Guinea-pig Substantia Nigra Research Articles

NK3 receptors mediate an increase in firing rate of midbrain dopamine neurons of the rat and the guinea pig

This in vitro study investigates and compares the effects of NK3 receptor ligands on the firing rate of rat and guinea pig midbrain dopamine neurons. The findings are discussed in the light of choosing suitable animal models for investigating pharmacological properties of NK3 receptor antagonists, which have been proposed to possess therapeutic activity in neuropsychiatric diseases like e.g. schizophrenia. In vitro midbrain slice preparations of both species were used to record (extracellularly) the firing rates of dopamine neurons located in the substantia nigra (SN) and ventral tegmental area (VTA). Furthermore, the effect of the D2 receptor agonist quinpirole on guinea pig SN and VTA dopamine neurons was investigated. The efficacy of quinpirole in inhibiting guinea pig dopamine neuron firing activity was much less as compared to that of rat dopamine neurons, suggesting a lower dopamine D2 autoreceptor density on the guinea pig neurons. The NK3 receptor agonist senktide induced in subpopulations of rat SN (55%) and VTA (79%) and guinea pig SN (50%) and VTA (21%) dopamine neurons an increase in firing rate. In responsive neurons this effect was concentration-dependent with EC₅₀ values of 3-5 nM (for both species). The selective NK3 receptor antagonist osanetant (100 nM) was able to partly block the senktide-induced increase in firing rates of dopamine neurons and shifted the concentration-response relation curves for senktide to the right (pA₂ values were ~7.5). The fractional block of the senktide responses by osanetant appeared to be larger in guinea pig dopamine neurons, indicating that osanetant is a more potent blocker of NK3 receptor-mediated responses with noncompetitive properties in the guinea pig.

Characterization of RO4583298 as a novel potent, dual antagonist with in vivo activity at tachykinin NK1 and NK3 receptors

Clinical results of osanetant and talnetant (selective-NK₃ antagonists) indicate that blocking the NK₃ receptor could be beneficial for the treatment of schizophrenia. The objective of this study was to characterize the in vitro and in vivo properties of a novel dual NK₁/NK₃ antagonist, RO4583298 (2-phenyl-N-(pyridin-3-yl)-N-methylisobutyramide derivative). RO4583298 in vitro pharmacology was investigated using radioligand binding ([³H]-SP, [³H]-osanetant, [³H]-senktide), [³H]-inositol-phosphate accumulation Schild analysis (SP- or [MePhe⁷]-NKB-induced) and electrophysiological studies in guinea-pig substantia nigra pars compacta (SNpc). The in vivo activity of RO4583298 was assessed using reversal of GR73632-induced foot tapping in gerbils (GFT; NK₁) and senktide-induced tail whips in mice (MTW; NK₃). RO4583298 has a high-affinity for NK₁ (human and gerbil) and NK₃ (human, cynomolgus monkey, gerbil and guinea-pig) receptors and behaves as a pseudo-irreversible antagonist. Unusually it binds with high-affinity to mouse and rat NK₃, yet with a partial non-competitive mode of antagonism. In guinea-pig SNpc, RO4583298 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurones with an apparent non-competitive mechanism of action. RO4583298 (p.o.) robustly blocked the GFT response, and inhibited the MTW. RO4583298 is a high-affinity, non-competitive, long-acting in vivo NK₁/NK₃ antagonist; hence providing a useful in vitro and in vivo pharmacological tool to investigate the roles of NK₁ and NK₃ receptors in psychiatric disorders.

Biochemical and pharmacological activities of SSR 146977, a new potent nonpeptide tachykinin NK3 receptor antagonist.

