Proteasome inhibitors protect against degeneration of nigral dopaminergic neurons in hemiparkinsonian rats

Parkinson's disease is characterized by dopaminergic neuronal death and the presence of Lewy bodies in the substantia nigra pars compacta (SNpc). alpha-Synuclein and ubiquitin are components of Lewy bodies, but the process of Lewy body formation and the relationship between inclusion formation and dopaminergic neuronal death have not been resolved. In this study, unilateral intranigral microinjection of 6-hydroxydopamine caused a significant loss of tyrosine hydroxylase-immunopositive neurons in both the substantia nigra and striatum and apomorphine-induced contralateral rotation. The co-administration of proteasome inhibitors, such as lactacystin or carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), significantly prevented both dopaminergic neurodegeneration and apomorphine-induced rotational asymmetry. Proteasome inhibitors markedly formed intracellular protein inclusions labeled by thioflavin-S in the SNpc. Inclusion-like immunoreactivities for alpha-synuclein and ubiquitin were detected after 4 weeks. These results suggest that proteasome plays an important role in both the early phase of dopaminergic neuronal death and inclusion body formation.     

Diazepam potentiates GABA-, but not adenosine-mediated, inhibition of neurons of the nigral pars reticulata

Experiments were conducted to assess the relative roles of gamma-aminobutyric acid (GABA) and adenosine in mediating the inhibition of neuronal activity by diazepam injected intravenously. Recent studies have shown that benzodiazepines inhibit, in a dose-dependent manner, the firing of neurons in the substantia nigra pars reticulata. In support of a predominantly GABAergic mechanism for this inhibitory action, a small dose of diazepam (50 micrograms/kg, i.v.), which itself had little effect on cell firing, significantly potentiated the inhibitory responses of neurons of the pars reticulata to muscimol, a potent GABA agonist given intravenously, and significantly and selectively potentiated the inhibition of reticulata neurons by GABA applied iontophoretically. In contrast to their extreme sensitivity to GABAergic inhibition, neurons of the pars reticulata were comparatively insensitive to systemically and iontophoretically administered adenosine-related drugs. However, in those instances when inhibitions could be achieved with iontophoretically applied adenosine-5'-monophosphate, the inhibitory responses were not significantly modified by a 50 micrograms/kg (i.v.) dose of diazepam. These findings, considered in light of differences in GABA and adenosine receptor densities within the substantia nigra, suggest that the benzodiazepine-induced inhibition of neurons of the nigral pars reticulata most likely involves potentiation of GABA but not adenosine-mediated influences.     

Intravenous GABA agonist administration stimulates firing of A10 dopaminergic neurons

Increasing intravenous doses of two γ-aminobutyric acid (GABA) agonists, muscimol and 4,5,6,7-tetrahydro-isoxazolo-[5,4-c]pyridin-3-ol (THIP), caused dose-related increases in extracellular, single unit activity of A₁₀ dopaminergic neurons of the ventral tegmental area. Muscimol, the more potent of the two compounds, stimulated firing at doses approximately 15–20 times lower than the doses of THIP required to elicit equivalent excitatory effects. The maximum stimulation achieved was approximately 150 and 140% of the baseline firing rate for muscimol and THIP, respectively.     

Hyperactivity induced by N-methyl-d-aspartate injections into nucleus accumbens: lack of evidence for mediation by dopaminergic neurons

