Inhibition of striatal acetylcholine release by endogenous serotonin

We have examined the hypothesis that endogenous serotonin (5-HT) exerts an inhibitory influence on the release of acetylcholine (ACh) in striatum. Striatal slices were prepared from adult rats, preincubated with [3H]choline, superfused, and exposed to electrical field stimulation. The stimulation-induced overflow of tritium into the superfusate was used as a measure of ACh release. We observed that fluoxetine, an inhibitor of 5-HT uptake, reduced ACh overflow in slices prepared from caudal striatum, an area of high 5-HT concentration, but not in slices from rostral striatum, an area of low 5-HT concentration. Moreover, methysergide, a 5-HT antagonist, increased ACh efflux in caudal but not rostral striatum. Finally, direct activation of 5-HT receptors with the 5-HT agonist, quipazine, inhibited stimulation-induced ACh overflow in both rostral and caudal striatum. These results suggest that endogenous 5-HT normally is capable of inhibiting striatal ACh release, and that the extent of the modulation is related to the degree of serotonergic innervation. In addition, 5-HT receptors capable of modulating ACh release are present in 5-HT-poor rostral striatum, as well as in 5-HT-rich caudal striatum.     

Regulation of NMDA-stimulated [C]GABA and [H]acetylcholine release by striatal glutamate and dopamine receptors

Striatal function is heavily influenced by glutamatergic and dopaminergic afferent input. To ultimately better understand how the N-methyl- -aspartate (NMDA) antagonist, phencyclidine (PCP), alters striatal function, we sought to determine how NMDA receptor function is influenced by activation of other glutamatergic receptors and by dopaminergic receptors. To this end, we used NMDA-stimulated efflux of [¹⁴C]GABA and [³H]acetylcholine (ACh) from striatal slices to assess the influence of these receptors on NMDA function. NMDA-stimulated [¹⁴C]GABA release was more sensitive to NMDA and glycine antagonists than was [³H]ACh release, suggesting that different NMDA receptors regulate the release of these neurotransmitters. Furthermore, NMDA-stimulated [³H]ACh release was inhibited by a D₂ receptor mechanism whereas NMDA-stimulated [¹⁴C]GABA release was enhanced by D₁ receptor activation. NMDA and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) interact additively to evoke [³H]ACh release, and synergistically to evoke [¹⁴C]GABA release. An additive effect of NMDA and kainate (KA) was found on [¹⁴C]GABA release, but NMDA and KA acted in a less than additive manner in evoking [³H]ACh release. KA-stimulated [³H]ACh release was largely blocked by NMDA antagonists, suggesting mediation through activation of NMDA receptors, probably secondary to KA-induced glutamate release. A selective group II metabotropic receptor agonist inhibited NMDA-stimulated [¹⁴C]GABA and [³H]ACh release. On the other hand, NMDA-stimulated [¹⁴C]GABA release was potentiated by activation of group I metabotropic receptors. Thus, in addition to the differential modulation by D₁- and D₂-like receptors, the release of striatal neurotransmitters by NMDA receptor activation depends on the extent to which the other glutamate receptors, both ionotropic and metabotropic, are activated.     

Effects of estrogen on acetylcholine release in frontal cortex of female rats: involvement of serotonergic neuronal systems