SSR 146977 is a potent and selective antagonist of the tachykinin NK3 receptor. In Chinese hamster ovary cells expressing the human tachykinin NK3 receptor, SSR 146977 inhibited the binding of radioactive neurokinin B to NK3 receptors (Ki = 0.26 nM), senktide (10 nM) induced inositol monophosphate formation (IC50 = 7.8-13 nM), and intracellular calcium mobilization (IC50 = 10 nM). It antagonized [MePhe7]neurokinin B induced contractions of guinea pig ileum (pA2 = 9.07). Senktide (30 nM) induced firing rate increase of noradrenergic neurons in the guinea pig locus coeruleus and dopaminergic neurons in the guinea pig substantia nigra was also blocked by SSR 146977 (50 and 100 nM, respectively). In vivo, in the respiratory system, SSR 146977 inhibited bronchial hyperresponsiveness to acetylcholine, bronchial microvascular permeability hypersensitivity to histamine (doses of 0.1-1 mg/kg i.p.), and cough (doses of 0.03-1 mg/kg i.p.) provoked by citric acid in guinea pigs. In the central nervous system, SSR 146977 inhibited turning behaviour (ID50 = 0.2 mg/kg i.p. and 0.4 mg/kg p.o.) and prevented the decrease of locomotor activity (10 and 30 mg/kg i.p) mediated by the stimulation of NK3 receptors in gerbils. In guinea pigs, SSR 146977 antagonized senktide-induced acetylcholine release in the hippocampus (0.3 and 1 mg/kg i.p) and norepinephrine release in the prefrontal cortex (0.3 mg/kg i.p.). It also prevented haloperidol-induced increase of the number of spontaneously active dopamine A10 neurons (1 and 3 mg/kg i.p.).

Blockade of neurokinin3 receptors antagonizes drug‐induced population response and depolarization block of midbrain dopamine neurons in guinea pigs

In vivo extracellular recording techniques were used to investigate the effects of neurokinin3 (NK3) receptor blockade on the pharmacological activation of midbrain dopamine (DA) neurons in the guinea pig substantia nigra (A9) and ventral tegmental area (A10). The number of spontaneously active DA cells (population response) was largely increased in A10 and A9 by acute administration of haloperidol (1 and 0.5 mg/kg i.p., respectively) and this effect was dose-dependently prevented in both areas by the selective NK3 receptor antagonist SR142801 (0.3, 1, 3, and 1, 3, 10 mg/kg i.p., respectively). This compound, which was totally inactive by itself, also antagonized the increase of population response induced in A10 cells by the neurotensin receptor antagonist SR142948 (1 mg/kg i.p.) and in A9 cells by the NK2 receptor antagonist SR144190 (1 mg/kg i.p.). None of the effects of SR142801 were reproduced by SR142806, its (R)-enantiomer with 240-fold lower affinity for NK3 receptors. In addition, neither SR144190 (0.3 mg/kg i.p.) nor the NK1 receptor antagonist GR205171 (1 mg/kg i.p.) affected the haloperidol-induced response. The antagonistic effects of SR142801 (3 mg/kg i.p.) were also observed on the depolarization block-related decrease of A10 cell population response evoked by repeated administration (22 days) of haloperidol. Finally, SR142801 (3 mg/kg i.p.) prevented depolarization block induced in A10 cells by acute co-administration of SR142948 and haloperidol, both on population response and on single cell firing. These results on pharmacologically induced activation and depolarization block of dopamine neurons suggest that NK3 receptors play a key role in the midbrain DA function, presumably through activation by neurokinin B. Synapse 33:71–79, 1999. © 1999 Wiley-Liss, Inc.

Blockade of neurokinin3 receptors antagonizes drug-induced population response and depolarization block of midbrain dopamine neurons in guinea pigs.

In vivo extracellular recording techniques were used to investigate the effects of neurokinin3 (NK3) receptor blockade on the pharmacological activation of midbrain dopamine (DA) neurons in the guinea pig substantia nigra (A9) and ventral tegmental area (A10). The number of spontaneously active DA cells (population response) was largely increased in A10 and A9 by acute administration of haloperidol (1 and 0.5 mg/kg i.p., respectively) and this effect was dose-dependently prevented in both areas by the selective NK3 receptor antagonist SR142801 (0.3, 1, 3, and 1, 3, 10 mg/kg i.p., respectively). This compound, which was totally inactive by itself, also antagonized the increase of population response induced in A10 cells by the neurotensin receptor antagonist SR142948 (1 mg/kg i.p.) and in A9 cells by the NK2 receptor antagonist SR144190 (1 mg/kg i.p.). None of the effects of SR142801 were reproduced by SR142806, its (R)-enantiomer with 240-fold lower affinity for NK3 receptors. In addition, neither SR144190 (0.3 mg/kg i.p.) nor the NK1 receptor antagonist GR205171 (1 mg/kg i.p.) affected the haloperidol-induced response. The antagonistic effects of SR142801 (3 mg/kg i.p.) were also observed on the depolarization block-related decrease of A10 cell population response evoked by repeated administration (22 days) of haloperidol. Finally, SR142801 (3 mg/kg i.p.) prevented depolarization block induced in A10 cells by acute co-administration of SR142948 and haloperidol, both on population response and on single cell firing. These results on pharmacologically induced activation and depolarization block of dopamine neurons suggest that NK3 receptors play a key role in the midbrain DA function, presumably through activation by neurokinin B.

