The effect of microglia on embryonic dopaminergic neuronal survival in vitro: diffusible signals from neurons and glia change microglia from neurotoxic to neuroprotective

When embryonic dopaminergic neurons are transplanted into the adult brain, approximately 95% die within a few days. To assess whether microglia activated during transplantation might be responsible for this rapid death, we examined the effect of microglia on rat embryonic dopaminergic neurons in vitro. Conditioned medium from 7-day-old microglia was found to decrease the number of dopamine neurons surviving in primary culture, but activation of the microglia with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or Zymosan A did not increase the toxicity of the conditioned medium. We next tested the effect of coculturing microglia and dopaminergic neurons by placing microglia in semipermeable well inserts over the neuronal cultures. The presence of microglia now increased dopaminergic neuronal survival, microglial activation again having no effect. To increase yet further the possible interactions between microglia and neurons, the mesencephalic cells and microglia were mixed together and placed as a tissue in three-dimensional culture, and here again the presence of microglia increased dopaminergic neuronal survival with no effect of activation. Contact of microglia with the mesencephalic cells therefore converted them from being toxic to dopaminergic neurons to promoting their survival. The change in microglial effect from toxic to protective was caused by soluble molecules secreted by cells in the neuronal cultures, as conditioned medium derived from microglia-neuronal cocultures also had a dopaminergic neuron survival effect, indicating that microglia in cocultures behave differently from microglia removed from neuronal and glial influence. Microglia cocultured with either neurons or astrocytes downregulated inducible nitric oxide synthase (iNOS), indicating a decrease in the production of nitric oxide and possibly other toxic molecules. These findings indicate that in their natural environment, microglia are likely to be beneficial for the survival of embryonic dopaminergic grafts.     

锰对大鼠子代新生鼠黑质酪氨酸羟化酶的影响

为了研究锰对子代新生鼠黑质中酪氨酸羟化酶（ｔｙｒｏｓｉｎｅｈｙｄｒｏｘｙｌａｓｅ,ＴＨ）的影响,对雌性大鼠腹腔注射氯化锰（ＭｎＣｌ２·４Ｈ２Ｏ）,然后采用原位杂交组织化学技术检测其子代新生鼠黑质中ＴＨ．ｍＲＮＡ神经元。结果：对照组新生鼠黑质中观察到ＴＨ．ｍＲＮＡ神经元,各实验组均未观察到ＴＨ．ｍＲＮＡ神经元。提示：锰在基因水平上抑制了染锰者子代新生鼠黑质中ＴＨ活性,进而干扰和抑制了胺能神经递质的正常代谢     

Effects of acute and chronic administration of olanzapine in comparison to clozapine and haloperidol on extracellular recordings of substantia nigra reticulata neurons in the rat brain

Rationale: Previously, we have shown that the atypical antipsychotics clozapine and risperidone, unlike haloperidol, decreased the firing rate of substantia nigra reticulata (SNR) neurons. As the SNR receives substantial input from the striatum, an area where motoric side-effects of antipsychotics are thought to be mediated, the SNR might be an interesting brain structure with regard to motor side-effects. Objective: The newly developed atypical antipsychotic olanzapine was studied for its effects on the firing rate of SNR cells. In addition, to gain insight in the implications of our experimental setup for clinical use, responses upon clozapine, olanzapine and haloperidol were studied after chronic treatment. Methods: In chloralhydrate-anaesthetized male Wistar rats, extracellular recordings were made from SNR neurons upon intravenously (IV) administered cumulative doses of the antipsychotics. Naive rats and rats that were subcutaneously (SC) injected for 21 days with an antipsychotic were used. Results: Olanzapine (50–1600 mg/kg; IV), significantly inhibited the firing rate of the SNR neurons. Upon 21 days of treatment with a daily SC injection of 20 mg/kg clozapine, the challenge on day 22 with cumulative injections of clozapine (200–6400 mg/kg; IV) significantly inhibited the firing rate of the SNR neurons. Olanzapine (50–1600 mg/kg; IV) also significantly inhibited the SNR activity when pretreated with olanzapine in an SC administered dose of 1 mg/kg, but not 5 mg/kg. Haloperidol (12.5–800 μg/kg; IV) did not significantly affect the SNR activity in rats pretreated with SC administered 0.5 mg/kg haloperidol. Conclusions: Upon acute and chronic administration of clozapine and olanzapine versus haloperidol, differential effects on SNR neuronal firing could be obtained. The experimental setup seem to be valid for further studies into the mechanism of action of typical versus (relatively low doses of) atypical antipsychotics. The implications of the inhibitory effect of atypical antipsychotics on the SNR firing rate are presently unknown, but could be associated with the lower propensity to induced motoric side-effects. On the other hand, the SNR activity might also reflect non-motoric activity possibly related to negative symptoms. Received: 11 December 1998/Final version: 20 January 1999     

