Release of newly synthesized 3H-dopamine in the striatum: an adaptation of the push-pull cannula method to awake restrained and anesthetized rats

The release of newly synthesized 3H-dopamine (3H-DA) was measured in the rat striatum superfused, through a push-pull cannula, with a physiological medium enriched in 3H-tyrosine. The level of spontaneous 3H-DA release was dependent on the topographical localisation of the cannula in the striatum (anterior parts displayed higher levels than posterior ones) and on the anesthetic state (halothane anesthetized rats demonstrated higher levels than awake ones). Inhibition of DA inactivation processes by local application of benztropine (a DA reuptake inhibitor, 10(-6) M) or by IV administration of pargyline (a MAO inhibitor, 100 mg/kg) enhanced the detectable outflow of 3H-DA from the striatum in both halothane anesthetized and awake rats. Local application of D-amphetamine (10(-5) M) or acetylcholine (5 X 10(-5) M) in the presence of eserine (5 X 10(-5) M) evoked respectively a fivefold and a 30% increase in spontaneous 3H-DA release in halothane anesthetized rats. Inhibition of the firing of dopaminergic neurons by IV injection of gamma-hydroxybutyrate (400 mg/kg) produced a 30% decrease in striatal 3H-DA release. The present results demonstrate that the push-pull cannula method is suitable for the study of DA release in both the anesthetized and the awake rat.     

Apomorphine and haloperidol effects on striatal H-dopamine release in anesthetized, awake restrained and freely moving rats

The ability of apomorphine (APO) and haloperidol (HAL) to affect the spontaneous release of newly synthesized ³H-DA in the striatum was studied in halothane anesthetized, gallamine paralyzed, awake restrained and freely moving rats. The striatum was continuously superfused through a push-pull cannula with a physiological medium enriched in ³H-tyrosine. Basal levels of ³H-DA release were different in the four experimental models: highest in halothane anesthetized rats, intermediate in awake restrained and gallamine treated rats and lowest in freely moving rats. In all experimental models IV or SC injection of APO (1 mg/kg) inhibited the release of ³H-DA (30–50%) from 15 to 90 min following its administration. In awake restrained and freely moving rats, stereotyped behaviour was observed for one hour following the APO injection. In halothane anesthetized rats the inhibitory effect of APO on ³H-DA release was prevented by pretreatment with HAL (2 mg/kg IV). Injection of HAL (2 mg/kg IV or SC) failed to enhance the release of ³H-DA in anesthetized and awake restrained rats, whilst a long-lasting increase in ³H-DA release was observed in gallamine treated and freely moving animals (55% and 120% respectively). However, catalepsy was observed in both restrained and freely moving rats. It is concluded that the modifications of ³H-DA release produced by HAL but not those produced by APO are dependent on the experimental model used, a fact possibly related to the different sites of action of these two drugs.     

In vivo studies on the dopamine re-uptake mechanism in the striatum of the rat: Effects of benztropine, sodium and ouabain

We used the push-pull perfusion technique to study the in vivo changes in dopamine (DA) levels in the rat striatum in response to treatments which could affect DA re-uptake into the nigrostriatal DA terminals. Benztropine (10(-6) M), a potent DA uptake inhibitor induced a 1.7-fold increase in DA levels in the perfusates compared to basal levels. Perfusion with a Na+-free medium in which Na+ was replaced with either Tris-Cl or choline-Cl in equimolar proportions induced respectively 6.5- and 8.5-fold increases in DA levels in the perfusates. Perfusion of media containing NaCl:Tris-Cl (50:50) or NaCl:choline-Cl (50:50) did not significantly alter the levels of DA in the perfusates. Ouabain (10(-6) M) did not significantly alter DA levels but at a concentration of 10(-4) M, there was a 5.3-fold increase in DA levels in the perfusates compared to basal levels. These results thus demonstrate that the raised DA levels in the extracellular space in response to benztropine is due to the action of the drug in blocking the uptake of DA. The dependence of the uptake mechanism on the presence of Na+ in the external medium and hence on metabolic energy (Na pump) is clearly demonstrated. However, the massive elevation of DA levels under these conditions cannot be due solely to an inhibition of DA uptake but to the carrier-mediated DA exit from cytoplasmic stores resulting from a running down of the ionic gradient.     

