was accompanied by intense expression of glial fibrillary acidic protein-immunoreactive astrocytosis in the substantia nigra. There were also significant reductions in the number of tyrosine hydroxylase(50-60%)- and fluorogold (65-75%)-positive neurones in the substantia nigra. In contrast, tyrosine hydroxylase-immunoreactivity in the ventral tegmental area was not altered. Pre-treatment of animals with the iNOS inhibitor, S-methylisothiourea (10 mg kg(-1), i.p.), led to a significant reduction of lipopolysaccharide-induced cell death. Similar reduction of tyrosine hydroxylase-immunoreactivity and fluorogold-labelled neurones in the substantia nigra following lipopolysaccharide administration suggests dopaminergic cell death rather than down-regulation of tyrosine hydroxylase. We conclude that the expression of iNOS- and 3-nitrotyrosine-immunoreactivity and reduction of cell death by S-methylisothiourea suggest the effects of lipopolysaccharide may be nitric oxide-mediated, although other actions of lipopolysaccharide (independent of iNOS induction) cannot be ruled out.

GDNF reverses priming for dyskinesia in MPTP-treated, L-DOPA-primed common marmosets.

Parkinson's disease (PD) is associated with a progressive loss of dopamine neurons in the substantia nigra and degeneration of dopaminergic terminals in the striatum. Although L-DOPA treatment provides the most effective symptomatic relief for PD it does not prevent the progression of the disease, and its long-term use is associated with the onset of dyskinesia. In rodent and primate studies, glial cell line-derived neurotrophic factor (GDNF) may prevent 6-OHDA- or MPTP-induced nigral degeneration and so may be beneficial in the treatment of PD. In this study, we investigate the effects of GDNF on the expression of dyskinesia in L-DOPA-primed MPTP-treated common marmosets, exhibiting dyskinesia. GDNF or saline was administered by two intraventricular injections, 4 weeks apart, to MPTP-treated, L-DOPA-treated common marmosets primed to exhibit dyskinesia. Prior to GDNF or saline administration, all animals displayed marked dyskinesia when treated with L-DOPA. GDNF administration produced a significant improvement in motor disability and, following the second injection of GDNF, a significant improvement in the locomotor activity was observed. Following the administration of L-DOPA there was a greater reversal of disability and a reduction in the intensity of L-DOPA-induced dyskinesia in GDNF-treated animals compared to saline-treated controls. However, there was no significant difference in L-DOPA's ability to increase locomotor activity between GDNF-treated and saline-treated animals. GDNF treatment caused a significant increase in the number of tyrosine hydroxylase-positive neurons in the substantia nigra, but no change in [(3)H]mazindol binding to dopamine terminals was found in the striatum of GDNF-treated animals compared to saline-treated controls. In GDNF-treated animals a small but significant reduction in enkephalin mRNA was observed in the caudate nucleus but not in the putamen or the nucleus accumbens. Substance P mRNA expression was equally reduced in the caudate nucleus and the putamen of the GDNF-treated animals but not in the nucleus accumbens. Intraventricular administration of GDNF improved MPTP-induced disability and reversed dopamine cell loss in the substantia nigra. GDNF also diminished L-DOPA-induced dyskinesia, which may relate to its ability to partly restore nigral dopaminergic transmission or to modify the activity of striatal output pathways.

Glial cell line-derived neurotrophic factor concentration dependently improves disability and motor activity in MPTP-treated common marmosets.

Glial cell line-derived neurotrophic factor (GDNF) has previously reduced motor deficits and preserved nigral dopamine neurones in rhesus monkeys with a unilateral MPTP-induced lesion of substantia nigra. We now report on the ability of GDNF to reverse motor deficits induced by parenteral administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to common marmosets resulting in bilateral degeneration of the nigrostriatal pathway. Prior to GDNF administration, all MPTP-treated animals showed akinesia or bradykinesia, rigidity, postural instability and tremor. Intraventricular injection of GDNF (10, 100 or 500 microg) at 9 and 13 weeks post MPTP treatment resulted in a concentration dependent improvement in locomotor activity and motor disability which became significant after administration of 100 and 500 microg of GDNF. The most prominent improvements were in alertness, checking movements, and posture. It is concluded that intraventricular GDNF administration improves bilateral Parkinsonian motor disability following MPTP treatment and this may reflect an action of GDNF on remaining nigral dopaminergic neurones.