The effects of estrogen on cortically projecting cholinergic neurons were investigated using in vivo microdialysis to measure cortical basal acetylcholine (ACh) levels and serotonin (5-HT)-stimulated ACh release in frontal cortex of freely moving Wistar female rats. Bilateral ovariectomy (OVX) or sham operations were performed under anesthesia. Immediately after surgery, each OVX animal was subcutaneously implanted with pellet containing 0.1/0.5 mg of 17beta-estradiol (E(2)) or a vehicle. Nineteen days later, a transverse microdialysis probe was stereotaxically implanted in the frontal cortex (AP: +2.7 mm, DV: -2.5 mm relative to bregma). Two days later (21 days after beginning of estrogen treatment), in vivo microdialysis experimentation was conducted. Serum E(2) levels of animals with 0.1 and 0.5 mg-pellets were equivalent to those levels during diestrous and proestrous, respectively. Although the replacement of different amounts of E(2) produced significant changes in body weight, it failed to affect basal ACh levels in the frontal cortex. Systemically administered serotonin releasing agent, fenfluramine, significantly increased cortical ACh release in all animal groups. The fenfluramine's ability to increase ACh release was potentiated by E(2) replacement with a 0.5 mg-pellet. E(2)-induced enhancement was also observed when the selective 5-HT(1A) agonist, 8-hydroxy-2-(di-n-propylamino) tetralin, but not the 5-HT(2A/2C) agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, was administered. Therefore, the effect of estrogen on 5-HT-stimulated ACh release might be exerted partly via 5-HT(1A) receptors, and not via 5-HT(2) receptors. These results suggest that the positive effects of estrogen on cognitive functions might be mediated through the ACh-5-HT interactions.     

Effects of 250 mg% ethanol on monoamine and amino acid release from rat striatal slices

A single IP injection of 2.5 g ethanol/kg body weight into the rat increased the striatal levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) one hour later to 133 and 141% of control values, respectively. Blood alcohol concentrations at this time were approximately 250 mg%. The increased striatal tissue levels of DOPAC and HVA found after IP administration did not appear to be due to a direct effect of ethanol on the efflux of these two metabolites or on the release of dopamine (DA) since in vitro studies with striatal slices demonstrated that 250 mg% ethanol had no effect on the endogenous release of DOPAC, HVA, or DA. However, ethanol did enhance the K+-stimulated, Ca2+-dependent release of glutamate and aspartate from striatal slices to 168 and 214% of control values, respectively. The release of glutamate and aspartate from slices of midbrain (minus colliculi) was also increased by 250 mg% ethanol. On the other hand, the release of GABA, NE and 5-HT did not appear to be significantly altered by 250 mg% ethanol. The in vitro findings have led to the hypothesis that the elevated DOPAC and HVA levels observed in the striatum following an acute IP injection of 2.5 g/kg of ethanol are due to increased release of DA produced by the excitatory actions of glutamate (and/or aspartate) on dopaminergic neurons.     

Sex mediates dopamine and adrenergic receptor expression in adult rats exposed prenatally to cocaine

The extent of catecholaminergic receptor and respective behavioral alterations associated with prenatal cocaine exposure varies according to exogenous factors such as the amount, frequency, and route of maternal exposure, as well as endogenous factors such as specific brain regions under consideration and sex of the species. The goal of the current study was to use autoradiography to delineate possible moderators of dopaminergic and adrenergic receptor expression in adult rat offspring exposed to cocaine in utero. The current study demonstrated sex-dependent D1 receptor, alpha2, and noradrenergic transporter binding alterations in prelimbic, hippocampus, and anterior cingulate regions of adult rat brains exposed to cocaine during gestational days 8-21. Of further interest was the lack of alterations in the nucleus accumbens for nearly all receptors/transporters investigated, as well as the lack of alterations in D3 receptor binding in nearly all of the regions investigated (nucleus accumbens, prelimbic region, hippocampus, and cingulate gyrus). Thus, the current investigation demonstrated persistent receptor and transporter alterations that extend well into adulthood as a result of cocaine exposure in utero. Furthermore, the demonstration that sex played a mediating role in prenatal cocaine-induced, aberrant receptor/transporter expression is of primary importance for future studies that seek to control for sex in either design or analysis.     