Changes in dopamine and acetylcholine in striatum of the awake rat after chronic treatment with a dopamine uptake blocker

The effects of chronic treatment with a dopamine uptake blocker on dopamine and acetylcholine extracellular concentrations in striatum of the awake rat was studied. Male Wistar rats received daily injections (i.p.) of the dopamine uptake blocker nomifensine (10 mg/kg) during 22 days. Control group was injected with vehicle (saline). Microdialysis experiments were performed on days 1, 8, 15 and 22 of treatment. Nomifensine injections increased extracellular concentration of dopamine in striatum in all days of treatment without differences among days. In contrast, acetylcholine levels showed no changes in days 1 and 8 but increased in days 15 and 22 of treatment. These results shows that chronic treatment with a dopamine uptake inhibitor, nomifensine, has no effects on dopamine release but it increases acetylcholine release in striatum of the awake rat. These results would help to further understand the effects of chronic dopamine uptake inhibition.     

Acetylcholine release from fetal tissue homotopically grafted to the motoneuron-depleted lumbar spinal cord. An in vivo microdialysis study in the awake rat

Grafts of spinal cord (SC) tissue can survive and develop into the severed SC, but no conclusive data are available concerning the functional activity of transplanted neurons. In the present study, suspensions of prelabeled embryonic ventral SC tissue were grafted to the lumbar SC of rats with motoneuron loss induced by perinatal injection of volkensin. Eight to ten months post-grafting, acetylcholine (ACh) release was measured by microdialysis in awake rats, under either basal or stimulated conditions. In normal animals, baseline ACh output averaged 1.6 pmol/30 microl, it exhibited a 4-fold increase after KCl-induced depolarization or handling, and it was completely inhibited by tetrodotoxin administration. Moreover, ACh levels did not change following acute SC transection performed under anesthesia during ongoing dialysis, suggesting an intrinsic source for spinal ACh. Treatment with volkensin produced a severe (>85%) motoneuronal loss accompanied by a similar reduction in baseline ACh release and almost completely abolished effects of depolarization or handling. In transplanted animals, many motoneuron-like labeled cells were found within and just outside the graft area, but apparently in no case were they able to extend fibers towards the denervated muscle. However, the grafts restored baseline ACh output up to near-normal levels and responded with significantly increased release to depolarization, but not to handling. The present findings indicate that spinal neuroblasts can survive and develop within the motoneuron-depleted SC and release ACh in a near-normal, but apparently non-regulated, manner. This may be of importance for future studies involving intraspinal stem cell grafts.     

Agonists of A1 and A2A adenosine receptors attenuate methamphetamine-induced overflow of dopamine in rat striatum

The effect of adenosine receptor agonists on the release of striatal dopamine (DA), induced by repeated doses of methamphetamine (MTH), was evaluated. Rats received three injections of MTH (5 mg/kg i.p.) at 2-h intervals. The release of DA in the striatum was measured by a microdialysis in freely moving animals. The agonist of adenosine A₁ receptor, N⁶-cyclopentyladenosine (CPA) and the agonist of adenosine A_2A receptor, 2-[p-(carboxy-ethyl)phenylethylamino]-5′-N-ethylcarboxyamidoadenosine (CGS 21680), either of them being infused locally into the striatum at concentrations of 50 and 100 μM, produced decreases in the extracellular DA level during exposure to MTH, and a weaker effect on the levels of DOPAC and HVA. The above effects were reversed by the specific antagonists of adenosine A₁ and A_2A receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 3,7-dimethyl-1-propargylxanthine (DMPX), respectively. Our results indicate that both the A₁ and A_2A adenosine receptors appear to be involved in reducing the excessive release of DA in the striatum; furthermore, they suggest a neuroprotective role of adenosine in MTH neurotoxicity.     