Direct effects of 3,4-methylenedioxymethamphetamine (MDMA) on serotonin or dopamine release and uptake in the caudate putamen, nucleus accumbens, substantia nigra pars reticulata, and the dorsal raphé nucleus slices.

We examined the effects of pressure ejected 3, 4-methylenedioxymethamphetamine (MDMA) from a micropipette on direct chemically stimulated release, and on electrically stimulated serotonin (5-HT) or dopamine (DA) release in the caudate putamen (CPu), nucleus accumbens (NAc), substantia nigra pars reticulata (SNr), and the dorsal raphé nucleus (DRN) brain slices of rat, using fast cyclic voltammetry (FCV). MDMA is electroactive, oxidising at +1100 mV. When the anodic input waveform was reduced from +1.4 to +1.0 volt, MDMA was not electroactive. Using this waveform, pressure ejection of MDMA did not release 5-HT or DA in brain slices prepared from any of the nuclei studied. MDMA significantly potentiated electrically stimulated 5-HT release in the SNr and DA release in CPu. In the DRN or in the NAc, MDMA was without effect on peak electrically stimulated 5-HT or DA release. The rates of neurotransmitter uptake, expressed as t(1/2), were in all cases significantly decreased after MDMA. The results indicate that MDMA, unlike (+)amphetamine, is not as a releaser of DA or 5-HT, it is a potent inhibitor of both DA and 5-HT uptake.

MK-801 interaction with the 5-HT transporter: a real-time study in brain slices using fast cyclic voltammetry.

The effects of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine ((+)-MK-801) and a competitive NMDA antagonist, (+/-)-3-2-carboxypiperazin-4-yl-propyl-1-phosphonic acid (CPP) were compared in electrically evoked 5-HT release in the brain slices incorporating the substantia nigra pars reticulata (SNr) or the dorsal raphé nucleus (DRN) using fast cyclic voltammetry (FCV). Electrical stimulation of either the SNr or the DRN with 50 pulses at frequencies greater than 10 Hz generated signals that were indistinguishable from 5-HT. In the SNr, 0.6-60 microM MK-801 concentration dependently potentiated stimulated 5-HT release. CPP 20 microM or NMDA 100 microM had no effect on 5-HT release evoked by electrical stimulation. In the SNr, 1 microM fluvoxamine or 0.6-60 microM MK-801 potentiated electrically evoked release of 5-HT. Pre-exposure to 20 microM MK-801 inhibited the enhancing effects of 1 microM fluvoxamine on electrically evoked 5-HT release in the SNr. In the DRN, the presence of 1 microM fluvoxamine or 20 microM MK-801 weakly potentiated 5-HT release. In the presence of 1 microM methiothepin (a nonselective 5-HT1-2 antagonist), 1 microM fluvoxamine or 20 microM MK-801 were equipotent in potentiating the concentration of 5-HT released in response to electrical stimulation. The T1/2 values for 5-HT release following MK-801 or fluvoxamine administration were significantly increased. Potentiation of 5-HT release by MK-801 in the SNr and the DRN and lack of effect of either CPP or NMDA on 5-HT release or uptake argues against a role for NMDA receptors in modulation of 5-HT release. Inhibition of fluvoxamine induced potentiation of 5-HT signal in the presence of MK-801 suggests that MK-801 and fluvoxamine may interact at the level of the 5-HT transporter.

Differential release of dopamine by nitric oxide in subregions of rat caudate putamen slices.