Monoamine oxidase-dependent metabolism of dopamine in the striatum and substantia nigra of

Summary The effects on the substantia nigra of -dihydroergocryptine (DEK), a drug with strong dopaminomimetic activity, were tested with a severe 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in monkeys. Compared with monkeys treated with MPTP alone, the animals which received DEK plus MPTP showed reduced neuronal death in the substantia nigra. The reactive astrocytes were increased in number. Moreover, several axons which were immunopositive to phosphorylated neurofilament proteins and with features similar to those of control animals were seen in the pars compacta. The findings suggest that DEK preserves neuronal morphology and brain architecture.supported by Italian M.U.R.S.T. grants (to G.B.) and by Poli S. P. A., Rozzano, Milano     

Neuroprotective effects of α-dihydroergocryptine against damages in the substantia nigra caused by severe treatment with 1-methyl-4-phenyl-1,3,3,6-tetrahydropyridine

Summary The effects on the substantia nigra of -dihydroergocryptine (DEK), a drug with strong dopaminomimetic activity, were tested with a severe 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in monkeys. Compared with monkeys treated with MPTP alone, the animals which received DEK plus MPTP showed reduced neuronal death in the substantia nigra. The reactive astrocytes were increased in number. Moreover, several axons which were immunopositive to phosphorylated neurofilament proteins and with features similar to those of control animals were seen in the pars compacta. The findings suggest that DEK preserves neuronal morphology and brain architecture.supported by Italian M.U.R.S.T. grants (to G.B.) and by Poli S. P. A., Rozzano, Milano     

Effects of GABA,dopamine, and substance P on the release of newly synthesized 3H-5-hydroxytryptamine from rat substantia nigra in vitro

Summary The effects of GABA, substance P and dopamine on the release of newly synthesized ³H-5-HT were investigated, using slices of rat substantia nigra superfused with l-³H-tryptophan in vitro. GABA (50 M) had no inhibitory effect on the potassium-evoked-release of ³H-5-HT. Substance P (50 M) and eledoisin (50 M) stimulated the spontaneous release of ³H-5-HT. This effect seems to be indirect and is possibly mediated by dopaminergic neurones, since the dopamine antagonist drug -flupenthixol (1 M) abolished the substance P-evoked release of 5-HT. Furthermore, it was found that substance P (10 M) stimulated ³H-dopamine release from nigral slices in vitro and the dopaminergic agonist apomorphine (50 M) also stimulated ³H-5-HT release. Substance P may, therefore, activate nigral dopaminergic neurones which then release dopamine from their dendrites. The release of dopamine may in turn stimulate 5-HT release from terminals of the raphe-nigral pathway.     

Response sensitization and depression following long-term amphetamine treatment in a self-stimulation paradigm

The effects of long-term amphetamine treatment were evaluated on responding supported by self-stimulation of the substantia nigra. Rats repeatedly treated with d-amphetamine, and tested with a low dose of the drug that ordinarily has no behavioral effect, showed higher response rates than animals repeatedly treated with saline and tested with the same dose of amphetamine. In contrast, a depression in responding was observed among animals that received long-term amphetamine administration and were tested with saline. The effects of long-term amphetamine treatment on self-stimulation could not be explained by the intrusion of drug-induced competitive behaviors such as locomotor activity and stereotypy. The results were attributed to changes in dopamine neurotransmission following prolonged exposure to amphetamine and were also discussed in terms of an animal model for amphetamine psychosis and postamphetamine depression in man.     

Activation of striatal dopamine receptors induces pain inhibition in rats

Summary In the rat, elevating dopamine content in corpus striatum with electrical stimulation of substantia nigra or direct administration of apomorphine (50–200g) into the lateral cerebral ventricle or apomorphine (2–10g) into the caudate-putamen complex decreased pain sensitivity (as shown by an increase in the latency to hind-paw lick in the hot plate test). Furthermore, the decreased pain sensitivity after the central administration of apomorphine was antagonized by pretreatment with haloperidol (a dopamine antagonist). On the other hand, lowering dopamine content in corpus striatum with electrolytic destruction of substantia nigra and 6-hydroxydopamine lesions to the substantia nigra, as well as direct injection of haloperidol into the lateral cerebral ventricle or caudate-putamen complex increased pain sensitivity. The data indicate that activation of striatal dopamine receptors in rat brain induces pain inhibition.     