Magnitude of 5-HT 1B and 5-HT 1A receptor activation in guinea-pig and rat brain: evidence from sumatriptan dimer-mediated [ [formula omitted]]GTPγS binding responses

The present study reports on G-protein activation by recombinant 5-HT receptors and by native 5-HT 1A and 5-HT 1B receptors in guinea-pig and rat brain using agonist-stimulated [ 35 S ]GTPγS binding responses mediated by a new 5-HT ligand, a dimer of sumatriptan. Dimerization of sumatriptan increased the binding affinity for h 5-HT 1B (p K i: 9.22 vs. 7.79 for sumatriptan), h 5-HT 1D (9.07 vs. 8.08) and also h 5-HT 1A receptors (7.80 vs. 6.40), while the binding affinity for h 5-ht 1E (6.67 vs. 6.19) and h 5-ht 1F (7.37 vs. 7.78) receptors was not affected. Sumatriptan dimer (10 μM) stimulated [ 35 S ]GTPγS binding mainly in the superficial gray layer of the superior colliculi, hippocampus and substantia nigra of guinea-pig and rat coronal brain sections. This fits with the labelling by the 5-HT 1B/1D receptor antagonist [ 3 H ] GR 125743. The observed [ 35 S ]GTPγS binding responses in the substantia nigra are likely to be mediated by stimulation of the 5-HT 1B receptor subtype, since they were antagonized by the 5-HT 1B inverse agonist SB 224289 (10 μM), and not by the 5-HT 2A/1D antagonist ketanserin (10 μM). Quantitative assessment of the [ 35 S ]GTPγS binding responses in the substantia nigra of rat showed highly efficacious responses for both sumatriptan dimer and its monomer. In contrast, less efficacious agonist responses (51±10% and 35±13%, respectively) were measured in the guinea-pig substantia nigra. This may suggest that the G-protein coupling efficacy of 5-HT 1B receptors is different between the substantia nigra of both species. In addition, the sumatriptan dimer also activated guinea-pig and rat hippocampal 5-HT 1A receptors with high efficacy in contrast to sumatriptan. Therefore, dimerization of sumatriptan can be considered as a new approach to transform a partial 5-HT 1A agonist into a more efficacious agonist. In conclusion, the sumatriptan dimer stimulates G-protein activation via 5-HT 1B receptors besides 5-HT 1A receptors in guinea-pig and rat brain. The magnitude of the 5-HT 1B receptor responses is superior for sumatriptan and its dimer in rat compared to guinea-pig substantia nigra.

The Pharmacology of Physostigmine

The Pharmacology of Physostigmine

Association between the low threshold calcium spike and activation of NMDA receptors in guinea-pig substantia nigra pars compacta neurons.

The aim of this study was to examine the interaction between N-methyl-D-aspartate (NMDA) receptor activation and the low threshold calcium spike (LTS) of phasically firing neurons in the rostral part of the substantia nigra pars compacta (SNpc) in mid-brain slices. Bath perfusion of 10 microM NMDA gradually increased the LTS area and the effect reached a maximum after 6 min of perfusion. This enhancement of the LTS by NMDA was blocked both by a competitive and non-competitive NMDA receptor antagonist, 50 microM D-AP5 and 10 microM MK801, respectively, demonstrating that this effect of NMDA was mediated through NMDA receptors. Prolonged exposure to increasing concentrations of NMDA (0.1-100 microM) progressively decreased the LTS area. The higher doses led to an irreversible marked depolarization and decrease of the membrane resistance. These results suggest that the LTS of SNpc neurons can trigger a NMDA receptor-dependent response which may have physiological and pathological roles.

5-Hydroxytryptamine1A and 5-hydroxytryptamine1B receptors stimulate [35S]guanosine-5'-O-(3-thio)triphosphate binding to rodent brain sections as visualized by in vitro autoradiography.

