Maria C. Olianas

The striatal dopaminergic function is mediated by the inhibition of a nigral, non-dopaminergic neuronal system via a strio-nigral gabaergic pathway

Abstract The unilateral, intranigral administration of muscimol produced a spontaneous circling, contralateral to the injected side, lasting from 90 min to several h, according to the dose (5, 25, 50 ng/s.n.). This contralateral circling was potentiated by haloperidol (0.2 mg/kg) and antagonized by apomorphine. The bilateral injection of muscimol in the s.n. resulted in stereotyped movements which were resistant to haloperidol administration. Moreover, haloperidol (up to 5 mg/kg) failed to produce catalepsy in these bilaterally injected rats. Picrotoxin, injected unilaterally in the nigra in a dose of 20 ng, produced ipsilateral turning, which was potentiated by apomorphine (0.2 mg/kg) and antagonized by haloperidol administered in the contralateral striatum. The bilateral injection of the same dose of picrotoxin induced rigid catalepsy, which was not sensitive to apomorphine treatment (0.20 mg/kg). Both the contralateral turning and the stereotypes induced by the acute, intranigral administration of muscimol were very similar to the chronic effects elicited by intranigral kainic acid (k.a.) observed from 24 h to several days after treatment, a compound that destroys rather selectively the neurons of the pars reticulata. Conversely, the acute effect of the unilateral injection of k.a., which is supposed to be initially stimulatory, recalled the effect of intranigral picrotoxin. The following neuronal model is proposed: the nigral, k.a. sensitive neurons control posture in a manner opposite to striatal dopamine. The function of the nigro-striatal dopamine system is to inhibit these neurons by activating a strio-nigral GABAergic pathway.

SELECTIVE INCREASE OF BRAIN DOPAMINE SYNTHESIS BY SULPIRIDE

—Sulpiride (5–200 mg/kg) increases brain HVA and DOPAC levels, causes no change in dopamine concentration, does not interfere with the outflow of HVA from the CNS and enhances the disappearance of brain dopamine after inhibition of tyrosine hydroxylase. The compound influences neither 5‐HT nor NE metabolism. The central action of sulpiride differs from that of classic neuroleptics in that this drug stimulates dopamine turnover without producing catalepsy.

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 nigro-striatal dopamine. Treatment with D-amphetamine or apomorphine changed the contralateral into ipsilateral turning, while haloperidol 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.

Rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity: potent block by the selective m4 ligand muscarinic toxin 3 (MT3)

1 In rat striatal membranes, muscarinic toxin 3 (MT3), a selective ligand of the cloned m4 receptor subtype, antagonized the acetylcholine (ACh) inhibition of forskolin‐and dopamine D1 receptor‐stimulated adenylyl cyclase activities with pA2 values of 8.09 and 8.15, respectively. 2 In radioligand binding experiments, MT3 increased the Ki but did not change the Bmax value of [3H]‐ N‐methylscopolamine (3H]‐NMS) binding to rat striatal muscarinic receptors. The toxin displaced the major portion of the [3H]‐NMS binding sites with a Ki of 8.0 nM. 3 In rat myocardium, MT3 antagonized the ACh inhibition of adenylyl cyclase with a Ki value of 860 nM. 4 In rat cerebral cortical membranes prelabelled with [3H]‐myo‐inositol, MT3 counteracted the methacholine stimulation of [3H]‐inositol phosphates formation with a Ki value of 113 nM. 5 The present study shows that MT3 is a potent antagonist of the striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity. This finding provides strong evidence for the classification of these receptors as pharmacologically equivalent to the m4 gene product (M4). On the other hand, the weaker potencies of MT3 in antagonizing the muscarinic responses in cerebral cortex and in the heart are consistent with the reported lower affinities of the toxin for the cloned ml and m2 receptor subtypes, respectively.

