Enhancement of [m-methoxy3H]MDL100907 binding to 5HT2A receptors in cerebral cortex and brain stem of streptozotocin induced diabetic rats

5-Hydroxytryptamine_2A (5-HT_2A) receptor kinetics was studied in cerebral cortex and brain stem of streptozotocin (STZ) induced diabetic rats. Scatchard analysis with [³H] (±) 2,3dimethoxyphenyl-1-[2-(4-piperidine)-methanol] ([³H]MDL100907) in cerebral cortex showed no significant change in maximal binding (B_max) in diabetic rats compared to controls. Dissociation constant (K_d) of diabetic rats showed a significant decrease (p < 0.05) in cerebral cortex, which was reversed to normal by insulin treatment. Competition studies of [³H]MDL100907 binding in cerebral cortex with ketanserin showed the appearance of an additional low affinity site for 5-HT_2A receptors in diabetic state, which was reversed to control pattern by insulin treatment. In brain stem, scatchard analysis showed a significant increase (p < 0.05) in B_max accompanied by a significant increase (p < 0.05) in K_d. Competition analysis in brain stem also showed a shift in affinity towards a low affinity State for 5-HT_2A receptors. All these parameters were reversed to control level by insulin treatment. These results show that in cerebral cortex there is an increase in affinity of 5-HT_2A receptors without any change in its number and in the case of brain stem there is an increase in number of 5HT_2A receptors accompanied by a decrease in its affinity during diabetes. Thus, from the results we suggest that the increase in affinity of 5-HT_2A receptors in cerebral cortex and upregulation of 5-HT_2A receptors in brain stem may lead to altered neuronal function in diabetes.     

No Orcadian Rhythms of Serotoninergic,Alpha-, Beta-Adrenergic and Imipramine Binding Sites in Rat Brain Regions

B_max values of the specific binding of [³H]-WB 4101, [³H]-dihydroalprenolol, [³H]-spiperone and [³H]-imipramine to various rat brain regions were determined at 4 hr intervals over 24 hr under circadian conditions. No significant circadian rhythm of binding sites number was found for any receptor investigated in cerebral cortex, hypothalamus or brain stem. Some methodological issues are discussed.     

DEVELOPMENT OF THE BLOOD-BRAIN BARRIER FOR 6-HYDROXYDOPAMINE

The postnatal development of the blood-brain barrier for the neurotoxic action of 6-hydroxydopamine on central noradrenaline neurons has been investigated by recording the in vitro uptake of [³H]noradrenaline in slices from cerebral cortex, hypothalamus and spinal cord in rats treated with large doses of 6-hydroxydopamine at different ages. The [³H]noradranaline uptake was permanently and markedly reduced in all regions when the animals were treated at birth, certainly related to degeneration of noradrenaline neurons, caused by 6-OH-DA. In the cerebral cortex and hypothalamus an efficient protection against the effects of 6-OH-DA on [³H]noradrenaline uptake developed postnatally, while in the spinal cord this protection was never seen to become complete. The results obtained indicate a rapid formation of a blood-brain barrier for 6-OH-DA in the cerebral cortex between the 7th and 9th day after birth. In the hypothalamus the development of this barrier seemed to have a more gradual time-course, but appeared to be fully developed already at day 5 postnatally. Also in the spinal cord the barrier developed more gradually from birth to the adult age. It was observed, however, that both in the cerebral cortex and in the spinal cord, the blood-brain barrier developed, could not completely protect the central noradrenaline neurons from the neurotoxic actions of large doses of 6-OH-DA administered systemically to adult rats. Furthermore, the results obtained support the view that 6-OH-DA does not seem to apparently affect the outgrowth of remaining NA neurons which have not been destroyed by the 6-OH-DA treatment.     