To test the hypothesis that the motor hyperactivity associated with intra-accumbens injections of N-methyl-d-aspartate (NMDA) results from stimulation (direct or indirect) of nucleus accumbens dopaminergic mechanisms, the behavioral effects of intra-accumbens and intraventricular NMDA were compared to those of the prototypic dopaminergic releasing agent, amphetamine, and the competitive NMDA receptor antagonist, 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP). Drugs were injected into the right lateral ventricle, or bilaterally into the nucleus accumbens of rats. Locomotor activity was monitored electronically and by direct observation for 40 min prior to, and 1 hour after, drug treatment. Intra-accumbens injections of NMDA (0.4, 1.2 and 2.0 micrograms/side) produced dose-related increases in distance traveled, but had no significant effect on movement time or vertical movements. The NMDA-induced increase in distance traveled was temporally correlated with convulsive wild running, but not with exploratory behavior, suggesting that this increase may have been secondary to seizure-like activity. Intra-accumbens injections of amphetamine (10, 20 and 40 micrograms) or CPP (0.1 microgram) produced dose-related increases in all three measures. By the intraventricular route, the effects of NMDA were similar to those of intra-accumbens administration, whereas intraventricularly administered d-amphetamine had no effect. The behavioral effects of intra-accumbens NMDA cannot be explained by an NMDA receptor-mediated facilitation of dopaminergic neurotransmission; rather, this type of facilitation may be associated with competitive NMDA receptor antagonism.     

Separation of dopaminergic and serotonergic inhibitory mechanisms in the mediation of estrogen-induced lordosis behaviour in the rat

The administration of the putative 5-hydroxytryptamine1 (5-HT1) agonist 8-hydroxy-2(di-n-propylamino) tetralin (8-OH-DPAT) (0.0625-1.0 mg X kg-1) suppresses lordosis behaviour induced in ovariectomized female rats by daily treatment for 3-5 days with estradiol benzoate (1.25 micrograms/rat). A similar suppressive effect on the lordosis behaviour can be obtained by administration of the dopamine/serotonin agonist, lisuride (0.1-0.4 mg X kg-1), or after the administration of the dopamine (DA) agonists, apomorphine (0.2-0.8 mg X kg-1) or quinpirole (0.75-2.50 mg X kg-1). The suppressive effects on the lordosis behaviour by 8-OH-DPAT cannot be antagonized by the DA receptor antagonist haloperidol (0.2 mg X kg-1) neither with methiotepin (0.5 mg X kg-1), which is assumed to be a non-selective 5-HT receptor blocking agent, nor with pirenperone (0.25 mg X kg-1) which is assumed to be a 5-HT2 receptor blocking agent. However, a partial blockade of the lordosis suppressive effects of 8-OH-DPAT was obtained by treatment with (-)-pindolol, which is thought to be a partial 5-HT1 blocking agent, suggesting that 8-OH-DPAT exerts its suppressive effects on the lordosis behaviour through the 5-HT system. Haloperidol causes a complete blockade of the suppressive effects of apomorphine and quinpirole suggesting that these drugs exert their inhibitory effects on the lordosis behaviour by activating the DA system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substantia nigra as an out-put station for striatal dopaminergic responses: Role of a GABA-mediated inhibition of pars reticulata neurons

Summary Intranigral administration of kainic acid results in loss of pars reticulata neurons without damage to axons traversing or terminating within the nigra. Unilateral nigral lesions with kainic acid result in an ipsilateral turning upon administration of apomorphine, a dopamine (DA)-receptor agonist and in contralateral turning upon administration of haloperidol, a DA-receptor blocker. Destruction of post-synaptic structures in the striatum of the side contralateral to that injected with kainic acid results in a drastic reduction, abolition or even reversal of the turning effects elicited by apomorphine and haloperidol. Unilateral intranigral microinjection of nanogram amounts of the GABA-receptor antagonists picrotoxin and bicuculline elicits ipsilateral circling upon apomorphine administration. Kainic-induced lesion or micro-injection of picrotoxin or bicuculline in the nigra ipsilateral to a 6-OHDA-lesion of nigro-striatal DA-neurons results in reduction, abolition or reversal of the contralateral circling produced by apomorphine. The results indicate that the nigra pars reticulata is a station for dopaminergic impulses originating from the striatum and suggest that the turning behavior in response to striatal DA-receptor stimulation is due to a GABA-mediated inhibition of ipsiversive pars reticulata neurons.     