[35S]Guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding to G proteins was measured by in vitro autoradiography in guinea pig and rat brain sections after activation by 5-hydroxytryptamine (5-HT) receptor agonists. 5-Carboxamidotryptamine stimulated binding strongly in hippocampus and lateral septum and weakly in substantia nigra. This effect was blocked in the substantia nigra by the 5-HT1B/1D receptor antagonist GR-127,935 and in the former two regions by the 5-HT1A antagonist NAN-190. 5-HT1B/1D receptor agonists stimulated binding in substantia nigra and in areas containing 5-HT1A receptors. In guinea pig substantia nigra, 5-(nonyloxy)-tryptamine maximally stimulated [35S]GTPgammaS binding by 54%, with an EC50 value of 62 nM; at 100 microM, this agonist increased binding by approximately 200% in hippocampus (with a 2-fold weaker EC50 value). The distribution of [3H]8-OH-DPAT binding sites was identical to that of the [35S]GTPgammaS labeling stimulated by the 5-HT1A agonist (R)-8-hydroxy-2-dipropylaminotetralin [(R)-8-OH-DPAT)]. (R)-8-OH-DPAT, (S)-8-OH-DPAT, and buspirone stimulated [35S]GTPgammaS binding in hippocampus by 340%, 140%, and 78%, with EC50 values of 71, 51, and 132 nM. Enhanced [35S]GTPgammaS binding was not detected in the presence of 5-HT1F, 5-HT2, 5-HT4, and 5-HT7 receptor agonists. Because activation of mu-opioid, muscarinic M2, histamine H3, and cannabinoid receptors was also visualized successfully, these data suggest that only receptors coupled to pertussis toxin-sensitive G proteins can be seen by [35S]GTPgammaS binding autoradiography. This study also shows that different 5-HT receptors coupled to these proteins can show a wide range of [35S]GTPgammaS binding stimulation. Although the functional significance of these variations is unclear, this technique offers advantages over receptor autoradiography because it does not require high affinity radioligands and provides a measure of agonist efficacies in various brain regions.

Effects of drugs interfering with sodium channels and calcium channels on the release of endogenous dopamine from superfused substantia nigra slices.

The importance of voltage-dependent sodium channels and different types of voltage-sensitive calcium channels for depolarisation-induced release of endogenous dopamine from dendrites and cell bodies in superfused guinea pig substantia nigra slices was investigated. The stimulatory effect of veratridine (10 microM) on dopamine release was only marginally attenuated in Ca(2+)-free medium but was completely blocked by tetrodotoxin (1 microM) and by the dopamine reuptake inhibitor GBR 12909 (10 microM). Low extracellular concentration of Na+ stimulated the dopamine release. Potassium-evoked dopamine release was completely Ca(2+)-dependent, not blocked by GBR 12909 and partially blocked by tetrodotoxin. Nifedipine (20 microM), omega-conotoxin GVIA (0.5 microM), penfluridol (5 microM), and Ni2+ (20 microM) had no effect, amiloride (1 mM) attenuated and neomycin (350 microM), and omega-agatoxin IVA (1 microM) almost totally blocked the potassium-induced dopamine release. The results suggest that veratridine released dopamine mostly by reversing the dopamine transporter. High concentrations of potassium induced release of nigral dopamine by opening of voltage-sensitive calcium channels of P/Q type but not L-type, N-type and probably not T-type. The depolarisation evoked by high concentrations of potassium seems to open voltage-sensitive calcium channels both by the depolarisation induced by potassium per se and by the secondary depolarisation induced by opening of voltage-dependent sodium channels.

Tachykinin neurokinin-1 and neurokinin-3 receptor-mediated responses in guinea-pig substantia nigra: an in vitro electrophysiological study

