Effect of anticholinergic drugs on striatal acetylcholine release and motor activity in freely moving rats studied by brain microdialysis

We have studied effects of intraperitoneal administration of anticholinergic drugs on striatal acetylcholine release in association with motor activity in freely moving rats using brain microdialysis. A low dose of atropine (2.5 mg/kg) increased striatal acetylcholine release. A high dose of atropine (5 mg/kg) or scopolamine (2.5, 5 and 10 mg/kg) increased both striatal acetylcholine release and motor activity, while its quaternary ammonium compounds, atropine methylbromide (5 and 10 mg/kg) and methscopolamine bromide (5 and 10 mg/kg), increased striatal acetylcholine release without motor excitation. Scopolamine (2.5 mg/kg) produced no significant change in striatal acetylcholine content 4 hr after the injection followed by perfusion. These results suggest that anticholinergic drugs cause an increase in striatal acetylcholine release which does not always result in the increase of motor activity.     

Effects of drugs on tremor and increase in brain acetylcholine produced by oxotremorine in the rat

1. In rats the effect of drugs on oxotremorine tremor and oxotremorine-induced increase in brain acetylcholine has been investigated.2. Reserpine, (+/-)-alpha-methylmetatyrosine and diethyldithio-carbamic acid, drugs which have in common the ability to decrease tissue noradrenaline concentration, inhibited oxotremorine tremor without preventing the oxotremorine-induced increase in brain acetylcholine.3. (+/-)-p-chlorophenylalanine, a depletor of tissue 5-hydroxytryptamine, did not inhibit oxotremorine tremor.4. Phenoxybenzamine and propranolol inhibited oxotremorine tremor, and propranolol was without effect on oxotremorine-induced increase in brain acetylcholine.5. The toxicity of oxotremorine was increased by reserpine and phenoxybenzamine.6. The significance of these findings is discussed with regard to the mode of action of oxotremorine.     

Effects of apomorphine on the in vivo release of dopamine and its metabolites, studied by brain dialysis

The effect of apomorphine (0.05-0.5 mg/kg s.c.) on the release of endogenous dopamine and extracellular levels of the metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5HIAA) were examined in vivo by intracerebral dialysis. A dialysis tube was implanted stereotaxically through both caudate nuclei of rats and perfused with Ringer solution at a rate of 2 microliters/min. The amount of dopamine, DOPAC, HVA and 5HIAA in the perfusates was measured by HPLC with electrochemical detection. With the dialysis tube implanted into the striatum of anaesthetized rats it was possible to measure basal levels of dopamine and the metabolites in the perfusates; dopamine, 0.27 +/- 0.05 pmol/20 min (n = 15), DOPAC 43.3 +/- 2.57 pmol/20 min (n = 15), HVA 24.5 +/- 1.89 pmol/20 min (n = 15) and 5HIAA 13.9 +/- 1.77 pmol/20 min (n = 15). The % recoveries of the monoamines through the membrane were estimated to be 12% (dopamine), 21% (DOPAC), 23% (HVA) and 25% (5HIAA). Apomorphine 0.05-0.2 mg/kg decreased the spontaneous release of dopamine by a maximum of approximately 50%. When the dose of apomorphine was raised up to 0.5 mg/kg there was a 100% inhibition of dopamine release. Also, the extracellular levels of the metabolites DOPAC and HVA decreased following apomorphine administration; however there was no consistent change in 5HIAA. These findings indicate that the dopamine autoreceptors decrease dopamine release in vivo by 0-50% while larger decreases probably involve postsynaptic neurons engaging short- as well as long-loop reflexes.     

The use of tetrodotoxin for the characterization of drug-enhanced dopamine release in conscious rats studied by brain dialysis

Summary The effect of TTX (infused during brain dialysis of the striatum and nucleus accumbens) on the in vivo release of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid, was investigated. In addition it was studied whether the increase in the release of dopamine, induced by various pharmacological treatments, was sensitive to TTX infusion. The following drugs were studied: haloperidol, amphetamine, haloperidol co-administered with GBR 12909, morphine and MPP⁺. Dialysis was carried out in the striatum, except for morphine, which was studied in the nucleus accumbens. The infusion of TTX revealed three different types of pharmacologically enhanced dopamine release in conscious rats. First, action potential dependent dopamine release (exocytosis), which was observed in untreated animals as well as in animals treated with haloperidol, haloperidol + GBR 12909, and morphine. Second, action potential independent release (carrier-mediated) was established in the case of amphetamine. Third, action potential independent DA release, probably caused by neurotoxic reactions was observed during MPP+ infusion. Send offprint requests to B. H. C. Westerink     