Iontophoresis of amphetamine in the neostriatum and nucleus accumbens of awake, unrestrained rats

When administered systemically to ambulant animals, amphetamine (AMPH) has both excitatory and inhibitory effects on single-unit activity in the neostriatum and nucleus accumbens. To determine the extent to which these results reflect a direct action of the drug, AMPH was applied iontophoretically to neostriatal and accumbal neurons under naturally occurring behavioral conditions. AMPH dose-dependently (5–40 nA) inhibited the vast majority of spontaneously active units. The inhibition, which was evident at low ejection currents (5–10 nA), had relatively short onset (4–12 s) and offset (6–24 s) latencies, and was positively correlated with basal firing rate. Even stronger dose-dependent inhibitory responses were recorded when neurons having no or a very low rate of spontaneous activity were tonically activated by continuous, low-current applications of glutamate (Glu). Systemic injection of either SCH-23390 (0.1 mg/kg) or haloperidol (0.2 mg/kg), relatively selective D₁ and D₂ receptor antagonists, respectively, blocked the AMPH-induced inhibition. Prolonged AMPH iontophoresis (2–3 min; 5–30 nA) inhibited both spontaneous impulse activity and Glu-induced excitations, resulting in a complete blockade of the Glu response at relatively high AMPH ejection currents (≥20 nA). Taken together, these results suggest that although dopamine is largely responsible for the inhibitory effects of iontophoretic AMPH, dopamine alone cannot account for the complex response of neostriatal and accumbal neurons to systemic AMPH administration.     

Persistent sensitization of dopamine neurotransmission in ventral striatum (nucleus accumbens) produced by prior experience with (+)-amphetamine: a microdialysis study in freely moving rats

In humans the repeated use of amphetamine (AMPH) produces a hypersensitivity to the psychotogenic effects of AMPH that persists for months to years after the cessation of drug use. To explore the neurobiological basis of this phenomenon the long-term effects of dextroamphetamine [+)-AMPH) on brain monoamines and behavior were studied in an animal model of AMPH psychosis. An escalating dose pretreatment regimen (from 1 to 10 mg/kg over 5 weeks) was used to mimic the pattern of drug use associated with the development of addiction and AMPH psychosis. The effect of pretreatment with AMPH on dopamine (DA) release in the ventral striatum (nucleus accumbens) was determined by measuring the extracellular concentrations of DA and DA metabolites using in vivo microdialysis, both before and after an AMPH challenge. The postmortem tissue concentrations of DA, serotonin and their metabolites were measured to determine if this treatment was neurotoxic. Escalating doses of (+)-AMPH were not neurotoxic, and 25-30 days after the cessation of drug treatment animals showed relatively normal levels of spontaneous motor activity across the day-night cycle. However, AMPH pretreatment produced robust behavioral sensitization. Animals showed a marked hypersensitivity to the motor stimulant effects of an AMPH challenge, even after 15-20 days of withdrawal. Most importantly, this hyperdopaminergic behavioral syndrome was accompanied by significantly elevated DA release in the ventral striatum. In contrast, AMPH pretreatment had no effect on the basal extracellular concentrations of DA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous microdialysis and amphetamine infusion in the nucleus accumbens and striatum of freely moving rats: Increase in extracellular dopamine and serotonin

To test the effects of systemic and local amphetamine on dopamine and serotonin release in freely moving rats, guide cannulas were implanted in the nucleus accumbens and ventral striatum for removable 200 mu microdialysis probes. Comparing 45 min samples before and after IP amphetamine (2 mg/kg), dopamine (DA) in dialysate from the accumbens increased from a baseline of 3 pg/20 microliters to 11 pg/20 microliters whereas dopamine metabolites, DOPAC and HVA decreased. This was probably due to block of DA reuptake and inhibition of monoamine oxidase, MAO. Accumbens serotonin increased from a baseline of 8 to 11 pg/20 microliters. Changes in the ventral striatum were similar. In the second experiment, microdialysis was performed before and after local injection of amphetamine (4 micrograms) to reveal effects of amphetamine in the terminal area only instead of the whole brain. DA in the accumbens increased from 11 to 147 pg per sample; serotonin, from 11 to 107. The effect was even larger in the ventral striatum. DA increased from 12 to 409 pg/20 microliters; serotonin, 3 to 139. To avoid handling the rat or disturbing brain tissue in experiment three, amphetamine was infused via the microdialysis probe during ongoing dialysis sampling. Perfusate containing amphetamine was switched into the flow line which allowed roughly 10% or 4 micrograms to diffuse out into the extracellular space. During this 20 min, extracellular DA in the accumbens increased from a baseline of 10 pg/20 microliters to 300 pg/20 microliters sample. Serotonin increased from barely detectable to 60 pg/20 microliters. DOPAC decreased significantly; HVA and 5HIAA drifted lower.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrastriatal injection of [H]dopamine through a chronic cannula to produce rotation: Distribution and concentration of the tracer in specific brain regions