The effects of tachykinin receptor agonists and antagonists were investigated using intra- and extracellular recordings on spontaneously firing nigral neurons in guinea-pig brain slices. In 70 of 76 electrophysiologically identified dopaminergic neurons, a concentration-dependent increase in firing rate was induced by the selective neurokinin-3 tachykinin agonist senktide and by the natural tachykinin agonists neurokinin B and substance P, with ec50 values of 4.7, 31.2 and 12 200nM respectively. These responses were inhibited in a concentration- and time-dependent manner by the selective non-peptide neurokinin-3 receptor antagonist SR 142801 (1–100nM; n=23), but neither by its S-enantiomer SR 142806 (100nM; n=4) nor by selective antagonists of neurokinin-1 (SR 140333) or neurokinin-2 (SR 48698) receptors (both at 100nM; n=3). The selective neurokinin-1 agonist [Sar9,Met(O2)11]substance P (30–100nM; n=23) and the selective neurokinin-2 agonist [Nle10]neurokinin A(4–10) (30–100nM; n=13) were without any effect on dopaminergic cells. In 13 of 21 electrophysiologically identified, presumably GABAergic neurons located in the pars compacta of the substantia nigra, excitatory responses were evoked concentration dependently by substance P and [Sar9,Met(O2)11]substance P, with ec50 values of 18.6 and 41.9nM respectively. These responses were inhibited by SR 140333 (100nM; n=3), but neither by its R-enantiomer SR 140603 nor by SR 142801 (both at 100nM; n=3). Senktide and [Nle10]neurokinin A(4–10) (both at 30–100nM; n=10) were without effect on these presumed GABAergic neurons. A small population (12%) of pars compacta neurons was insensitive to any of the three selective tachykinin agonists. In the nigral pars reticulata, 12 neurons were recorded which had an electrophysiological profile similar to that of presumed GABAergic neurons in the pars compacta. Of these 12 cells, seven did not respond to any of the selective tachykinin agonists tested, while five were excited by senktide in a concentration-dependent manner (ec50=98.5nM). Although this value was significantly higher than that found for dopaminergic neurons in the pars compacta, senktide-evoked responses were inhibited by SR 142801 (100nM; n=3).We conclude that, in the guinea-pig substantia nigra, tachykinins evoke excitatory responses in both dopaminergic and non-dopaminergic neurons; however, the sensitivity to tachykinin agonists (neurokinin-1 versus neurokinin-3) depends on both neuronal type and localization.

6-Hydroxy dopamine lesions block a tolbutamide-sensitive K+ conductance in the guinea-pig substantia nigra

A population of neurons in the anterior substantia nigra pars compacta of the guinea-pig display an outward K + conductance, which can be activated by a brief period of hypoxia, and blocked in a dose-dependent manner by tolbutamide (100–500 μm): these features are those of an ATP-sensitive K + channel (K ATP). Typically hyperpolarisation of the cell is accompanied by a mean decrease in input resistance (IR) of 41.29±3.69 M Ω (SEM), a mean decrease in resting membrane potential (RMP) of 8.58±2.41 mV and a cessation of action potential generation. These cells do not show positive immunoreactivity for tyrosine hydroxylase (TH), but they are present in slices that contain TH and release dopamine (DA). Given the intimacy of these cells with nigral DA systems, and given the fact that they have D 2 receptors and respond to dopaminergic (DAergic) agents, it was of interest to see if lesions of DAergic neurons had any effect on the above response. The neurotoxin 6-hydroxy dopamine (6-OHDA) was injected into the median forebrain bundle to destroy the DAergic neurons of the nigrostriatal pathway. Unilateral lesion of this pathway produced a significant reduction in the above conductance, in the lesioned substantia nigra pars compacta (SNpc) (mean IR decrease of only 17.99±4.44 M Ω (and a mean RMP decrease of 1.40±1.76 mV), whilst the hypoxia induced change in potential on the non-lesioned side was still marked and indistinguishable from that in control animals. The K ATP channel would therefore appear to be under the influence of the DAergic neurons of the SNpc.

THE SPONTANEOUS RELEASE OF ACETYLCHOLINESTERASE IN RAT SUBSTANTIA NIGRA IS ALTERED BY LOCAL CHANGES IN EXTRACELLULAR LEVELS OF DOPAMINE

Acetylcholinesterase release in the guinea-pig substantia nigra has been previously investigated ‘on-line’, using a sensitive chemiluminescent system. Since histological observations suggest that there is a difference in acetylcholinesterase distribution in the rat substantia nigra compared to that of the guinea-pig, the first aim of the present study was to use this chemiluminescent method to characterise acetylcholinesterase release in this brain region of the freely moving rat, and the second was explore the relationship between acetylcholinesterase release and dopamine systems in this region. Accordingly, acetylcholinesterase release in the rat substantia nigra was studied under basal conditions of spontaneous release and following the local administration of (a) elevated potassium ions (30, 45, 60 mM), (b) a stimulator of dopamine/acetylcholinesterase release—D-amphetamine (10−7, 10−6 and 10−5 M), (c) an inhibitor of dopamine uptake—GBR12909 (10−7, 10−6 and 10−5 M). Spontaneous release of acetylcholinesterase in this brain region of the rat appears to be comparable with that observed in the guinea-pig, despite the smaller number of acetylcholinesterase-containing neurones. Furthermore, not only elevated potassium ions, but d-amphetamine as well as GBR12909, all produced significant increases in the percentage spontaneous release of acetylcholinesterase. Thus, the release of acetylcholinesterase in this region may be triggered by levels of dopamine outside of the neurone. Copyright © 1996 Elsevier Science Ltd

Blockade of dopamine storage, but not of dopamine synthesis, prevents activation of a tolbutamide-sensitive K+ channel in the guinea-pig substantia nigra.