Effect of chronic administration of methamphetamine on the responsiveness of substantia nigra zona reticulata neurons to GABA or a GABA agonist in rats

Summary The effects of chronic administration of methamphetamine on the responsiveness of neurons of the substantia nigra zona reticulata (SNR) to gammaaminobutyric acid (GABA) or to a GABA receptor agonist were examined. Neuronal activity was recorded from the SNR of rats that had been pretreated twice daily, for 6 consecutive days, with saline or with 5 mg/kg methamphetamine. Intravenous administration of the GABA receptor agonist, muscimol, caused a dose-dependent decrease in the unit activity of the SNR neurons and the SNR neurons became less sensitive to the depressant effects of the drug after chronic treatment with methamphetamine. Iontophoretic application, with increasing currents, of GABA produced a progressive inhibition of unit activity in control animals, an effect that was significantly reduced in rats pretreated with methamphetamine. These results support the hypothesis that long-term administration of methamphetamine increases the activity of the striatonigral GABA system and thereby reduces the sensitivity of postsynaptic GABA receptors in the SNR.     

Intranigral kainic acid: Evidence for nigral non-dopaminergic neurons controlling posture and behavior in a manner opposite to the dopaminergic ones

The unilateral, intranigral administration of kainic acid (k.a.) produced a syndrome characterized by early sequelae of contra- and ipsilateral circling and by a chronic contralateral turning associated with moderate loss of neurons in the pars reticulata. The acute contralateral circling seems to be related to dopaminergic nigro-neostriatal neuron stimulation, since it was prevented by previous intranigral injections of 6-OHDA. The acute ipsilateral circling and the chronic contralateral turning, on the other hand, seem to be independent of the integrity of the dopaminergic system and may be due to an initial stimulation, followed by destruction, of a nigral neuronal system which mediates turning behavior in a manner opposite to that of nogro-striatal dopamine. Treatment with D-amphetamine or apomorphine changed the contralateral into ipsilateral turining, while haliperidol potentiated the contralateral turning. Bilateral injection of k.a. into the nigra resulted in chronic stereotyped sniffing and gnawing, which were not inhibited by haloperidol. Moreover, haloperidol did not produce catalepsy in these animals. It is suggested that the intranigral k.a. injection destroyed a neuronal system antagonistic to dopamine and resulted in a reduction of the response to DA-receptor stimulation of the c. striatum.     

Iptakalim alleviates rotenone-induced degeneration of dopaminergic neurons through inhibiting microglia-mediated neuroinflammation.

Inhibition of microglia-mediated neuroinflammation has been regarded as a prospective strategy for treating neurodegenerative disorders, such as Parkinson's disease (PD). In the present study, we demonstrated that systematic administration with iptakalim (IPT), an adenosine triphosphate (ATP)-sensitive potassium channel (K(ATP)) opener, could alleviate rotenone-induced degeneration of dopaminergic neurons in rat substantia nigra along with the downregulation of microglial activation and mRNA levels of tumor necrosis factor-alpha (TNF-alpha) and cyclooxygenase-2 (COX-2). In rat primary cultured microglia, pretreatment with IPT suppressed rotenone-induced microglial activation evidenced by inhibition of microglial amoeboid morphological alteration, declined expression of ED1 (a marker for activated microglia), and decreased production of TNF-alpha and prostaglandin E2 (PGE(2)). These inhibitory effects of IPT could be reversed by selective mitochondrial K(ATP) (mitoK(ATP)) channel blocker 5-hydroxydecanoate (5-HD). Furthermore, pretreatment with IPT prevented rotenone-induced mitochondrial membrane potential loss and p38/c-jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) activation in microglia, which might in turn regulate microglial activation and subsequent production of TNF-alpha and PGE(2). These data strongly suggest that the K(ATP) opener IPT may be a novel and promising neuroprotective drug via inhibiting microglia-mediated neuroinflammation.     