Fast cyclic voltammetry was used to measure NO and dopamine (DA) simultaneously in rat caudate putamen (CPu) slices. Analysis of electrochemical signals obtained from mixtures of DA and NO showed that subtraction of either the DA or the NO component revealed the contribution of the other component. Application of such data manipulation to signals obtained in CPu slices indicated that DA and NO components contributed to electrochemical signals. Following electrical stimulation (>1 s), site-dependent NO-like signals were observed. Pressure ejection of NMDA yielded a biphasic electrochemical signal. The first phase (lasting 10-20 s) had electrochemical characteristics of DA and was observed only during the first application of NMDA. The second phase developed more slowly, was of longer duration (1-3 min), and had electrochemical characteristics of a mixture of DA and NO. Generation of the NO-like signal by NMDA was antagonised by L-N-monomethylarginine. Pressure ejection of NO into CPu slices caused dose- and site-dependent DA release. More DA was released in the dorsolateral than in the dorsomedial CPu. Pressure ejection of DA did not generate NO. It is concluded that electrically stimulated DA release is not mediated by prior release of NO.

Real-time measurement of stimulated 5-hydroxytryptamine release in rat substantia nigra pars reticulata brain slices.

Fast cyclic voltammetry at a carbon fibre microelectrode was used to measure 5-HT signals following electrical or chemical stimulation in rat substantia nigra pars reticulata slices. Chemical stimulation with (+)-amphetamine or veratrine gave signals which were indistinguishable from those of exogenous 5-HT. Electrical stimulation of sufficient duration gave voltammetric signals which were characteristic of 5-HT. Release of dopamine was not detected following either chemical or electrical stimulation. The 5-HT signals were attenuated by TTX and enhanced by fluvoxamine. It was not possible to demonstrate regulation of 5-HT release in the SNr by 5-HT1B autoreceptors using CGS 12066A or methiothepin. Signal following electrical stimulation were not enhanced by either benztropine or GBR12909, or modified in the presence of either quinpirole or sulpiride. We conclude that 5-HT release can be detected voltammetrically in the SNr; 5-HT release is likely to be from axon terminals, but somatodendritic DA release could not be detected.

The presence and the effects of neuropeptide Y in rat anococcygeus muscle.

Isolated anococcygeus muscle from male rats was examined for the presence of neuropeptide Y-immunoreactive nerves and for the effects of neuropeptide Y on its tone and its contractile/relaxant responses to electrical field stimulation, acetylcholine, guanethidine and noradrenaline. Using peroxidase anti-peroxidase immunohistochemistry in stretch preparation of the anococcygeus, neuropeptide Y-immunoreactive nerve fibres were observed, in abundance, running along both vascular as well as non-vascular smooth muscle cells. Neuropeptide Y (> 250 nM) evoked phentolamine and tetrodotoxin-resistant contractile response. Neuropeptide Y, even in subspasmogenic concentrations, potentiated contractions evoked by acetylcholine, guanethidine and noradrenaline. Electrical field stimulation (trains of 3-4 pulses, 0.1 ms, 10 Hz) of the isolated anococcygeus preparation produced robust, phentolamine and tetrodotoxin sensitive contractions. Neuropeptide Y (< 10 nM) exerted a biphasic effect on the electrical field stimulation-evoked contractions; an early potentiation was followed by a delayed and progressive inhibition. Neuropeptide Y (> 10 nM) caused a concentration-dependent potentiation of electrical field stimulation-evoked contraction alone, matching its potentiation of noradrenaline-evoked contraction. Electrical field stimulation (5 pulses, 0.1 ms, 10 Hz) of guanethidine (50 microM)-contracted anococcygeus induced a relaxant response and neuropeptide Y (1-100 nM) exerted a concentration-related slight and variable effect on the electrical field stimulation-evoked relaxant response (1 nM, augmentation; 10 nM, no effect; 100 nM, reduction). It is concluded that rat anococcygeus muscle has a rich neuropeptide Y-containing innervation and neuropeptide Y is mostly stored within adrenergic nerves. The main functional roles of neuropeptide Y in the anococcygeus muscle are likely to be post-junctionally mediated facilitation and prejunctionally mediated inhibition of adrenergic motor transmission.

The role of ventral tegmental dopamine neurons in locomotor sensitization following quinpirole or (+)-amphetamine: ex vivo voltammetric evidence.