The substantia nigra has one of the highest levels of ATP-sensitive K+ channel in the brain. Since this channel is controlled by cell metabolism, the aim of this study was to see how closely it is associated with nigral dopamine systems, which are decreased in Parkinson's disease. In a sub-population of neurons within the rostral substantia nigra pars compacta of the guinea-pig, a brief period of hypoxia resulted in a tolbutamide (100-500 microM) sensitive hyperpolarisation [input resistance (IR) decrease from 144.88 +/- 14.04 M omega pre-hypoxia to 105.91 +/- 13.25 M omega during hypoxia]. Maximal blockade of this decrease was seen in presence of 500 microns tolbutamide [IR decrease only from 161.35 +/- 32.82 M omega to 155.02 +/- 34.29 M omega]. Reserpine (which depletes dopamine stores) but not alpha-methyl-para-tyrosine (which decreases de novo synthesis of dopamine) caused a marked attenuation of this hyperpolarisation [IR decrease only from 163.32 +/- 44.42 M omega pre-hypoxia to 154.42 +/- 50.97 M omega during hypoxia]. This observation suggests that blockade of dopamine storage, but not of de novo synthesis, leads to a loss of responsiveness of certain mid-brain neurons to hypoxia, rendering them potentially more susceptible to subsequent degeneration. The possible link between nigral dopamine systems and ATP-sensitive K+ channels is discussed.

Differential Release of Acetylcholinesterase in vivo, from the Guinea Pig Substantia Nigra Compared to the Caudate Putamen Following Dopamine Depletion

In the substantia nigra acetylcholinesterase may have a novel role unrelated to acetylcholine but linked instead to dopamine. Using a sensitive chemiluminescent system, we have investigated the effects of dopamine depletion on the in vivo release of acetylcholinesterase in both the substantia nigra and the caudate putamen. Dopamine levels in the caudate putamen were significantly depleted compared to the non-lesioned side, using either of two different toxins for dopaminergic nigrostriatal cells: 6-hydroxydopamine (1 or 3 weeks prior to study) or N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (1 week prior to study). Spontaneous release of acetylcholinesterase from the substantia nigra was significantly reduced following all three pretreatments; however, in the caudate putamen a significant reduction in the spontaneous release of acetylcholinesterase, compared to controls, was only seen in animals studied 1 week after the administration of 6-hydroxydopamine. In all control groups, application of potassium ions (60 mM) evoked a significant release of acetylcholinesterase in the substantia nigra ( p < 0.05) and this effect persisted in the surviving neurones following a partial lesion by neurotoxin pre-treatment. The results from this study are discussed in the light of a regulatory mechanism for acetylcholinesterase release from the striatum, which may come into operation depending on the extent of destruction of dopaminergic nigrostriatal neurones. Copyright © 1996 Elsevier Science Ltd.

Involvement of the NMDA receptor in a non-cholinergic action of acetylcholinesterase in guinea-pig substantia nigra pars compacta neurons.

Evidence is accumulating that a soluble, secretory form of acetylcholinesterase may have novel, non-cholinergic functions in certain brain regions, such as the substantia nigra. In this study, application of human recombinant acetylcholinesterase (rhAChE) to pars compacta neurons in the rostral substantia nigra resulted in a sustained hyperpolarization that was not only mimicked by application of N-methyl-D-aspartate (NMDA) but also blocked by the NMDA receptor antagonists MK8O1 and 2-amino-5-phosphonopentanoic acid. Neither the rhAChE- nor the NMDA-induced hyperpolarization was seen when the calcium chelator BAPTA was injected into the neuron; hence the effect is mediated by accumulation of intracellular calcium. This intracellular calcium appears sufficient to compromise neuronal metabolism since the rhAChE-induced hyperpolarization was reversed by application of the K-ATP channel antagonist tolbutamide. Butyrylcholinesterase, a protein of similar molecular weight to acetylcholinesterase, which also hydrolyses acetylcholine, had no effect whatsoever. The results suggest that, independent of its normal catalytic function, acetylcholinesterase can act via the NMDA receptor complex to enhance calcium entry into nigral neurons and jeopardize cell metabolism. This non-classical action of acetylcholinesterase might thus be an important factor in the mechanisms underlying parkinsonian neurodegeneration.