[I]Epibatidine-labelled nicotinic receptors in the extended striatum and cerebral cortex: lack of association with serotonergic afferents

In rat extended striatum, most nicotinic cholinoceptors are likely to be presynaptic. A previous report suggested that DA and 5-HT afferents each account for at least 30% of nicotinic binding sites in the striatum. To explore this question further, rats received unilateral infusions of the neurotoxins 5,7-dihydroxytryptamine, 6-hydroxydopamine or vehicle into the medial forebrain bundle, and were sacrificed 3 weeks later. Denervation was quantified by [¹²⁵I]RTI-55 autoradiography, using separate assay conditions that revealed DA and 5-HT transporters (i.e. DAT and SERT). Nicotinic cholinoceptors were quantified by [¹²⁵I]epibatidine autoradiography. Infusion of 6-hydroxydopamine depleted DAT but not SERT labelling in all striatal areas (i.e. caudate-putamen, nucleus accumbens core and shell, olfactory tubercle). The serotonergic neurotoxin 5,7-dihydroxytryptamine depleted SERT and, to a lesser extent, DAT labelling. Both neurotoxins reduced [¹²⁵I]epibatidine binding in striatal areas. Multiple linear regression analysis showed that these reductions in [¹²⁵I]epibatidine binding were entirely associated with loss of DAT rather than SERT. The DAT-associated proportion of total [¹²⁵I]epibatidine binding was 36±2% (caudate-putamen), 28±3% (accumbens core), 27±4% (accumbens shell) and 44±5% (olfactory tubercle). Cortical [¹²⁵I]epibatidine binding was unaltered by 5,7-dihydroxytryptamine lesions that reduced SERT labelling by 46 to 73%. In all brain areas, even small (3.4 to 8.8%) SERT-associated reductions in [¹²⁵I]epibatidine binding would have been detected as statistically significant. In conclusion, we report the failure to detect nAChRs on 5-HT terminals in extended striatum or cerebral cortex, using a sensitive [¹²⁵I]epibatidine autoradiographic assay.     

Physiological release of striatal acetylcholine (in vivo): effect of somatostatin on dopaminergic-cholinergic interaction

The effects of somatostatin (SOM) on the release of acetylcholine (ACh) and dopamine (DA) from striatum of freely moving rats were studied by transversal microdialysis. Acetylcholine (ACh) and dopamine (DA) were detected by high performance liquid chromatography (HPLC) with electrochemical detection. Somatostatin (0.1, 0.5 and 1 microM), administered locally through the microdialysis probe to the striatum, was able to release dose-dependently ACh from the cholinergic neurons of the striatum. The increase in the extracellular levels of ACh produced by 1 microM SOM in the striatum reached a maximum of 200%. ACh-releasing effect of SOM was completely inhibited by tetrodotoxin indicating that neuronal firing is involved in its effect. Local infusion of sulpiride, 10 microM, D(2) receptor antagonist, potentiated (about 100%) the SOM (1 microM)-induced release of ACh. SOM, 1 microM, was more effective in enhancing the release of ACh in the striatum (two-fold increase) after degeneration of the nigrostriatal DA pathway with 6-hydroxydopamine (6-OHDA) (250 microg/animal, i.c.v.). The D(2) receptor agonists bromcriptine, 10 microM, or apomorphine, 10 microM, completely antagonize SOM-induced release. SOM, 1 microM, enhanced the release of DA (about 400%). These findings indicate that SOM is capable of releasing both ACh and DA in the striatum, however, its effect on ACh release is partially masked unless the D(2) receptor-mediated tonic inhibitory effect of released DA from the nigro-striatal pathway is attenuated.     

5-HT2 receptor regulation of acetylcholine release induced by dopaminergic stimulation in rat striatal slices

