Silvana Consolo

Modulatory functions of neurotransmitters in the striatum: ACh/dopamine/NMDA interactions

The striatum is viewed as a structure performing fast neurotransmitter-mediated operations through somatotopically organized projections to medium-size spiny neurons. This view is contrasted with another view that depicts the striatum as a site of diffuse modulatory influences mediated by cholinergic interneurons and by dopamine and N-methyl-D-aspartate receptors. These two operational and organizational modes both contribute, through their mutual interaction, to the function of basal ganglia. Detailed knowledge of the neural mechanisms by which such interactions take place and are expressed into behaviour, can provide new insight into the physiopathology and new clues for therapy of disorders of basal ganglia.

Determination of endogenous acetylcholine release in freely moving rats by transstriatal dialysis coupled to a radioenzymatic assay: effect of drugs.

Abstract: The technique of intracerebral dialysis in combination with a sensitive and specific radioenzymatic method was used for recovery and quantification of endogenous extracellular acetylcholine from the striata of freely moving rats. A thin dialysis tube was inserted transversally through the caudate nuclei, and the tube was perfused with Ringer solution, pH 6.1, at a constant rate of 2 μl min−1. The perfusates were collected at 10‐min intervals. In the presence of 1 and 10 μM physostigmine, acetylcholine release was 4.5 ± 0.02 and 7.3 ± 0.3 pmol/10 min, respectively (not corrected for recovery). The latter concentration of the acetylcholinesterase inhibitor was used in all experiments. Under basal conditions, acetylcholine output was stable over at least 4 h. A depolarizing K+ concentration produced a sharp, reversible 87% increase in acetylcholine output. Both the basal and K+‐stimulated release were Ca2+ dependent. The choline uptake inhibitor hemicholinium‐3 (20 μg intracerebroventricularly) reduced striatal acetylcholine output to 35% of the basal value within 90 min. Scopolamine (0.34 mg/kg s.c.) provoked a sharp enhancement of acetylcholine release of ∼63% over basal values, whereas oxotremorine (0.53 mg/kg i.p.) transiently reduced acetylcholine release by 54%. These results indicate the physiological and pharmacological suitability of transstriatal dialysis for monitoring endogenous acetylcholine release.

Cholinergic-dopaminergic interaction in the striatum: The effect of 6-hydroxydopamine or pimozide treatment on the increased striatal acetylcholine levels induced by apomorphine, piribedil andD-amphetamine

Apomorphine (1 and 2 mg/kg), piribedil (15 and 60 mg/kg) and d-amphetamine (5 and 10 mg/kg) increased rat striatal acetylcholine levels without affecting choline. Pretreatment with pimozide (0.5 mg/kg) completely antagonized the effect of apomorphine and piribedil and by itself markedly decreased striatal acetylcholine levels. d-Amphetamine signigicantly antagonized the effect of pimozide. Nine days after pretreatment with 6-hydroxydopamine plus pargyline, striatal dopamine was decreased by 78% while acetylcholine and choline levels remained unaltered. Under these conditions, the effect of d-amphetamine was completely abolished while apomorphine and piribedil were just as active as in the vehicle-treated group. The results suggest that d-amphetamine acted indirectly to increase striatal acetylcholine levels probably through the release of dopamine and/or noradrenaline, while apomorphine and piribedil acted directly at dopamine receptor sites.

Galanin reduces carbachol stimulation of phosphoinositide turnover in rat ventral hippocampus by lowering Ca2+ influx through voltage-sensitive Ca2+ channels.

Abstract: The 29‐amino‐acid peptide galanin (GAL) caused concentration‐dependent inhibition of the accumulation of 3H‐inositol phosphates (3H‐InsPs) induced by the muscarinic agonist carbachol (CARB; 10‐3‐10‐5M) in the presence of 5 mM lithium, specifically in tissue miniprisms from rat ventral hippocampus. The inhibitory effect of GAL involved the mono‐, bis‐, tris‐, and tetrakisphosphates formed during activation for 2 min of phospholipase C by CARB (1 mM) in the absence of lithium. GAL (1 μM) did not affect α‐adrenergic or serotonergic type 2 receptor‐mediated phosphoinositide (PI) breakdown in the same tissue. GAL by itself neither acted on basal levels of 3H‐InsPs nor affected muscarinic receptors in binding studies. Blockade of the T‐, N‐, and L‐types of voltage‐sensitive calcium channel (VSCC) with 200 μM Cd2+ reduced muscarinic receptor‐mediated PI breakdown by 50% and abolished the inhibitory effect of GAL (1 μM). Reduction of the extracellular Ca2+ concentration from 1.3 mM to 0.49 μM abolished the GAL inhibition of CARB‐stimulated PI hydrolysis. Ca2+ influx promoted by 18 mM K+ depolarization or by 1 μM Bay K 8644, a selective agonist of the L‐type VSCC, prevented the inhibitory effect of GAL. Blockade of the L‐type VSCC with nifedipine (1 μM) potentiated the inhibitory effects of GAL without affecting muscarinic stimulation of PI breakdown. The neurotoxin ω‐conotoxin (2 μM), a blocker of both L‐ and N‐types of VSCC, by itself reduced CARB‐mediated breakdown of PIs by ∼25%, and when it was added before GAL (1 μM) there was no summation of the two individual inhibitory effects, a result suggesting a common site of action for GAL and ω‐conotoxin. The data presented thus indicate that GAL modulation of muscarinic stimulation of the phospholipase C activity is mediated by a reduction of Ca2+ entry through VSCCs, presumably of the N type.