Nimodipine protects dopaminergic neurons against inflammation-mediated degeneration through inhibition of microglial activation

Nimodipine, a calcium channel blocker, has been used mainly in the therapy of cardiovascular diseases. Recently, its indications have been extended experimentally to a wider range of disorders especially some central nervous system (CNS) disorders. In this study, we investigated whether nimodipine is neuroprotective to inflammation-mediated neurodegenerative diseases. Pretreatment with nimodipine reduced the degeneration of dopaminergic (DA) neurons induced by LPS in mesencephalic neuron-glia cultures in a dose-dependent manner. The neuroprotective effect of nimodipine was attributed to the inhibition of microglial activation, since nimodipine significantly inhibited the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and prostaglandin E₂ (PGE₂) from LPS-stimulated microglia. Moreover, nimodipine was not neuroprotective to 1-methyi-4-phenylpyridinium (MPP⁺)-induced DA neurotoxicity in the absence of microglia. Mechanistic study showed that nimodipine failed to protect the degeneration of neurons in neuron-glia cultures from mice lacking functional NADPH oxidase (PHOX), a key enzyme for extracellular superoxide production in immune cells. Taken together these results suggest that nimodipine is protective to DA neurodegeneration via inhibiting the microglial-mediated oxidative stress and inflammatory response. Thus, nimodipine may be a potential therapeutic agent for the treatment of inflammation-related neurodegenerative disorders such as Parkinson's disease.     

The role of GABA receptors in the control of nigrostriatal dopaminergic neurons: dual-probe microdialysis study in awake rats.

A microdialysis probe implanted into the substantia nigra was used to infuse gamma-aminobutyric acid-ergic (GABAergic) compounds onto cell bodies/dendrites of dopaminergic neurons, while a second microdialysis probe was used to record the extracellular concentrations of dopamine and 3,4-dihydroxy-phenylacetic acid (DOPAC) in the ipsilateral striatum. The GABAA receptor agonist muscimol (10 mumol/l) increased the release of dopamine in the ipsilateral striatum to 120% of the control values. The GABAB receptor agonist, (Z)-3[(aminoiminomethyl)-thiol]-prop-2- enoic acid (500 mumol/l), was without effect. Infusion of the GABAA receptor antagonists, bicuculline (50 mumol/l) and picrotoxin (50 mumol/l), stimulated the release of dopamine in the ipsilateral striatum to 160 and 130% of the controls, respectively. The GABAB receptor agonist, baclofen (10 and 50 mumol/l), strongly inhibited the release of striatal dopamine, whereas infusion of the GABAB receptor antagonist, 2-hydroxy-saclofen (100 mumol/l), was without effect. The results indicate that, in the substantia nigra, GABAA as well as GABAB receptors participate in controlling the activity of dopaminergic neurons.     

Morphine dependence and dopaminergic activity: tests of circling responses in rats with unilateral nigral lesions

1 Rats with unilateral electrolytic lesions involving both parts of the substantia nigra show dose-related, ipsilateral circling responses to apomorphine which are stable over time. 2 In non-tolerant rats, morphine (up to 10 mg/kg) does not elicit any circling behaviour but as tolerance develops to morphine, initially 10 mg/kg daily and then 100 ng/kg daily for about 4 months, the rats show a progressive tendency to walk more towards the side of the lesion. This behaviour is qualitatively different from apomorphine-induced circling. 3 When apomorphine (0 to 1.0 mg/kg) and morphine (10 or 100 mg/kg) are tested together, the total amounts of 'circling' are increased in an additive manner. However, after 22 h withdrawal from morphine there is a more marked increase in apomorphine-induced circling which is related to the level of dependence. 4 It is suggested that the sensitivity of striatal dopamine receptors is not altered by morphine dependence and that the increased response to apomorphine in abstinence probably reflects changes in the modulating actions of other neurotransmitter systems in the striatum.     