Effects of cholinergic drugs on extracellular levels of acetylcholine and choline in rat cortex, hippocampus and striatum studied by brain dialysis

A brain dialysis technique was used to investigate the effects of cholinergic drugs on acetylcholine (ACh) release and on the extracellular choline levels. Scopolamine (0.5 mg/kg s.c.) markedly increased ACh release in frontal cortex, hippocampus and corpus striatum. Conversely, it significantly decreased choline levels in extracellular spaces of these three regions. Oxotremorine (0.5 mg/kg i.p.) induced no significant ACh release in these three regions, probably due to the presence of highly concentrated physostigmine in the perfusate, while it induced an increase of the choline levels in both frontal cortex and hippocampus but not in corpus striatum. Nicotine (0.5 mg/kg s.c.) significantly increased the ACh release in these three regions with no change in choline levels. Nicotine had a biphasic effect on ACh release in frontal cortex and hippocampus but not in corpus striatum. It should be noted that all such cholinergic drugs, in our time course determinations, yielded certain differences among these brain regions for both the magnitude and the response pattern of ACh and choline levels. The present investigation appears to indicate that the in vivo brain dialysis technique can be useful for probing cholinergic neurotransmission of cholinergic drugs via presynaptic terminals.     

Effects of REMS deprivation on striatal dopamine and acetylcholine in rats.

Changes in the concentration of striatal dopamine (DA) and acetylcholine (ACh) in rats deprived of REM sleep for 10 days were compared with those obtained after a 4 day deprivation procedure. Animals placed on small (7 cm dia.) islands surrounded by water were completely deprived of REM sleep but able to obtain some slow-wave sleep. Concentration of striatal DA was significantly increased after 4 days and 10 days of REM sleep deprivation by 73 and 133%, respectively when compared to controls. Levels of ACh in the striatum were significantly enhanced (by 28%) after 10 day, but failed to show significant change after 4 day REM sleep deprivation procedure. The short term locomotor activity was significantly higher in REM sleep-deprived animals. Our data indicate that REM sleep deprivation results in marked alterations of both cholinergic and dopaminergic mechanisms in the rat striatum.     

Effects of physostigmine on brain acetylcholine content and release

The content and release of acetylcholine (ACh) from cat brain and ACh content of rat brain was measured using the frog rectus and leech muscle bioassays before and after physostigmine 100 $\text{μg}\text{kg}$i.v. or i.p. The drug elevates steady state levels of brain ACh of awake animals especially in the neocortex, caudate nucleus and hippocampus but much less so in the lateral geniculate and superior colliculus. Physostigmine was shown to produce neocortical release of ACh through a subcortical mechanism in brainstem transected cats and to decrease ACh neocortical content in pentobarbital anesthetized cats and rats. The neocortical release of ACh seems to be mediated primarily through a subcortical brain mechanism which is activated by physostigmine. It is postulated that the ascending reticular formation is involved. Local application of scopolamine (1 $\text{μg}\text{ml}$) into the collecting cup did not antagonize the effects of 100 $\text{μg}\text{kg}$ of physostigmine i.v. as much as l $\text{mg}\text{kg}$ of scopolamine i.v. On the other hand, 2 $\text{mg}\text{kg}$ of mecamylamine given i.v. not only did not antagonize physostigmine-induced neocortical release of ACh, but seemed to enhance it. The results indicate a complex action of physostigmine on the cholinergic system of the brain.     

Enhancement by oxotremorine of acetylcholine release from the rat phrenic nerve.

Oxotremorine (10.5 micron) produced a paralytic effect on twitch responses of rat diaphragm in vitro to direct and indirect stimulation. 2 The paralytic effect of oxotremorine was absent when the diaphragm was stimulated directly in the presence of hemicholinium-3 (0.42 mM), at a time when twitch responses to indirect stimulation ceased completely. 3 Oxotremorine, at two different pharmacologically active doses, strikingly increased the resting as well as electrically evoked release of acetylcholine into the bathing fluid from the phrenic nerve-diaphragm preparation. 4 This presynaptic effect of oxotremorine may explain its pharmacological effects at the cholinergic synapses studied so far.     