As others have shown, rats rotated contralaterally following unilateral intrastriatal injections of dopamine. However, the concentration of the injection solution necessary to elicit rotation in our and others' studies was high, 50 μg/0.5 μl (10,000 times endogenous concentration) and the latency to turn was 20–60 min. Thus, we injected [³H]dopamine ([³H]DA) to determine the concentration of the injected DA in the striatum and to determine if the long latency to turn was related to spread of DA over time to distal nuclei such as the globus pallidus, subthalamic nucleus and substantia nigra. Microinjections (0.5 μl) of [³H]DA were made through a chronic striatal cannula in pargyline-treated freely moving rats. Determinations of [³H]DA concentrations in individual brain areas were made at 20 min and 60 min following intrastriatal injection. The major accumulation of [³H]DA and its metabolites was in the dorsal striatum, near the cannula tip, and in the sensorimotor cortex where the cannula passed through to the striatum. The concentration of [³H]DA in the dorsal striatum was only 10–100 times the endogenous concentration, depending upon the cannula injection system used. The 60 min latency to rotate could not be attributed to spread of DA to the subthalamic n., entopeduncular n. and substantia nigra. Rotation following an intrastriatal injection of DA was dependent upon the dorsal striatal tissue concentration of DA. The ventral striatum and globus pallidus were also possibly involved. The highest striatal [³H]DA concentrations were, at most, 10 times greater than the concentrations of systemically injectedl-[³H]DOPA, and, at the lower concentrations which produced twisting and weak rotation, the DA concentration was similar to that seen with systemicl-DOPA.     

Involvement of cholinergic neurons in the release of dopamine elicited by stimulation of μ-opioid receptors in striatum

The involvement of striatal cholinergic neurons in the release of dopamine (DA) elicited by the (μ-opioid receptor agonist DAGO [d-Ala², NMePhe⁴-Gly⁵(ol)]enkephalin) was explored. The striatal release of DA was measured by microdialysis in rats anesthetized with chloral hydrate. When infused in the striatum, through the microdialysis probe, DAGO increased the extracellular levels of DA. The previous injection in striatum of AF 64-A, a toxin for cholinergic neurons, or the concomitant infusion of the M₂-muscarinic antagonist methoctramine abolished the effect of DAGO on the DA release. It is concluded that stimulation of μ-opioid receptors, by inhibiting the acetylcholine release which stimulates tonically M₂-muscarinic receptors likely associated with dopaminergic nerve endings, indirectly increases the striatal DA release.     

Intrastriatal injection of [3H]dopamine through a chronic cannula to produce rotation: Distribution and concentration of the tracer in specific brain regions

As others have shown, rats rotated contralaterally following unilateral intrastriatal injections of dopamine. However, the concentration of the injection solution necessary to elicit rotation in our and others' studies was high, 50 μg/0.5 μl (10,000 times endogenous concentration) and the latency to turn was 20–60 min. Thus, we injected [³H]dopamine ([³H]DA) to determine the concentration of the injected DA in the striatum and to determine if the long latency to turn was related to spread of DA over time to distal nuclei such as the globus pallidus, subthalamic nucleus and substantia nigra. Microinjections (0.5 μl) of [³H]DA were made through a chronic striatal cannula in pargyline-treated freely moving rats. Determinations of [³H]DA concentrations in individual brain areas were made at 20 min and 60 min following intrastriatal injection. The major accumulation of [³H]DA and its metabolites was in the dorsal striatum, near the cannula tip, and in the sensorimotor cortex where the cannula passed through to the striatum. The concentration of [³H]DA in the dorsal striatum was only 10–100 times the endogenous concentration, depending upon the cannula injection system used. The 60 min latency to rotate could not be attributed to spread of DA to the subthalamic n., entopeduncular n. and substantia nigra. Rotation following an intrastriatal injection of DA was dependent upon the dorsal striatal tissue concentration of DA. The ventral striatum and globus pallidus were also possibly involved. The highest striatal [³H]DA concentrations were, at most, 10 times greater than the concentrations of systemically injectedl-[³H]DOPA, and, at the lower concentrations which produced twisting and weak rotation, the DA concentration was similar to that seen with systemicl-DOPA.     