Evidence of an interaction between serotoninergic and cholinergic neurons in the corpus striatum and hippocampus of the rat brain.

Abstract The existence of an interaction between serotoninergic and cholinergic neurons in the brain has been investigated by studying the effects of quipazine and d -fenfluramine on regional brain acetylcholine in various experimental conditions. Quipazine, at a dose of 10 mg/kg, i.p., significantly increased the levels of acetylcholine in the striatum and hippocampus but not in the telencephalon and brain stem. The striatal increase was not significantly modified by electrolytic lesions placed in the midbrain raphe nuclei, an important site of origin of serotonin-containing neurons in the brain. On the other hand, pretreatment with serotonin antagonists such as methergoline and cinanserin or with parachlorophenylalanine, a serotonin synthesis blocker, prevented the increase of striatal acetylcholine induced by quipazine. Impairment of nigrostriatal dopaminergic mechanisms by local application of 6-hydroxydopamine or by pretreatment with alpha-methylparatyrosine did not modify the effect of quipazine on acetylcholine. The quipazine-induced increase in hippocampal acetylcholine was instead completely blocked by an electrolytic lesion of the nucleus medianus raphe. d -Fenfluramine also significantly increased striatal acetycholine, this effect being completely prevented by parachlorophenylalanine pretreatment. These findings are compatible with the hypothesis that serotoninergic neurons originating in the raphe nuclei may normally serve to inhibit cholinergic neurons in two areas of the rat brain, i.e. the corpus striatum and the hippocampus.

Increase in striatal acetylcholine by picrotoxin in the rat: evidence for a gabergic-dopaminergic-cholinergic link.

Picrotoxin, 2 mg/kg i.p., a GABA receptor blocking agent, increased rat striatal acetylcholine content by approximately 70% without altering the levels of this amine in the cerebral hemispheres, mesencephalon, diencephalon, hippocampus and cerebellum. Striatal choline levels were concomitantly decreased by about 25%. This dose of picrotoxin also increased striatal homovanillic acid levels by about 30%, an effect which was not antagonized by pretreatment with the dopamine receptor stimulating agent, piribedil. Picrotoxin did not affect striatal choline-O-acetyltransferase or cholinesterase activity after in vitro incubation. The action of picrotoxin on striatal acetylcholine levels was partially antagonized by pimozide and completely blocked by alpha-methyl-para-tyrosine pretreatment while the intraventricular injection of 6-hydroxydopamine was without effect. Convulsions were not prevented by any of these treatments. The results are interpreted as follows: picrotoxin released dopamine through disinhibition of the dopaminergic neurons as a result of blockade of gabergic receptors. The increased dopaminergic activity inhibited cholinergic neurons and lead to an increase in acetylcholine content. The data thus provide evidence for a possible gabergic (inhibitory)--dopaminergic (inhibitory)-cholinergic link terminating in the striatum.

Acetylcholine, choline and choline acetyltransferase activity in the developing brain of normal and hypothyroid rats.

Abstract— Acetylcholine, choline and choline acetyltransferase activity were measured in the whole brains of normal and hypothyroid rats during development. At 1 day postpartum, brain acetylcholine was 73 per cent of adult levels. Propylthiouracil‐induced hypothyroidism up to age 20 days did not alter brain acetylcholine concentrations, but at 30 days resulted in significantly decreased levels. At day 1, brain choline was 20 per cent higher than adult levels and decreased between days 8 and 10. In hypothyroid rats this phenomenon did not occur until days 15–20. At day 1 postnatally, choline acetyltransferase activity was only 7 per cent of adult levels, then between days 5 and 20 rose to 77 per cent of adult levels. Beginning at day 8, hypothyroidism resulted in significantly decreased enzyme levels. This effect could be reversed at day 17 by concurrent tri‐iodothyronine substitution therapy. In hypothyroid rats, maximum brain choline acetyltransferase activity was 30 per cent less than normal adult levels.