Role of External and Internal Calcium on Heterocarrier-Mediated Transmitter Release

Abstract: Release-regulating heterocarriers exist on brain nerve endings. We have investigated in this study the mechanisms involved in the neurotransmitter release evoked by GABA heterocarrier activation. GABA increased the basal release of [³H]acetylcholine and [³H]noradrenaline from rat hippocampal synaptosomes and of [³H]dopamine from striatal synaptosomes. These GABA effects, insensitive to GABA receptor antagonists, were prevented by inhibiting GABA uptake but not by blocking noradrenaline, choline, or dopamine transport. Lack of extracellular Ca²⁺ or addition of tetrodotoxin selectively abolished the GABA-evoked release of [³H]noradrenaline, leaving unaffected that of [³H]acetylcholine or [³H]dopamine. 1,2-Bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) or vesamicol attenuated the release of [³H]acetylcholine elicited by GABA. Reserpine, but not BAPTA-AM, prevented the effect of GABA on [³H]dopamine release. Autoreceptor activation inhibited the GABA-evoked release of [³H]noradrenaline but not that of [³H]acetylcholine or [³H]dopamine. It is concluded that (a) the release of [³H]noradrenaline consequent to activation of GABA heterocarriers sited on noradrenergic terminals meets the criteria of a conventional exocytotic process, (b) the extracellular [Ca²⁺]-independent releases of [³H]acetylcholine and [³H]dopamine appear to occur from vesicles possibly through involvement of intraterminal Ca²⁺, and (c) autoreceptor activation only affects heterocarrier-mediated vesicular release linked to entry of extracellular Ca²⁺.     

THE EFFECT OF LIMB ISCHAEMIA ON THE TURNOVER OF NORADRENALINE IN THE HYPOTHALAMUS AND BRAIN STEM OF THE RAT

The effect of ischaemic limb injury on the turnover of noradrenaline in the hypothalamus and brain stem has been studied in rats. There are theoretical reasons for thinking that these regions are activated in trauma and previous work showed that during limb-ischaemia the concentration of noradrenaline in the hypothalamus decreased by 27 per cent. The tourniquets were applied to both hind-limbs 1 h after the injection of [¹⁴C]-tyrosine when the labelling of the noradrenaline was maximal. During 4 h limb ischaemia the endogenous tyrosine concentration in the plasma decreased while that in the hypothalamus first rose and then fell. Changes in a similar direction in the brain stem were not statistically significant. Limb ischaemia did not affect the decline in the specific activity of the plasma or tissue tyrosine. It was concluded that the injury increased the utilization of tyrosine by the body. During the 4 h bilateral hind-limb ischaemia the rate of decline of [¹⁴C]noradrenaline was significantly increased in the brain stem but not in the hypothalamus. Conditions in the brain stem were sufficiently close to ‘steady-state’ to be able to conclude that the injury increased the metabolism of noradrenaline in the brain stem. Conditions in the hypothalamus were too complicated for definite conclusions to be drawn. The possible reasons for this and the limitations of this method for studying noradrenaline turnover are discussed.     

Polyamine turnover in different regions of rat brain

The dynamics of the formation and disappearance of polyamines in rat brain have been examined after intraventricular administration of a tracer dose of [³H]putrescine. After 2 days [³H]putrescine was no longer detectable in any brain region examined. [³H] Spermidine and [³H] spermine were formed in all brain areas. In the midbrain, hypothalamus and cerebellum (regions which manifested the greatest initial accumulation of tritium) the specific radioactivity of spermidine declined with a half-life of 16-19 days. However, in areas with a low initial accumulation of tritium (the medulla-pons, internal capsule, cerebral cortex and corpus striatum) the specific radioactivity of spermidine changed very little between 2 and 19 days after the putrescine administration. Levels of [³H]spermine increased continuously in all brain areas for a 14-day period after the putrescine injection.     

Heterogeneity of Benzodiazepine Receptors: Experimental Differences Between [3H]Flunitrazepam and [3H]Ethyl-β-carboline-3-carboxylate Binding Sites in Rat Brain Membranes