Injection of the protein kinase C inhibitor Ro31-8220 into the nucleus accumbens attenuates the acute response to amphetamine: tissue and behavioral studies

The ability of amphetamine to produce heightened locomotor activity is thought to be due to its ability to enhance dopamine release from mesolimbic dopamine neurons. The mechanism by which amphetamine increases dopamine release is not well understood, but is thought to involve exchange diffusion with synaptosomal dopamine through the dopamine transporter. We recently reported that amphetamine-mediated dopamine release in the striatum is also dependent on protein kinase C activity. In the current study, we investigated the role of protein kinase C activity in the acute neurochemical and behavioral response to amphetamine in the nucleus accumbens. Consistent with previous results in the striatum, amphetamine-stimulated dopamine release from nucleus accumbens tissue was inhibited by the specific protein kinase C inhibitor Ro31-8220, but not by the relatively inactive analog bisindoylmaleimide V. In addition, the effects of protein kinase C activity on the acute behavioral response to amphetamine was examined by injecting Ro31-8220 into the nucleus accumbens 15 min prior to intra-accumbens amphetamine. Pretreatment with Ro31-8220 attenuated the motor-stimulant effects of intra-accumbens amphetamine relative to control subjects pretreated with vehicle. Bisindoylmaleimide V did not significantly inhibit the motor-stimulant effects of intra-accumbens amphetamine. These results suggest that the action of amphetamine in the nucleus accumbens in increasing dopamine release and locomotor activity is dependent on protein kinase C activity.     

Dopamine in the rabbit retina and striatum: Diurnal rhythm and effect of light stimulation

Summary In rabbits, dopamine levels in the retina, but not in the caudate nucleus, showed clear diurnal rhythm, with high values seen in the light phase. Thirty min exposition of dark-adapted rabbits to day-light produced no changes in dopamine levels in the retina. In rabbits treated with -methyl-p-tyrosine, the same light exposition decreased the retinal amine level by 18%, while stimulation with intensive, flickering light significantly decreased the retinal dopamine content by 36%. Experiments performed at noon and midnight, under light or dark conditions, showed the retinal dopamine levels to be very similar in groups kept either at light or dark, irrespective of the time of the day, although in animals deprived of light the amine levels were clearly lower than in those exposed to light, both at noon and midnight. Under all experimental conditions there were no significant changes in dopamine level and utilization in the caudate nucleus. The isolated and superfused retina (preloaded with [³H]-dopamine), when stimulated with flashes of white light (2 Hz, 10 min), released [³H]-radioactivity in a Ca²⁺-dependent manner. It is concluded that in rabbits, light enhances dopamine levels and utilization selectively in the retina, and the observed diurnal changes in the amine metabolism are dependent on the presence or absence of light, and not on the time of the day. The proposed physiological role(s) of the retinal dopaminergic mechanisms is discussed.     

Monoamine neurotransmitters and amino acids in the cerebrum and striatum of immature rats with kaolin-induced hydrocephalus

Hydrocephalus is characterized by enlargement of the cerebral ventricles. The behavioral disturbances are, in some cases, rapidly reversible by surgical treatment suggesting that there may be a functional impairment of neurons. Hydrocephalus was induced in 3-week old rats by kaolin injection into the cisterna magna. Parietal cerebrum and striatum content of monoamine neurotransmitters and amino acids were assayed by high performance liquid chromatography (HPLC), 1, 2, or 4 weeks after induction of hydrocephalus. The ventricles exhibited progressive enlargement which was partially reversed by surgical treatment. Cerebral water content was increased at all stages. Increased levels of cerebral aspartate and glutamate suggest that there is the potential for excitatory neurotoxicity. The increase in cerebral taurine correlated negatively with the increase in water content. Cerebral concentrations of norepinephrine and serotonin, and its metabolite 5-HIAA, were increased at 1 and 2 weeks suggesting an increase in their turnover during the early stages of ventricular dilatation. Dopamine and its metabolite DOPAC were transiently diminished in the striatum at 1 and 2 weeks, respectively, suggesting that axonal projections from the brainstem may be impaired. We conclude that the effect of hydrocephalus on amino acids and monoamines varies regionally. Due to increased water content, there may be dilution effects in whole tissue, therefore, it is important to make determinations on the basis of protein content.     