Endogenous Dopamine Facilitates Striatalln Vivo Acetylcholine Release by Acting on D1 Receptors Localized in the Striatum

Abstract: Intrastriatal application of the D1 antagonist SCH 23390 by two procedures, reverse dialysis (20 μM) and local injection (0.45 nmol per striatum), elicited a reduction in acetylcholine (ACh) release superimposable on that induced by systemic administration. The novel selective D1 antagonist SCH 39166 produced a similar decreasing effect on striatal ACh release on local injection (0.45 nmol per striatum). On the other hand, local application of SCH 23390 into the frontal cortices (0.45 nmol per side) failed to alter striatal ACh overflow, indicating that the drug does not diffuse out of its injection site to any significant extent. The dopamine release inducer d‐amphetamine (2 mg/kg s.c.) and the dopamine uptake inhibitor cocaine raised ACh release like the D1 agonists. These effects were completely blocked by 10 μM SCH 23390 applied by reverse dialysis. The results suggest that D1 receptors regulating ACh release are located in the striatum.

The Cerebral Cortex and Parafascicular Thalamic Nucleus Facilitate In vivo Acetylcholine Release in the Rat Striatum through Distinct Glutamate Receptor Subtypes

Electrical stimulation (ten pulses of 0.5 ms, 10 V applied over 10 s at 10 Hz, 140 pA) delivered bilaterally to the prefrontal cortex or the parafascicular thalamic nucleus of freely moving rats facilitated acetylcholine release in dorsal striata, assessed by trans‐striatal microdialysis. The facilitatory effects were blocked by coperfusion with 5 μM tetrodotoxin, suggesting that the release was of neuronal origin. The response of the striatal cholinergic neurons to prefrontal cortical stimulation was short‐lived and required a longer period of stimulation (20 min) than the response to thalamic stimulation (4 min) to reach maximal effect. The α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)/kainate glutamatergic receptor antagonist 6,7‐dinitroquinoxaline‐2,3‐dione [DNQX; 12 nmol per side, intracerebroventricularly (i.c.v.)] and the AMPA antagonist 6‐nitro‐7‐sulphamoylbenzo(f)quinoxaline‐2,3‐dione (NBQX; 12 nmol per side, i.c.v. or 12.8 μM infused into the striatum), but not the NMDA‐type receptor antagonist MK‐801 (0.2 mg/kg, i.p.), abolished the facilitatory effect on striatal acetylcholine release evoked by stimulation of the prefrontal cortex. By contrast, DNQX or NBQX did not prevent the increase in striatal acetylcholine release evoked by parafascicular nucleus stimulation, but MK‐801, in accordance with previous results, did so. MK‐801 by itself lowered striatal acetylcholine output while DNQX and NBQX did not. The results provide in vivo evidence that the cerebral cortex facilitates cholinergic activity in the dorsal striatum apparently through the non‐tonic activation of AMPA‐type glutamatergic receptors while the parafascicular nucleus does this through tonic activation of NMDA receptors. Both glutamate receptor types are probably located in the striatum. The overall results suggest that the two pathways operate independently to regulate striatal cholinergic activity through distinct mechanisms.

Role of vesicular dopamine in the in vivo stimulation of striatal dopamine transmission by amphetamine: evidence from microdialysis and Fos immunohistochemistry.

The role of vesicular and newly synthesized dopamine in the action of amphetamine was investigated by studying the effect of reserpine and alpha-methyl-p-tyrosine pretreatment on amphetamine-induced changes in extracellular dopamine and acetylcholine, estimated by brain microdialysis, and on c-fos expression, estimated by quantitative immunohistochemistry of the Fos antigene, in the dorsal caudate-putamen of rats. Blockade of dopamine synthesis by alpha-methyl-p-tyrosine pretreatment (1 or 2 h) only partially prevented the increase in extracellular dopamine concentrations elicited by 0.5 and 2 mg/kg s.c. of amphetamine. Inactivation of vesicular amine uptake by reserpine pretreatment (3 h) reduced the increase in extracellular dopamine by 2 mg/kg but not by 0.5 mg/kg of amphetamine. Combined pretreatment with reserpine (3 h) and alpha-methyl-p-tyrosine (1 h) drastically reduced the increase in extracellular dopamine by both doses of amphetamine (0.5 and 2 mg/kg s.c.). alpha-Methyl-p-tyrosine pretreatment reduced c-fos expression stimulated by amphetamine (2 mg/kg) in the dorsomedial and dorsolateral caudate-putamen while reserpine pretreatment reduced it only in the dorsolateral caudate-putamen. Amphetamine (2 mg/kg s.c.) stimulated acetylcholine release but this effect was not modified by reserpine or alpha-methyl-p-tyrosine pretreatment. The results indicate that blockade of dopamine synthesis, by itself, is insufficient to prevent the stimulation of dopamine transmission by amphetamine and, conversely, that inactivation of vesicular dopamine significantly reduces amphetamine effects at pre- and postsynaptic levels. Therefore, vesicular dopamine appears to contribute to the stimulation of dopamine transmission elicited by amphetamine in the dorsal caudate-putamen.