Abstract: This study was designed to analyze possible differences in the binding of [³H]flunitrazepam ([³H]FNZP) and [³H]ethyl - β - carboline - 3 - carboxylate ([³H]β-CCE), to rat brain membranes, in various experimental conditions. In cerebral cortex, hippocampus, cerebellum, and orain stem the number of binding sites for [³H]β-CCE was higher than for [³H]FNZP; both were displaced by clonazepam. Until the 7th day of postnatal brain development the numbers of [³H]FNZP and [³H]β-CCE sites were equivalent; but later on, the β-carboline sites increased to a higher level. Noradrenergic denervation by 6-hydroxydopamine was followed in the hippocampal formation. Already after 2 days, there was a decrease in [³H]FNZP sites, which reached 70% of control after 14 days. Similar results were obtained with DSP-4 denervation. This change was only in B_max and not in K_D, In contrast, the [³H]β-CCE sites did not change with denervation. Neonatal injection of l - 2,4,5 - trihydroxyphenylalamine or DSP-4 produced in the adult a decrease in [³H]FNZP sites in the cerebral cortex, in parallel with the noradrenergic denervation. On the other hand, there was an increase in the cerebellum and brain stem, in correspondence with the hyperinnervation by sprouting. In these rats, the number of sites for [³H]β-CCE did not change in the different brain regions. With 0.1% Triton X-100, applied to synaptosomal membranes, [³H]FNZP binding was reduced by 35%, while that of [³H]β-CCE was not significantly changed. These results suggest that there is heterogeneity of binding sites for benzodiazepine receptors in rat brain. A tentative interpretation of the experiments involving noradrenergic denervation and hyperinnervation, as well as those with Triton X-100, is that [³H]FNZP binds to pre- and postsynaptic receptors, while [³H]β-CCE binds mainly to postsynaptic benzodiazepine receptors.     

6-AMINODOPAMINE-INDUCED DEGENERATION OF CATECHOLAMINE NEURONS

the effects of 6-aminodopamine on central and peripheral catecholamine neurons using fluorescence histochemical and isotope techniques have been investigated. Systematic administration of 6-aminodopamine (20 mg/kg intraveneously) produced a rapid (within 1 h) and long-lasting depletion of endogenous noradrenaline in adrenergic nerves of mouse atrium and iris with a concomitant loss of [³H]noradrenaline uptake. The effects were dosedependent. Accumulations of noradrenaline in non-terminal axons were observed histochemically, indicating that 6-aminodopamine induces neuronal damage. Desipramine completely blocked the 6-aminodopamine induced noradrenaline depletion and reduction in [³H]noradrenaline uptake, indicating that 6-aminodopamine has to be taken up by the axonal ‘membrane pump’ to produce its effects. Themonoamine oxidase inhibitor, nialamide, potentiated the effect of 6-aminodopamine on [³H]noradrenaline uptake. 6-Aminodopamine did not affect the cell bodies of the adrenergic neurons and there was a reappearance of adrenergic nerves and recovery of [³H]noradrenaline uptake. 6-Aminodopamine does not seem to pass the blood-brain barrier after systemic injection. Intraventricular injection of 6-aminodopamine in rats led to a considerable reduction in endogenous whole brain noradrenaline and [³H]noradrenaline uptake in slices from cerebral cortex and hypothalamus. Similar, but less pronounced effects were observed on dopamine neurons in the caudate nucleus. Histochemically, pronounced accumulations of transmitter were observed in the axons of the catecholamine neurons. The results obtained favour the view that 6-aminodopamine is able to produce an acute and selective degeneration of catecholamine neurons similar to that seen after the neurotoxicagent, 6-hydroxydopamine. Both compounds seemed to be approximately equally potent in their neurotoxicity, although 6-aminodopamine seemed to be more generally toxic.     

Regional brain GABA metabolism and release during hepatic coma produced in rats chronically treated with carbon tetrachloride

Hepatic coma was induced in rats chronically treated with CCl₄, by means of a single injection of ammonium acetate. The activities of glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), as well as the synaptosomal uptake and release of [³H]GABA, were measured in the following brain areas of the comatose rats: cortex, striatum, hypothalamus, hippocampus, midbrain and cerebellum. Hepatic coma was associated with a general decrease of GAD activity, whereas GABA-T activity was diminished only in the hypothalamus, striatum and midbrain. During hepatic coma, the K⁺-stimulated [³H]GABA release was notably diminished in the striatum and cerebellum, whereas a significant increase was observed in the hippocampus. [³H]GABA uptake increased in most regions after CCl₄ treatment, independently of the presence of coma. The results indicate that GABAergic transmission seems to be decreased in most cerebral regions during hepatic coma.     