Motor effects following application of putative excitatory amino acid antagonists to the region of the mesencephalic dopamine cell bodies in the rat

Summary The excitatory amino acid antagonists 2-amino-5-phosphonovalerate and -D-glutamylglycine have been applied focally to the ventral tegmental area and both the pars compacta and pars reticulata of the substantia nigra of the rat. The injections were performed under halothane anaesthesia so that behavioural effects could be observed 5 min afterwards. Bilateral application of either antagonist to the ventral tegmental area and the pars compacta of the substantia nigra induced enhanced locomotor activity in an open field. This effect was blocked by pretreatment of the animals with a low dose of the dopamine receptor antagonist fluphenazine. Bilateral application of either antagonist to the pars reticulata of the substantia nigra produced sedation and a reduction in locomotor activity. Unilateral injection of either of the excitatory amino acid antagonists into the pars reticulata or pars compacta of the substantia nigra both resulted in contraversive circling behaviour The effect of intranigral (both pars compacta and reticulata) 2-APV and -DGG was accompanied by a significant increase in concentrations of both 3,4-dihydroxyphenylacetic acid (to 158–160% of control following injection into pars compacta, and 134–146% of control injected into pars reticulata) and homovanillic acid (to 161–166% of control following injection into pars compacta, and 186–210% of control injected into pars reticulata) in the ipsilateral, striatum. Pretreatment of these animals with fluphenazine (0.3 mg/kg) antagonized this circling behaviour.These results indicate that antagonism, of excitatory amino acid receptors in the region of the midbrain of the rat leads to specific behavioural effects, which may in part be mediated through the ascending dopaminergic projections.Abbreviations used 2-APV 2-amino-5-phosphonovaleric acid - -DGG -D-glutamylglycine - SN substantia nigra - VTA ventral tegmental area - HVA homovanillic acid - DOPAC 3,4-dihydroxyphenylacetic acid A preliminary communication of this work was presented to the British Pharmacological Society in Bradford, April 1981     

The effects of GBR 12909, a dopamine re-uptake inhibitor,on monoaminergic neurotransmission in rat striatum,limbic forebrain,cortical hemispheres and substantia nigra

Summary In order to investigate the physiological importance of the membrane pump in eliminating released dopamine (DA) we have studied the effects of the putative selective dopamine re-uptake inhibitor, GBR 12909, on synthesis and metabolism of monoamines in the rat striatum, limbic forebrain, cortical hemispheres and substantia nigra (SN). The effects of the drug on the firing rate of catecholamine containing neurons in the SN and locus coerulus (LC) were also investigated. For comparison we have investigated the effects of desipramine and maprotiline. As a measure of the synthesis of noradrenaline (NA), DA and 5-hydroxytryptamine (5-HT) we determined the 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) accumulation after inhibition of aromatic l-amino acid decarboxylase by 3-hydroxy-benzylhydrazine (NSD 1015). As indirect measurements of DA and NA release in vivo, we have assessed pargyline-induced 3-methoxytyramine (3-MT) and normetanephrine (NM) accumulation and disappearance rates of DA and NA after inhibition of their synthesis by -methyl-p-tyrosine (-MT). Administration of GBR 12909 (2.5, 5, 10, 20 or 40 mg/kg) decreased the NSD 1015-induced DOPA accumulation in the striatum and in the limbic forebrain. In contrast, only minor effects of the drug were seen on the DOPA accumulation in the cortical hemisphere and on the cerebral 5-HTP accumulation. GBR 12909 increased the 3-MT accumulation in the striatum, limbic forebrain and the cortical hemispheres, an effect that was even more pronounced in haloperidol-pretreated animals. However, GBR 12909 did not alter the 3-MT accumulation in the SN either when given alone or when given to haloperidol-pretreated rats. In haloperidol-pretreated rats GBR 12909 markedly enhanced the DA disappearance in the striatum and in the limbic forebrain, but not in the SN. Furthermore, GBR 12909 did not significantly affect the firing rate of dopaminergic neurons in the SN or that of noradrenergic neurons in the LC. Taken together, our results support the notion that GBR 12909 is a specific DA uptake inhibitor without a transmitter releasing action. In addition, our findings indicate that DA re-uptake is of physiological importance in the elimination of DA from the synaptic cleft in the striatum, limbic forebrain and cortical hemispheres, but not in the SN. Furthermore, a large part of the DA taken up by the dopaminergic terminals in the striatum and in the limbic forebrain seems to be re-incorporated into the storage vesicles. Send offprint requests to H. Nissbrandt at the above address