Evidence that adenosine A2 and dopamine autoreceptors antagonistically regulate tyrosine hydroxylase activity in rat striatal synaptosomes

Incubation of rat striatal synaptosomes with the adenosine receptor agonist 2-chloroadenosine (2-CADO) produced a concentration-dependent increase of dopamine (DA) synthesis (about 50% of control value). The effect was not additive with the stimulation produced by either 10 microM forskolin or 2 mM dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with 2-CADO produced an activation of tyrosine hydroxylase (TH) which withstood washing and lysing of the tissue. This activation was largely independent of the presence of Ca2+ ion in the preincubation medium and, when analyzed as a function of different concentrations of the pterin cofactor 6-methyl-5,6,7,8-tetrahydropterin (0.08-0.4 mM), it was associated with an apparent increase in the Vmax of the enzyme. Quinpirole, a selective D2 DA receptor agonist, reduced control synaptosomal DA synthesis and caused a persistent inhibition of TH activity. When added together with 2-CADO, quinpirole depressed the stimulation of DA synthesis and TH activity produced by the adenosine analog. The effect of quinpirole was stereospecifically antagonized by the D2 DA antagonist sulpiride. Quinpirole also inhibited the activation of TH elicited by a submaximal concentration of forskolin, but not that produced by dibutyryl cyclic AMP. The inhibitory effect of quinpirole on basal and 2-CADO-stimulated TH activities was mimicked by DA. These results indicate that presynaptic DA autoreceptors and adenosine A2 receptors interact antagonistically in controlling DA synthesis in rat striatal synaptosomes presumably by exerting opposite inputs on a presynaptic adenylate cyclase system.

Mixed Agonist–Antagonist Properties of Clozapine at Different Human Cloned Muscarinic Receptor Subtypes Expressed in Chinese Hamster Ovary Cells

We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarinic receptor subtype. In the present study, we investigated whether the drug could elicit similar effects at the cloned human m1, m2, and m3 muscarinic receptor subtypes expressed in the Chinese hamster ovary (CHO) cells. Clozapine elicited a concentration-dependent stimulation of [3H]inositol phosphates accumulation in CHO cells expressing either the m1 or the m3 receptor subtype. Moreover, clozapine inhibited forskolin-stimulated cyclic AMP accumulation and enhanced [35S] GTPγS binding to membrane G proteins in CHO cells expressing the m2 receptor. These agonist effects of clozapine were antagonized by atropine. The intrinsic activity of clozapine was lower than that of the full cholinergic agonist carbachol, and, when the compounds were combined, clozapine potently reduced the receptor responses to carbachol. These data indicate that clozapine behaves as a partial agonist at different muscarinic receptor subtypes and may provide new hints for understanding the receptor mechanisms underlying the antipsychotic efficacy of the drug.

Evidence that a nigral gabaergic-chrolinergic balance controls posture

The intranigral injection of kainic acid (k.a.) (3.5 nM/s.n.) produced a lesion which resulted in a decreased muscarinic receptor binding capacity and in a decreased choline acetyl transferase (CAT) activity confined to the pars reticulata. The unilateral, intranigral injection of carbachol in the substantia nigra (s.n.) produced turning, ipsilateral to the injected side, of dose-related intensity, which was antagonized by scopolamine given either i.p. or intranigrally together with carbachol. The bilateral, intranigral injection of carbachol produced rigid catalepsy, highly resistant to apomorphine administration and antagonized by scopolamine. On the other hand, the catalepsy produced by intranigral picrotoxin was much more sensitive to apomorphine and was disrupted by systemic scopolamine administration. Intranigral scopolamine per se produced either contralateral turning or stereotyped movements consistently, when injected unilaterally or bilaterally, respectively. In addition, scopolamine injected bilaterally in the s.n. but not in the caudate nucleus (c.n.), at the concentration of 64 nM side, was able to antagonize the haloperidol-induced catalepsy and to prevent the tremors and the muscular rigidity produced by arecoline. This effect of scopolamine was surmountable with a higher dose of arecoline. Finally, intranigral muscimol (0.44 nM/s.n.) prevented the occurrence of the parkinsonian syndrome produced by systemic arecoline. It is concluded that the muscarinic receptors present in the s.n. pars reticulata play a role in the control of posture opposite to that of the nigral GABA receptors.

Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor

1 Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 muscarinic receptor artificially expressed in Chinese hamster ovary (CHO) cells. In the present study we have investigated whether clozapine could activate the rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity, considered as pharmacologically equivalent to the m4 gene product. In addition, we have examined the effect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells. 2 In rat striatum, clozapine (1 nM–10 μM) caused a slight inhibition of forskolin‐stimulated adenylyl cyclase activity, which was not counteracted by 10 μM atropine. On the other hand, clozapine antagonized the inhibitory effect of acetylcholine with a pA2 value of 7.51. Moreover, clozapine (1 μM) failed to inhibit dopamine D1 receptor stimulation of adenylyl cyclase activity, but counteracted the inhibitory effect of carbachol (CCh). Clozapine displaced [3H]‐N‐methylscopolamine ([3H]‐NMS) bound to striatal M4 receptors with a monophasic inhibitory curve and a pKi value of 7.69. The clozapine inhibition was not affected by the addition of guanosine‐5′‐O‐(thio)triphosphate (GTPγS). 3 In intact CHO cells, clozapine inhibited forskolin‐stimulated cyclic AMP accumulation with an EC50 of 31 nM. This effect was antagonized by atropine. CCh produced a biphasic effect on cyclic AMP levels, inhibiting at concentrations up to 1 μM (EC50=50 nM) and stimulating at higher concentrations (EC50=7 μM). Clozapine (0.3–5 μM) antagonized the CCh stimulation of cyclic AMP with a pKi value of 7.47. Similar results were obtained when the adenylyl cyclase activity was assayed in CHO cell membranes. 4 In CHO cells pretreated with the receptor alkylating agent 1‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (10 μM), the maximal inhibitory effect of clozapine on cyclic AMP formation was markedly reduced, whereas the CCh inhibitory curve was shifted to the right with no change in the maximum. 5 As in rat striatum, in CHO cell membranes the displacement of [3H]‐NMS binding by clozapine yielded a monophasic curve which was not affected by GTPγS. 6 Clozapine (10 nM–10 μM) had a small stimulant effect (∼20%) on the binding of [35S]‐GTPγS to CHO cell membranes, whereas CCh caused a 250% increase of radioligand binding. Moreover, clozapine (50 nM–5 μM) antagonized the CCh‐stimulated [35S]‐GTPγS binding with a pA2 value of 7.48. 7 These results show that at the striatal M4 receptors clozapine is a potent and competitive antagonist, whereas at the cloned m4 receptor it elicits both agonist and antagonist effects. Thus, clozapine behaves as a partial agonist, rather than as a full agonist, at the m4 receptor subtype, with intrinsic activity changing as a function of the coupling efficiency of the receptor to effector molecules.

Inhibition of acetylcholine muscarinic M1 receptor function by the M1‐selective ligand muscarinic toxin 7 (MT‐7)

MT‐7 (1–30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M1 receptor, inhibited the acetylcholine (ACh)‐stimulated [35S]‐guanosine‐5′‐O‐(3‐thio)triphosphate ([35S]‐GTPγS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M1 receptor subtype. MT‐7 failed to affect the ACh‐stimulated [35S]‐GTPγS binding in membranes of CHO cells expressing either the M2, M3 or M4 receptor subtype. In N1E‐115 neuroblastoma cells endogenously expressing the M1 and M4 receptor subtypes, MT‐7 (0.3–3.0 nM) inhibited the carbachol (CCh)‐stimulated inositol phosphates accumulation, but failed to affect the CCh‐induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38‐stimulated cyclic AMP accumulation. In both CHO/M1 and N1E‐115 cells the MT‐7 inhibition consisted in a decrease of the maximal agonist effect with minimal changes in the agonist EC50 value. In CHO/M1 cell membranes, MT‐7 (0.05–25 nM) reduced the specific binding of 0.05, 1.0 and 15 nM [3H]‐N‐methylscopolamine ([3H]‐NMS) in a concentration‐dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT‐7 (3 nM) decreased the dissociation rate of [3H]‐NMS by about 5 fold. CHO/M1 cell membranes preincubated with MT‐7 (10 nM) and washed by centrifugation and resuspension did not recover control [3H]‐NMS binding for at least 8 h at 30°C. It is concluded that MT‐7 acts as a selective noncompetitive antagonist of the muscarinic M1 receptors by binding stably to an allosteric site.

Increase in large neutral amino acid transport into brain by insulin.

The administration of oral glucose to fasted rats produced a decline of all large neutral amino acid levels in serum, including that of the free fraction of tryptophan. In addition to this well known effect, it also decreased the brain concentrations of leucine, isoleucine and valine, while increasing those of tryptophan, tyrosine and phenylalanine. The total concentration of large neutral amino acids in serum was decreased by 44%, while it was slightly increased in brain. Analogous results were obtained in 4 rats injected with exogenous insulin. Moreover, the administration of either glucagon or isoproterenol to rats force‐fed with glucose produced a decline in total serum tryptophan concentration proportional to that of the rise in FFA, while it increased free serum tryptophan and brain tryptophan levels. It can be concluded that insulin stimulates the transport of large neutral amino acids from blood to brain and that the level of free serum tryptophan also controls the entry of tryptophan into the brain under the influence of insulin.