Regional release of aromatic amines from tissues of the rat brain in vitro

Abstract— Radioactive hydroxylated phenylethylamines were released in vitro by electrical stimulation of minces of brain tissues from several anatomically discrete areas, while labelled urea and amphetamine were poorly released from all regions. Release of [³H]norepinephrine occurred in the order hypothalamus > caudate nucleus ≥ cerebral cortex, thus in parallel with the distribution of endogenous norepinephrine. In contrast, [³H]tyramine was poorly released from cortical tissues but readily released from minces of caudate nucleus or hypothalamus. [³H]Octopamine was released from all areas, but was most readily released from thecaudatenucleus. Results for cerebral cortex were similar to those for coronal slices or minces of whole brain; release occurred in the order: norepinephrine > octopamine > tyramine in all three preparations. We suggest that certain β-hydroxylated phenolic phenylethyl-amines may be released from norepinephrine- or dopamine-containing nerve endings in the brain, and that their non-β-hydroxylated congeners may be released from neurons in which endogenous amines are not β-hydroxylated.     

Arachidonic Acid Inhibits Choline Uptake and Depletes Acetylcholine Content in Rat Cerebral Cortical Synaptosomes

The effects of arachidonic acid on [3H]choline uptake, on [3H]acetylcholine accumulation, and on endogenous acetylcholine content and release in rat cerebral cortical synaptosomes were investigated. Arachidonic acid (10-150 microM) produced a dose-dependent inhibition of high-affinity [3H]choline uptake. Low-affinity [3H]choline uptake was also inhibited by arachidonic acid. Fatty acids inhibited high-affinity [3H]choline uptake with the following order of potency: arachidonic greater than palmitoleic greater than oleic greater than lauric; stearic acid (up to 150 microM) had no effect. Inhibition of [3H]choline uptake by arachidonic acid was reversed by bovine serum albumin. In the presence of arachidonic acid, there was an increased accumulation of choline in the medium, but this did not account for the inhibition of [3H]choline uptake produced by the fatty acid. Arachidonic acid inhibited the synthesis of [3H]acetylcholine from [3H]choline, and this inhibition was equal in magnitude to the inhibition of high-affinity [3H]choline uptake produced by the fatty acid. A K+-stimulated increase in [3H]acetylcholine synthesis was inhibited completely by arachidonic acid. Arachidonic acid also depleted endogenous acetylcholine stores. Concentrations of arachidonic acid and hemicholinium-3 that produced equivalent inhibition of [3H]choline uptake also produced equivalent depletion of acetylcholine content. In the presence of eserine, arachidonic acid had no effect on acetylcholine release. The results suggest that arachidonic acid may deplete acetylcholine content by inhibiting high-affinity choline uptake and subsequent acetylcholine synthesis. This raises the possibility that arachidonic acid may play a role in the impairment of cholinergic transmission seen in cerebral ischemia and other conditions in which large amounts of the free fatty acid are released in brain.     

Muscarinic receptor activation inhibits both release and synthesis of noradrenaline in rat hippocampal synaptosomes

Rat hippocampal synaptosomes were used to investigate the effects of muscarinic cholinergic drugs on the release of [³H]noradrenaline and the rate of noradrenaline synthesis. Oxotremorine and acetylcholine caused depression of K⁺-evoked release of [³H]noradrenaline; these effects were reversed by atropine and pirenzepine. Muscarinic agonists depressed 2-chloroadenosine- and isoprenaline-stimulated noradrenaline synthesis but had no effect on 8-Br-cyclic AMP-stimulated synthesis. Oxotremorine also depressed the K⁺-acceleration of noradrenaline synthesis. The action of pirenzepine suggests that the inhibition of release and synthesis are mediated by separate muscarinic receptor subtypes.     

Actions of a monoclonal antibody Tor 23 on rat brain presynaptic cholinergic processes

Tor 23 is a monoclonal antibody, generated against cholinergic terminals of theTorpedo californica, that has been found to bind to the extracellular surface of cholinergic neurons in a variety of tissues. This study shows that Tor 23 inhibits: 1) high affinity [³H]hemicholinium-3 binding to detergent-solubilized membranes prepared from rat neocortices; 2) high affinity [³H]choline uptake in rat neocortical and striatal P₂ preparations; and 3) [³H]acetylcholine synthesis in isolated nerve terminals. Tor 23 does not appear to affect low affinity [³H]choline uptake or [³H]acetylcholine release. These results are consistent with the hypothesis that Tor 23 may bind to nerve terminal high affinity choline transporters in the rat brain.     