The involvement of dopamine in nociception: the role of D1 and D2 receptors in the dorsolateral striatum

Determination of the neuroanatomical and neurochemical factors that contribute to nociception is an essential element in the study and treatment of pain. Several lines of evidence have implicated nuclei and neurotransmitters within the basal ganglia in nociception. For example, previous studies have shown that dopamine receptors in the striatum are involved in acute nociception, however, it remains to be determined if dopamine receptors in the dorsolateral striatum are involved in persistent nociception. The purpose of the present study was therefore to determine whether activation or antagonism of dopamine receptors in the dorsolateral striatum influences the nociceptive responses of rats in the formalin test, a model of persistent pain. It was found that micro-injection of the non-selective dopamine antagonist haloperidol into the dorsolateral striatum increases formalin-induced nociception whereas injection of the non-selective dopamine agonist apomorphine reduces formalin-induced nociception. Injection of the D₁ antagonist SCH23390 or the D₁ agonist SKF38393 does not affect formalin-induced nociception. In contrast, injection of the D₂ antagonist eticlopride enhances formalin-induced nociception, whereas injection of the D₂ agonist quinpirole reduces formalin-induced nociception. These results provide additional evidence that dopamine receptors in the striatum are involved in nociception. Furthermore, this study strongly suggests that D₂, but not D₁, dopamine receptors in the dorsolateral striatum are involved in modulation of persistent nociception.     

Extracellular glutamate and dopamine measured by microdialysis in the rat striatum during blockade of synaptic transmission in anesthetized and awake rats

We investigated the effect of high dose tetrodotoxin (TTX) on microdialysis measurements of extracellular striatal glutamate and dopamine in normal female rats. Both halothane-anesthetized rats with acutely implanted microdialysis probes and awake rats with microdialysis probes implanted for 24 h were tested. Glutamate levels in awake rats were 45% higher than those of anesthetized rats. Extracellular glutamate remained TTX-insensitive irregardless of TTX concentration, anesthesia, or time lapsed after probe implantation. In contrast, TTX reduced dialysate dopamine in all TTX concentrations tested. We speculate that the lower glutamate levels in anesthetized rats reflect the effect of anesthesia. Because glutamate is involved, either as a reactant or a product in a variety of reactions critical to intermediary metabolism in the brain, basal dialysate glutamate levels might indirectly reflect brain metabolism. Further, we conclude that extracellular glutamate collected during non-stimulated conditions is TTX-insensitive. The fact that glutamate levels are TTX-independent does not rule out that glutamate is synaptic in origin but rather demonstrates that it is not nerve impulse-dependent. However, the brain interstitial glutamate pool accessible to the microdialysis probe during control conditions is most likely isolated from the synapse, and therefore does not impose a neurotoxic potential.     

Estimation of the number of somatostatin neurons in the striatum: An in situ hybridization study using the optical fractionator method

Somatostatin-containing neurons of the striatum constitute fewer than 5% of the total neuronal population. Their involvement in the feedforward inhibition of the spiny projection neurons, the modulation of other interneurons, and the regulation of regional blood flow indicates that this small population of neurons plays an important role in the processing of information in the striatum. As a first step in developing a quantitative structural framework within which a more rigorous analysis can be made of the functional circuitry of the striatum, we used modern unbiased stereological techniques to make estimates of the total number of neurons expressing mRNA for somatostatin in the striatum of rats. The strategy developed involved the application of the optical fractionator technique to relatively thick tissue sections that were hybridized in situ with a relatively short oligonucleotide probe conjugated to a nonradioactive reporter molecule. The approach is generally applicable to other subpopulations of in situ hybridized cells in other parts of the brain and can provide a link between molecular neurobiology and stereology. The mean total number of neurons on one side of the striatum was estimated to be 21,300. An analysis of the sampling scheme indicated that counting no more than 200 neurons in a systematic sample of not more than 15 sections per individual results in an estimate with a precision that is more than sufficient for comparative and experimental studies. The issues that must be considered when analyzing in situ hybridized tissue with modern stereological methods, the interpretive caveats inherent in the resulting data, and the unique perspectives provided by data like that presented here for striatal somatostatin neurons are discussed. © 1996 Wiley-Liss, Inc.     