Mechanism of block by ZD 7288 of the hyperpolarization-activated inward rectifying current in guinea pig substantia nigra neurons in vitro.

1. The effects of the novel bradycardic agent 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD 7288) (Zeneca) were investigated on the hyperpolarization-activated cationic current (Ih) in guinea pig substantia nigra pars compacta neurons in vitro, using a single-microelectrode current-clamp/voltage-clamp technique. 2. Under current-clamp conditions, injection of large negative current pulses (0.1-0.5 nA, 400 ms) evoked a slow depolarizing "sag" in the electrotonic potential due to activation of the slow inward (anomalous) rectifier. In voltage-clamp recordings, hyperpolarizing voltage steps from a holding potential of -60 mV (close to resting potential) elicited slow inward current relaxations with kinetic properties similar to those seen for other neuronal Ihs. 3. ZD 7288 (10-100 microM) produced a consistent abolition of the electrotonic potential sag with no effect on membrane potential or spike properties. Under voltage clamp, Ih amplitude was clearly reduced in a time- and concentration-dependent manner (apparent half-maximum blocking concentration = 2 microM); full block of Ih was typically achieved after 10-15 min of exposure to 50 microM ZD 7288, with no significant recovery observed after 1 h of washing. 4. A similar (although more rapid) block of Ih was seen after application of 3-5 mM Cs+ (partially reversible after 30 min of washing). 5. Partial block of Ih by 10 microM ZD 7288 was accompanied by a reduction in the maximum amplitude of the Ih activation curve, a small negative shift in its position on the voltage axis, and a linearization of the steady-state current-voltage relationship. The estimated Ih reversal potential, however, remained unaffected. 6. In 10 microM ZD 7288, the time course of Ih activation and deactivation was significantly slowed (within the range of -70 to -120 mV for the activation time constant and -70 to -90 mV for the inactivation time constant). 7. Blockade of Ih by ZD 7288 or Cs+ was independent of prior Ih activation (i.e., non-use dependent). 8. Intracellular loading with ZD 7288 also abolished the sag in the electrotonic voltage response and Ih relaxations, suggesting an intracellular site of action. By contrast, intracellular Cs+ had no effect on Ih properties. 9. Block of Ih by ZD 7288 (but not Cs+) was relieved by prolonged cell hyperpolarization, manifested as a slowly developing (half-time approximately 20 s) inward current at a holding potential of -100 mV. 10. We propose that ZD 7288, when applied externally, may behave as a "lipophilic" quaternary cation, capable of passing into the cell interior to block Ih channels in their closed state; this compound may thus prove a useful research tool, in place of Cs+, for studying the properties and significance of Ih currents in controlling neuronal function.

Inhibition of the ATP‐sensitive potassium channel in the guinea pig substantia nigra by BMS 181100 is not mediated by a sigma‐binding site

Quantitative autoradiography of brain tissue has revealed a high density of binding sites for the K-ATP channel antagonists, the sulphonylureas, and for sigma-ligands in the substantia nigra (SN). In view of the high density of the two binding sites in the SN the possibility has been investigated that the K-ATP channel and the sigma-binding site are functionally linked. The K-ATP channel-mediated membrane hyperpolarisation and decrease in input resistance produced by hypoxia and by the metabolic inhibitor, cyanide, in rostral substantia nigra pars compacta neurons are antagonised by the sigma-ligand BMS 181100. In addition, BMS 181100 antagonises activation of the K-ATP channel by diazoxide; cromakalim is found to be without effect in these neurons. Antagonism of the cyanide-induced hyperpolarisation is dose dependent and is observed at concentrations of the drug which have no observable effect on the resting membrane properties of the neurons. By contrast, the nonselective sigma ligands 1,3-di-O-tolylguanidine (10 microM) and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (100 microM), and the selective sigma 1-ligand (+)-pentazocine (5-10 microM) have no effect on the cyanide-induced hyperpolarisation. 5-HT (50-100 microM) and the selective 5-HT1A receptor agonist 8-OH-DPAT (50 microM) also fail to antagonise the cyanide-induced hyperpolarisation. The antagonism of the cyanide-induced hyperpolarisation by BMS 181100 persists in the presence of tetrodotoxin (1 microM) and in the presence of high concentrations of (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, but not under conditions of reduced calcium (0.1-0.2 mM) and raised magnesium (6 mM) concentrations, which block synaptic transmission. It is concluded that in substantia nigra phasic neurons the sigma-binding site does not regulate activation of the ATP-sensitive channel. However, BMS 181100 antagonises K-ATP channel activation in these neurons independently of sigma-binding sites and 5-HT receptors. This action of BMS 181100 is TTX insensitive and Ca2+ dependent.