Characteristics of the catecholamine and histamine receptor sites mediating accumulation of cyclic adenosine 3',5'-monophosphate in guinea pig brain

—Five areas of guinea pig brain were examined to determine the properties of the receptor sites mediating increases in [³H]adenosine 3′,5′-monophosphate (cyclic AMP). Both epinephrine and histamine were effective in causing increases in cyclic AMP in slices derived from cerebral cortex, hippocampus or amygdala, but not in diencephalon or brainstem. Stimulation of slices of cerebral cortex by either epinephrine or histamine resulted in a small, but reproducible, decrease in specific radioactivity of the [³H]-cyclic AMP produced, as did stimulation of the hippocampus by epinephrine. The catecholamine receptor was an α-adrenergic receptor in all three areas where epinephrine was effective; α-adrenergic stimulation, but not β-adrenergic stimulation, increased levels of [³H]-cyclic AMP. Furthermore, α-, but not β-adrenergic blocking agents, prevented the epinephrine- induced increase of both [³H]- and total cyclic AMP in cerebral cortex and hippocampus. Only antihistaminic agents were capable of antagonizing the histamine-induced increase of both [³H]- and total cyclic AMP in these two brain areas. The catecholamine receptor in the amygdala also appeared to be an α-adrenergic receptor. The effects of histamine and epinephrine together were far greater than the sum of effects of either hormone alone in both cerebral cortex and hippocampus.     

High affinity transport of choline into synaptosomes of rat brain

—The accumulation of [³H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [³H]choline concentrations varying from 0.5 to 100 μm . The accumulation of [³H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K_m. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective K_m. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [³H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [³H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [³H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.     

Decreased 5-HT2C receptor binding in the cerebral cortex and brain stem during pancreatic regeneration in rats

The purpose of this study was to investigate the role of central 5-HT_2C receptor binding in rat model of pancreatic regeneration using 60–70% pancreatectomy. The 5-HT and 5-HT_2C receptor kinetics were studied in cerebral cortex and brain stem of sham operated, 72 h pancreatectomised and 7 days pancreatectomised rats. Scatchard analysis with [³H] mesulergine in cerebral cortex showed a significant decrease (p < 0.05) in maximal binding (B_max) without any change in K_d in 72 h pancreatectomised rats compared with sham. The decreased B_max reversed to sham level by 7 days after pancreatectomy. In brain stem, Scatchard analysis showed a significant decrease (p < 0.01) in B_max with a significant increase (p < 0.01) in K_d. Competition analysis in brain stem showed a shift in affinity towards a low affinity. These parameters were reversed to sham level by 7 days after pancreatectomy. Thus the results suggest that 5-HT through the 5-HT_2C receptor in the brain has a functional regulatory role in the pancreatic regeneration.     

Characterization of [3H]cholecystokinin octapeptide binding to mouse brain synaptosomes: Effects of neuroleptics

The presence of high concentrations of both dopamine and cholecystokinin (CCK) in the striatum and in various limbic structures suggests that the CCK may not only influence dopaminergic transmission, but it also may be relevant to the psychopathology of schizophrenia and to the therapeutic effects of neuroleptics. By using a synaptosomal fraction isolated from the mouse cerebral cortex and [propionyl-³H]CCK8-sulphate ([³H]CCK8S) as a ligand, a single binding site for [³H]CCK8 with aK _d value of 1.04 nM and aB _max value of 42.9 fmol/mg protein was identified. The competitive inhibition of [³H]CCK8S binding by related peptides produced an order of potency of CCK8-sulphated (IC50=5.4 nM)>CCK8-unsulfated (IC50=40 nM) and >CCK4 (IC50=125 nM). The regional distribution of [³H]CCK8S binding in the mouse brain was highest in the olfactory bulb (34.3±5.6 fmol/mg protein) > cerebral cortex > cerebellum > olfactory tubercle > striatum > pons-medulla > mid brain > hippocampus > hypothalamus (12.4±2.1 fmol/mg protein). The repeated administration of haloperidol (2.5 mg/kg/tid) increased the binding of [³H]CCK8S in cerebral cortex from 31.8±1.7 to 38.9±5.2 fmol/mg protein. The varied distribution of CCK8S receptors may signify nonuniform functions for the octapeptide in the brain.     