In vivo and in vitro studies on the regulation of cholinergic neurotransmission in striatum, hippocampus and cortex of aged rats

Young (3 months) and senescent (23 months) rats were challenged with oxotremorine both in vivo, to determine its effects on acetylcholine content in hemispheric regions, and in vitro, to assess its action on K⁺-evoked release of ACh from brain synaptosomes. The drug failed to inhibit KCl-induced [³H]ACh release from the P₂ fraction of striatal and hippocampal homogenates of the senescent animals, whereas it was less efficient in increasing striatal ACh content. In contrast, oxotremorine was still able to stimulate an increase in ACh in the hippocampus and cerebral cortex of the aged rats to the same extent as it did in the young ones. The [³H]ACh output from striatal synaptosomes was lower in old rats with respect to young ones at low KCl depolarizing concentrations but was equal in the two groups at a high depolarizing concentration. In the hippocampus of the senescent rats, the release was significanly lower at each concentration of KCl used, resulting in a parallel downward-shift in the concentration-release plot. We also measured cholinergic muscarinic receptor binding in rat hemispheric regions using the radioligand [³H]dexetemide, a classical non-selective muscarinic receptor antagonist. It was found, in conformity with some of the literature, that receptor binding was decreased by about 32% in striatum of aged female rats as compared to younger rats. Changes were not observed in cortex and hippocampus. Analysis of the binding data indicated that the observed decrease in specific ligand binding was due to a decrease in the number of binding sites without a change in affinity. The results favor, once again, the cholinergic hypothesis for geriatric dysfunction. The possibility that the functional state of the cholinergic neuron depends also on a balance between the cholinergic system and other neurotransmitters known to regulate cholinergic activity is discussed.     

Dopamine-releasing action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP) in the neostriatum of the rat as demonstrated in vivo by the push-pull perfusion technique: dependence on sodium but not calcium ions

This study examined the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its metabolite, 1-methyl-4-phenylpyridine (MPP+) on the levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in push-pull perfusates of the striatum in chloral hydrate-anaesthetized rats. In control animals the levels of DA and DOPAC remained stable for at least 6 h and responded rapidly to a depolarizing stimulus of 25 mM K+. This K+-induced DA release was Ca2+-dependent since no stimulation was observed when the striatal sites were perfused with high K+ in a Ca2+-free medium containing 2 mM EGTA thus verifying that the striatal sites were functionally active. MPTP (0.025 and 0.05 microgram/microliter) stimulated DA release and inhibited DOPAC output in a dose-related manner. MPP+ (0.01, 0.025 and 0.05 microgram/microliter) produced a more robust dose-dependent increase in DA levels in the perfusates; however, the level of suppression of DOPAC was similar to that in response to MPTP. The effect of MPP+ on DA release was attenuated by 10(-6) M benztropine, the DA re-uptake blocker and completely inhibited by 10 micrograms/kg i.p. benztropine and 10(-4) M ouabain, the Na+, K+-ATPase (Na pump) inhibitor. However, although these substances prevented the MPP+-induced release of DA, the levels of DOPAC in the perfusates did not recover and remained completely suppressed suggesting that MPP+ may inhibit extraneuronal rather than intraneuronal monoamine oxidase (MAO). Perfusion of the striatal sites with a Ca2+-free medium containing 2 mM EGTA did not prevent the MPP+-induced DA release indicating that MPP+ does not release DA from the striatal DA terminals by the Ca2+-dependent process of exocytosis. The responses of DA and DOPAC to 25 mM K+ were markedly suppressed in animals treated with MPTP and MPP+, these effects being most severe with the highest dose of MPP+. Moreover, this suppression of the K+-induced responses persisted in animals perfused with MPP+ in the presence of benztropine or ouabain, thus suggesting that MPP+ may have potent deleterious membrane effects. These studies have provided the first direct in vivo demonstration of the action of MPTP and MPP+ and the neuropharmacological basis of this action on DA metabolism in the rat striatum. The results show that the elevated levels of DA in the striatal perfusates are due to a direct action of MPTP and MPP+ on the nigrostriatal DA terminals and cannot be fully accounted for solely by their inhibition of MAO activity and/or inhibition of DA re-uptake.(ABSTRACT TRUNCATED AT 250 WORDS)