Differential effects of the neuropeptide galanin on striatal acetylcholine release in anaesthetized and awake rats

1. In the present study the mechanisms were examined by which the neuropeptide galanin modulates the extracellular concentrations of striatal acetylcholine (ACh) in enflurane anaesthetized and in freely moving male rats by use of in vivo microdialysis and high performance liquid chromatography.
2. The perfusion of galanin through the microdialysis probe (0.3 nmol μl⁻¹, flow rate: 2 μl min⁻¹) caused a statistically significant increase in the basal striatal ACh levels in anaesthetized but a decrease in awake animals. No significant effect was revealed after a low dose (0.1 nmol μl⁻¹, flow rate: 2 μl min⁻¹) of galanin perfusion. Both the stimulating and inhibitory effects of galanin on basal ACh release were reversible.
3. The muscarinic antagonist scopolamine (0.1 mg kg⁻¹, subcutaneously (s.c.)) caused a significant increase in ACh release in both anaesthetized and awake animals.
4. The combination of galanin plus scopolamine attenuated the stimulant effect on ACh release caused by scopolamine alone in awake animals.
5. The putative galanin receptor antagonist M35 at 0.3 nmol μl⁻¹ but not at 0.1 nmol μl⁻¹ caused a significant reduction (20%) in ACh release, supporting the view that M35 at higher concentrations behaves as a partial agonist at the galanin receptor. When M35 (0.1 nmol μl⁻¹) was co-infused with galanin (0.3 nmol μl⁻¹) the galanin-evoked decrease in ACh release was completely blocked.
6. Taken together, these results indicate that galanin affects basal ACh release via stimulation of galanin receptors within the striatum. The mechanism involved is dependent on the anaesthesia procedure which may act via enhancement of γ-aminobutyric acid_A (GABA_A) mediated transmission within striatal and/or output neurones. In addition, anaesthesia may also decrease the activity of glutamatergic striatal afferents. The results with M35 indicate that the role of galanin perfused in striatum is permissive in the normal rat. Furthermore, galanin is a potent inhibitory modulator of basal ACh release also in the striatum, as recently was shown in the ventral hippocampus in awake animals.

     

Effects of scopolamine on extracellular acetylcholine and choline levels and on spontaneous motor activity in freely moving rats measured by brain dialysis

The present study demonstrates the feasibility of measuring acetylcholine (ACh) and choline in perfusate samples collected by in vivo brain dialysis in the frontal cortex and hippocampus of freely moving rats in which spontaneous motor activity could be measured simultaneously. Systemically administered scopolamine increased the output of ACh about 10-fold and 20-fold in the frontal cortex and hippocampus, respectively. By contrast, scopolamine decreased the choline level in the extracellular fluid about 2-fold in both brain regions, possibly owing to enhanced choline uptake into the presynaptic nerve terminals. Scopolamine also increased spontaneous motor activity over the same time course as the changes in ACh and choline. These results indicate that the in vivo brain dialysis technique applied to freely moving rats may be useful in investigating ACh turnover and in studying the relation between cholinergic transmission and behavioral functions.     

Neurotransmitters extraction by local intracerebral dialysis in anesthetized rats

A new technique for in vivo neurotransmitter extraction was developed. Perfusion U shaped cannulae were chronically implanted in the caudate or the thalamus of rats. A segment of the cannula was a piece of dialysis tube of molecular weight cut off 1000. Measurable amounts of dopamine and norepinephrine were recovered from the perfusion fluid. Intraperitoneal amphetamine 10 mg/kg increased dopamine output from the caudate but not from the thalamus. The potential applications of this technique are discussed.     

Effect of oxotremorine on the acetylcholine content of whole brain and various brain regions in the pigeon

Oxotremorine (0·125 mg/kg) produces a significant increase in total acetylcholine content in whole pigeon brain. The contribution of different regions to this increase varies. The largest increase occurs in the nucleus basalis (paleostriatum augmentatum), a region which is highly involved in motor control. The mechanism by which oxotremorine increases the acetylcholine content of brain and the causal relationship between the rise in acetylcholine content and tremor are discussed.     

Modification of brain acetylcholine release by morphine and its antagonists in normal and morphine-dependent rats

1 The spontaneous release of acetylcholine (ACh) from the cerebral cortex of control and morphine-dependent rats was investigated. The rate of resting output of ACh in morphine-dependent animals was lower than that in the control animals.2 Administration of naloxone and nalorphine to morphine-dependent rats was followed by a significant rise in the release of cortical ACh. In control rats no such increase in the release of ACh occurred after similar injections of narcotic antagonists.3 Injections of morphine produced a consistent decrease in the rate of spontaneous release of cortical ACh in the control rats, but similar injections in the dependent rats did not produce a decrease in the rate of cortical ACh release.4 The relevance of these results with regard to development of the narcotic abstinence syndrome is discussed.     