Effects of thyroid status on the characteristics of alpha 1-, alpha 2-, beta, imipramine and GABA receptors in the rat brain.

The effects of a chronic treatment with L-triiodothyronine (T3; 100 mg/rat/day s.c. for 7 days) or with propylthiouracil (PTU; 50 mg/rat/day for 35 days by stomach tube) on the characteristics of alpha 1, alpha 2, beta, imipramine and GABA binding sites in different brain areas of the adult rat have been studied. T3-treatment caused an increase in the number of [3H]dihydroalprenolol and a decrease in the number of [3H]muscimol binding sites in the cerebral cortex. PTU-treatment caused a decrease in the number of [3H]prazosin, [3H]yohimbine and [3H]dihydroalprenolol binding sites in the cerebral cortex, while the number of [3H]imipramine binding sites was reduced in the cerebral cortex and hypothalamus, and increased in the hippocampus. Affinity constants were never modified. Concurrent experiments showed that the "in vitro" addition of T3 and PTU did not influence the binding of any of the ligands employed to control rat brain membranes. The present data further support the view that neurotransmission in the CNS is influenced by the thyroid status.     

Calcium-dependent [3H]acetylcholine release and muscarinic autoreceptors in rat cortical synaptosomes during development

A number of presynaptic cholinergic parameters (high affinity [³H]choline uptake, [³H]acetylcholine synthesis, [³H]acetylcholine release, and autoinhibition of [³H]acetylcholine release mediated by muscarinic autoreceptors) were comparatively analyzed in rat brain cortex synaptosomes during postnatal development. These various functions showed a differential time course during development. At 10 days of age the release of [³H]acetylcholine evoked by 15 mM KCl from superfused synaptosomes was Ca²⁺-dependent but insensitive to the inhibitory action of extrasynaptosomal acetylcholine. The muscarinic autoreceptors regulating acetylcholine release were clearly detectable only at 14 days, indicating that their appearance may represent a criterion of synaptic maturation more valuable than the onset of a Ca²⁺-dependent release.     

THE RELEASE IN VIVO OF [3H]ACETYLCHOLINE FROM CAT CAUDATE NUCLEUS AND CEREBRAL CORTEX BY ATROPINE, PENTYLENETETRAZOL, K+-DEPOLARIZATION AND ELECTRICAL STIMULATION

Abstract— In the present experiments, the resting and stimulus evoked release of newly synthesized [³H]acetylcholine from the caudate nucleus, the cerebral cortex and the cerebellar cortex into the perfusate of the push-pull cannula was studied in the unanesthetized, midpontine, pretrigeminally transected cat following infusion at the push-pull site of [³H]choline. Separation of the metabolites in the perfusate revealed that after 20 min, approximately 20% of the recovered radioactivity in the sample was in a lipid fraction, about 10% was found to be phosphorylcholine and around 3% was observed to be incorporated into acetylcholine. The rest of the recovered radioactivity remained as choline. Electrical stimulation applied directly to the caudate nucleus, local potassium depolarization, atropine and pentylenetetrazol were all observed to result in a significant and stimulus dependent increase in the levels of [³H]acetyIchoIine, but not [³H]choline or [¹⁴C]urea in the effluent of the push-pull cannula located in the caudate nucleus. A similar release of newly synthesized [³H]acetylcholine was observed following atropine and potassium stimulation in the cerebral but not the cerebellar cortex. The specificity of this evoked increase in the levels of [³H]acetylchoiine is substantiated by obtaining the release with stimuli having different modes of action, by the absence of stimulus evoked changes in the levels of other water-soluble elements found in the perfusate and by the absence of an observable release of [³H]acetylcholine in perfusion experiments involving the cerebellum, a tissue not thought to have strong cholinergic innervation. The percentage increases in release of [³H] acetylcholine over baseline levels evoked by the various methods closely corresponded to those reported in the literature for authentic acetylcholine. This was taken to suggest that the neuronal pools containing endogenous acetylcholine and those containing newly synthesized acetylcholine, if not identical, were disposed to behave in the same manner following the activation of the synapse.