Behavioural sensitization to the locomotor stimulating effects of (+)-amphetamine or quinpirole was induced in rats by intermittent drug administration. Following expression of sensitization, locomotor activity scores on day 9 were: vehicle 87 +/- 9, (+)-amphetamine 1441 +/- 227 and quinpirole 2078 +/- 214. Electrically stimulated dopamine release was measured on day 12 in ventral tegmental slices using fast cyclic voltammetry. Dopamine release was significantly elevated in the (+)-amphetamine- and quinpirole-treated groups when compared to vehicle-treated controls over a wide range of stimulation frequencies (5-200 Hz) and pulses (1-200). Quinpirole (1 microM) in the perfusion fluid attenuated dopamine release following 40-pulse, 200-Hz electrical stimulation, by 31.6 +/- 2.8% in the ventral tegmental area of the vehicle-treated group, by 14.8 +/- 5.6% in the (+)-amphetamine-treated group and 8 +/- 7.3% in the quinpirole-treated group. This study shows that dopamine release is increased in the ventral tegmental area following sensitization with either a direct or indirectly acting dopamine agonist. The findings that dopamine release was elevated at all stimulation frequencies in sensitized animals, and that quinpirole only attenuated this release at the highest stimulation frequency, would suggest that in addition to D2 autoreceptor subsensitivity, other mechanisms contribute to the enhanced release of dopamine in these animals.

Real-time effects of N-methyl-D-aspartic acid on dopamine release in slices of rat caudate putamen: a study using fast cyclic voltammetry.

The functional role of N-methyl-D-aspartic acid (NMDA) glutamate receptors in the real-time regulation of single electrical pulse (1 p)-stimulated endogenous dopamine release was investigated in slices of rat caudate putamen using fast cyclic voltammetry at a carbon fibre electrode. In the presence of Mg2+, 20 microM NMDA had a weak effect on background signals but did not affect 1 p-stimulated dopamine release. Removal of Mg2+ increased the background and doubled 1 p-stimulated dopamine release. In the absence of Mg2+, 20 microM NMDA caused a transient "release" of dopamine and decreased the background signal. The 1 p-stimulated dopamine release was subsequently reduced. In the presence of 1 microM (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), superfusion with 20 microM NMDA did not cause a transient "release" of dopamine, and 1 p-stimulated dopamine release was not subsequently attenuated. In the presence of 1 microM tetrodotoxin, 1 p-stimulated dopamine release was abolished, but 20 microM NMDA still caused a transient "release" of dopamine. Removal of Ca2+ from the artificial CSF abolished 1 p-stimulated dopamine release and resulted in a decline in the baseline but did not affect dopamine "release" when 20 microM NMDA was added. The dopamine release-inducing effect of 20 microM NMDA was less pronounced in sites in the caudate putamen where dopamine release increased with frequency of electrical stimulation (hot spots) than in sites where there was little frequency-dependent dopamine release (cold spots). Subsequent 1 p-stimulated dopamine release was less attenuated in cold spots than in hot spots. We conclude that in the absence of Mg2+, NMDA induces release of dopamine by acting at CPP-sensitive NMDA receptors in a Ca(2+)-independent manner. This transient release depletes dopamine from a storage site from which dopamine is released by 1 p electrical stimulation. These real-time observations of the effects of NMDA on electrical stimulus-independent and -dependent dopamine release may explain the apparently conflicting observations of the effects of NMDA on dopamine release made in previous studies. They also indicate that dopamine release and storage are heterogeneous at different sites in the rat caudate putamen.

Comparison of somatodendritic and axon terminal dopamine release in the ventral tegmental area and the nucleus accumbens.

Fast cyclic voltammetry at a carbon fibre microelectrode was used to measure dopamine release following electrical or chemical stimulation in rat brain slices incorporating either the ventral tegmental area or the core region of the nucleus accumbens. Electrical or chemical stimulation gave clear voltammetric signals which corresponded to dopamine; less dopamine was released in the ventral tegmental area than in the nucleus accumbens. In contrast to the nucleus accumbens, electrically stimulated dopamine release in the ventral tegmental area was not sensitive to tetrodotoxin, was not modified by the presence of dopamine uptake inhibitors, or agonist or blockers acting at dopamine D2 autoreceptors.