Effects of acetylcholine and atropine on the release of 14C-acetylcholine formed from U-14C-glucose in rat brain cortical and striatal prisms

Summary A perfusion technique has been used to study the mechanisms involved in the control of acetylcholine (ACh) release from rat brain cortical and striatal slices submitted to KCl depolarization. Tissues were superfused continuously with U-¹⁴C-glucose, a very efficient precursor of the acetyl moiety of ACh, and with eserine to prevent the hydrolysis of the ¹⁴C-ACh released in the superfusion medium.This radioenzymatic approach was more flexible than those in which endogenous ACh outflows were estimated using a biological or a radioenzymatic method. Moreover, the modulation of the ester release process could be studied using the physiological ligand of the presynaptic muscarinic receptor, i.e. ACh itself. The continuous synthesis of ¹⁴C-ACh from U-¹⁴C-glucose, throughout the superfusion experiment, allowed the rate of ¹⁴C-ACh release to reach a steady state, under resting conditions (5.6 mM KCl) as well as under conditions of stimulation by high concentrations of KCl. This method was very convenient for pharmacological studies as compared to other methods in which the efflux of ³H-ACh from performed intratissular stores of ³H-ACh was measured.Using this technique, it was shown that atropine (10 M) enhanced the rate of ¹⁴C-ACh spontaneous release. Curiously, extraneously added ACh (0.6 mM) had a similar effect. When cortical prisms were submitted to a brief 31 mM KCl pulse stimulation (3 min), the negative feedback regulation process was not fully triggered. Indeed, atropine, which prevents the interaction of the released ACh with the regulatory muscarinic receptor, significantly stimulated the ¹⁴C-ACh outflow only when brain tissues were submitted to a sustained 31 mM KCl depolarisation, or when the ACh concentration in the superfusion fluid was raised (0.6 mM). These results suggest that the muscarinic negative control of ACh release from brain cholinergic terminals becomes fully operative only when the nerve endings are depolarized and when the ACh concentration within the synapse exceeds a threshold, i.e. during intense functioning of the cholinergic synapse.Abbreviations AChE acetylcholinesterase - ChAc cholineacetyltransferase (EC 2.3.1.6)     

The effects of atropine and oxotremorine on acetylcholine release in rat phrenic nerve-diaphragm preparations.

1 Atropine (10(-5) M) enhanced the release of [3H]-acetylcholine from rat isolated hemidiaphragms, previously incubated with [3H-methyl]-choline, stimulated via their phrenic nerves. 2 Oxotremorine (10(-5) M) did not affect the stimulated release of [3H]-acetylcholine but antagonized the facilitatory effects of atropine (10(-5) M). 3 It is suggested that there are presynaptic inhibitory muscarinic receptors that modulate the release of acetylcholine in the phrenic nerves of the rat.     

Effect of atropine and oxotremorine on the release of acetylcholine from the frog spinal cord

Summary The in vitro frog spinal cord has been used to study acetylcholine (ACh) release and spinal root potentials. The preparation bathed in an eserine-containing medium spontaneously released ACh into the bathing fluid. This release was enhanced by atropine in a dose-related and long-lasting manner and transiently by oxotremorine. The release rate of ACh was increased by low frequency ventral root stimulation; this increase was found to be proportionally the same after application of atropine. Oxotremorine did not modify the electrically-evoked ACh release but blocked or reduced the effect of atropine. It is concluded that the stimulatory action of atropine on ACh output cannot be entirely explained by an interaction of atropine with presynaptic cholinergic receptors and that other indirect mechanisms (via interneurones) may play a role in this effect.     

Involvement of nicotinic and muscarinic acetylcholine receptors on striatal HgCl2-induced dopamine release in freely moving rats

The possible role of acetylcholine receptors on the HgCl₂-induced dopamine (DA) release from rat striatum was investigated by using in vivo brain microdialysis technique after administration of selective nicotinic and muscarinic receptor antagonists, mecamylamine and atropine, respectively. Intrastriatal infusion of 1 mM HgCl₂ increased striatal DA to 1717.2 ± 375.4% respect to basal levels. Infusion of 1 mM HgCl₂ in 1 mM mecamylamine pretreated animals produced an increase on striatal DA levels 58% less than that induced in non-pretreated animals. In the case of atropine, this treatment reduced 62% the effect produced by HgCl₂ as compared to non-pretreated rats. These data show that acetylcholine receptors could participate on HgCl₂-induced dopamine release since administration of nicotinic and muscarinic receptor antagonists reduces HgCl₂ effects on DA release.