Folate induced-hypermotility response after bilateral injection into the nucleus accumbens of the rat. Possible mediation through dopaminergic mechanisms

Folic acid (FA) and certain of its reduced congeners produce excitatory effects when applied to neuronal tissue. Recent evidence has suggested that folates have other biological properties in common with the excitatory amino acids. The purpose of this study was to determine the activity of folate compounds in a system sensitive to excitatory amino acids. Bilateral injection of folic acid into the nucleus accumbens resulted in a marked increase in locomotor activity at doses of 2.5 and 5 micrograms. Larger doses resulted in behavioral responses, such as body tremor and labored breathing, which interfered with the locomotor response. Similarly, 5-formyltetrahydrofolic acid (FTHF) produced a marked hypermotility response after bilateral injection into the nucleus accumbens (2.5-25 micrograms), while dihydrofolic acid, tetrahydrofolic acid, and 5-methyltetrahydrofolic acid were ineffective. Pretreatment with reserpine (10 mg/kg, i.p.) markedly reduced the hypermotility response elicited by folic acid and FTHF as did pretreatment with haloperidol in both peripheral (0.8 mg/kg) and direct (5 micrograms) injection into the nucleus accumbens. In addition, injection of muscimol (30 ng), which depresses hypermotility induced by dopamine and amphetamine, produced a significant decrease in the hypermotility response produced by folic acid. In contrast, pretreatment with phentolamine (5 mg/kg, i.p.) or propranolol (4 mg/kg, i.p.) did not decrease folic acid or FTHF-induced responses. These results suggest that folic acid and FTHF produce an increase in locomotor activity by facilitating dopaminergic neurotransmission in the nucleus accumbens, possibly by inducing the release of dopamine from the nerve terminals. Thus, these folates have effects similar to those of the excitatory amino acids when injected into the nucleus accumbens.     

Effects of chronic diazepam exposure on GABA sensitivity and on benzodiazepine potentiation of GABA-mediated responses of substantia nigra pars reticulata neurons of rats

This study examined the effects of chronic diazepam treatment on GABA sensitivity of substantia nigra pars reticulata neurons and on the ability of benzodiazepines to enhance GABAergic responses of these neurons in rats. Chronic diazepam exposure failed to significantly alter the sensitivity of reticulata neurons to microiontophoretically applied GABA. However, following chronic diazepam treatment for 1 day, or 1, 3 or 7-11 weeks, reticulata neurons showed tolerance to additional systemically or iontophoretically applied benzodiazepines and displayed an increased firing rate following injection of Ro 15-1788. These changes were not apparent 24 h after cessation of chronic treatment. Thus, tolerance to the effects of benzodiazepines on reticulata neurons appeared to develop after a single day of diazepam exposure and to dissipate by 24 h after cessation of treatment. When compared to our previous studies on dorsal raphe neurons, these results demonstrate regional differences in neuronal responses to chronic diazepam exposure, which may help elucidate neural systems which are involved in tolerance to the various functional aspects of benzodiazepines.     

Sigma-receptors: implication for the control of neuronal activity of nigral dopamine-containing neurons.

Using extracellular single unit recording techniques, we analyzed the significance of sigma-receptors in the neuronal control of dopamine (DA) neurons in the zona compacta of the substantia nigra. Administration of racemic trans-9-methoxy-4-benzyl-1,2,3,4,4a,5,6,10b-octahydrobenzo[f]quinoline (HW 173), a drug displaying high and specific affinity for sigma-receptors, was not associated with any change in the firing rate of DA neurons. Furthermore, pretreatment with the drug did not affect the dose-response curve for the inhibitory effects of the DA receptor agonist apomorphine or the mixed DA agonist/sigma-receptor ligand (+)-3-(3-hydroxyphenyl)-N-1-propyl)piperidine ((+)-3-PPP). In contrast to the effects of HW 173 on the DA cell firing rate, the firing pattern of the DA cells was significantly changed by the drug. The data suggest that sigma-receptors do not play a pivotal role in the control of the firing rate of DA neurons in the substantia nigra. Rather, they may have a function in the regulation of the firing pattern of these neurons.