Release of acetylcholinesterase from guinea-pig substantia nigra: Effects of tryptaminergic drugs and dorsal raphé nucleus stimulation

The guinea-pig substantia nigra receives a 5-hydroxytryptaminergic (5-HTergic) projection from the dorsal raphé nucleus. In this study we have attempted to identify the 5-HT receptor subtype mediating release of acetylcholinesterase (AChE) from nigral neurones, measured by assay of perfusate obtained via chronically implanted push-pull cannulae. The effects of direct nigral application of 5-HT and the following receptor agonists were tested: 5-carboxamidotryptamine, sumatriptan, α-methyl-5-HT, 2-methyl-5-HT and 5-methoxytryptamine. Application of submicromolar concentrations of 5-HT, 2,5,-dimethoxy-4-iodoamphetamine and α-methyl-5-HT significantly enhanced release of AChE, whereas 5-carboxamidotryptamine, sumatriptan, 2-methyl-5-HT and 5-methoxytryptamine were innefective at a similar concentration range. Electrical stimulation (50 Hz, 20–300 μA) of the dorsal raphé nucleus evoked release of AChE from the substantia nigra, and induced a rotational behavioural effect for the duration of stimulation. Pretreatment with 5,7-dihydroxytryptamine inhibited both DRN-evoked release of AChE and animal rotation. The 5-HT receptor antagonists ketanserin and ritanserin (10 −7 − 10 −6M), when applied to the substantia nigra, inhibited raphé-stimulated AChE release. Drugs which inhibited raphé-stimulated release of AChE had no effect on concomitant animal rotation, indicating that the behavioural events are mediated via distinct processes, unrelated to those mediating nigral AChE release. The data suggest that evoked release of AChE from the substantia nigra by stimulation of the dorsal raphé nucleus may be mediated in part via a 5-HT 2 receptor type. The 5-HT 1D agonists 5-carboxamidotryptamine (10 −6M) and sumatriptan (10 −5M) also inhibited raphé-evoked AChE release, suggesting a possible presynaptic autoinhibitory role for 5-HT 1D receptors on raphé-nigral nerve terminals.

In vitro and in vivo activity of 1-(1-naphthyl)piperazine at terminal 5-HT autoreceptors in guinea-pig brain

The effect of 1-(1-naphthyl)piperazine (NP) on the 5-HT terminal autoreceptor modulating 5-HT release was investigated in vitro and in vivo. In vitro 5-HT release was measured in slices of guinea-pig substantia nigra and hypothalamus prelabelled with 3H-5-HT, superfused with Krebs solution and depolarized electrically. NP, at 0.1 and 1 mumol/l, did not modify the calcium-dependent release of 3H-5-HT elicited by electrical stimulation using a frequency of 5 Hz, however at 0.1 mumol/l NP shifted to the right the inhibition curve of the non-selective autoreceptor agonist, 5-carboxamidotryptamine, in both regions. In hypothalamus when using lower frequencies (1 Hz or 0.2 Hz) or under pseudo-one-pulse stimulation, NP decreased the release of 3H-5-HT at 1 mumol/l. In vivo microdialysis was used to measure extracellular levels of endogenous 5-HT in the substantia nigra of freely moving guinea-pigs. The endogenous release of 5-HT was tetrodotoxin (TTX)-sensitive, indicating a neuronal origin of this efflux. NP, administered through the microdialysis probe (1-100 mumol/l), increased the levels of extracellular 5-HT in concentration-dependent and TTX-sensitive manner. These results suggest that in vitro NP acts as a 5-HT autoreceptor partial (ant)agonist in the substantia nigra and hypothalamus of guinea-pigs, and as a full antagonist in vivo. However, NP administered systemically at 10 mg/kg i.p., did not modify the levels of extracellular 5-HT in the substantia nigra. This lack of systemic effect of NP probably results from its interaction at other receptors that modify 5-HT neurotransmission.(ABSTRACT TRUNCATED AT 250 WORDS)