The role of 5-hydroxytryptamine (5-HT) receptor subtypes in acetylcholine (ACh) release induced by dopamine or neurokinin receptor stimulation was studied in rat striatal slices. The dopamine D₁ receptor agonist SKF 38393 potentiated in a tetrodotoxin-sensitive manner the K⁺-evoked [³H]ACh release while SCH 23390, a dopamine D₁ receptor antagonist, had no effect. [³H]ACh release was decreased by the dopamine D₂ receptor agonist LY 171555 (quinpirole) and slightly potentiated by the dopamine D₂ receptor antagonist haloperidol. The selective neurokinin NK₁ receptor agonist [Sar⁹, met(O₂)¹¹]SP also potentiated K⁺-evoked release of [³H]ACh. GR 82334, a NK₁ receptor antagonist, blocked not only the effect of [Sar⁹, met(O₂)¹¹]SP but also the release of ACh induced by the D₁ receptor agonist SKF 38393. Among the 5-HT agents studied, only the 5-HT_2A receptor antagonists ketanserin and ritanserin were able to reduce the ACh release induced by dopamine D₁ receptor stimulation. Mesulergine, a more selective 5-HT_2C antagonist, showed an intrinsic releasing effect but did not affect K⁺-evoked ACh release induced by SKF 38393. Methysergide and methiothepin, mixed 5-HT_1/2 antagonists, as well as ondansetron, a 5-HT₃ receptor antagonist, showed an intrinsic effect on ACh release, their effects being additive to that of SKF 38393. 5-HT₂ receptor agonists were ineffective. However, the 5-HT₂ agonist DOI was able to prevent the antagonism by ketanserin of the increased [³H]ACh efflux elicited by SKF 38393, suggesting a permissive role of 5-HT_2A receptors. None of the above indicated 5-HT agents was able to reduce the ACh release induced by the selective NK₁ agonist. The results suggest that 5-HT₂ receptors, probably of the 5-HT_2A subtype, modulate the release of ACh observed in slices from the rat striatum after stimulation of dopamine D₁ receptors. It seems that this serotonergic control is exerted on the interposed collaterals of substance P-containing neurons which promote ACh efflux through activation of NK₁ receptors located on cholinergic interneurons.     

Mercuric chloride and p-chloromercuriphenylsulfonate exert a biphasic effect on the binding of the stimulant [3H]methylphenidate to the dopamine transporter

Mercuric chloride was found to have a biphasic effect on the binding of the radiolabeled stimulant [³H]methylphenidate to membranes from a crude synaptosomal preparation of rat striatal tissue. Binding was enhanced at low concentrations of HgCl₂, reaching a maximum of 62% above control values at 2.5 μ HgCl₂. It was inhibited in a dose-dependent manner at concentrations greater than 5 μ HgC1₂, with an IC₅₀ of 7.2 μ. The increase in binding observed at the low concentrations of HgC1₂ was shown by Scatchard analysis to be due to an increase in the affinity of [³H]methylphenidate for its binding site on the dopamine transporter, while a decrease in both affinity and B_max accompanied the reduction of [³H]methylphenidate binding observed at the higher concentrations of the inorganic mercury compound. The sodium salt of the organic mercurial p-chloromercuriphenylsulfonic acid also caused an increase in [³H]methylphenidate binding (159% above controls at 2,000 μ), followed by an immediate decrease in binding at higher concentrations of the reagent. Because both of these mercury-containing compounds have a high propensity for interacting with sulfhydryl groups, these data suggest that the cysteine residues in the dopamine transporter molecule may play an important role in the regulation of stimulant binding to the uptake complex. © 1994 Wiley-Liss, Inc.     

The interaction of acetylcholinergic and serotonergic neural systems on performance in a continuous non-matching to sample task

The interactions of cholinergic and serotonergic neural systems were investigated in a working memory, continuous non-matching-to-sample task in an operant chamber. Each rat was injected (i.p.) with scopolamine and/or methysergide. The serotonergic system was destroyed by p-chloroamphetamine (PCA) injection. Scopolamine, but not PCA, impaired response accuracy. The effects of methysergide and scopolamine together were not different from scopolamine alone. Scopolamine had a significantly smaller effect on choice accuracy in PCA-treated than in untreated rats.     

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.     

Opposing effects of narcotic gases and pressure on the striatal dopamine release in rats

Nitrogen-oxygen breathing mixtures, for pressures higher than 0.5 MPa, decrease the release of dopamine in the rat striatum, due to the narcotic potency of nitrogen. In contrast, high pressures of helium-oxygen breathing mixtures of more than 1-2 MPa induce an increase of the striatal dopamine release and an enhancement of motor activity, referred to as the high pressure nervous syndrome (HPNS), and attributed to the effect of pressure per se. It has been demonstrated that the effect of pressure could be antagonized by narcotic gas in a ternary mixture, but most of the narcotic gas studies measuring DA release were executed below the threshold for pressure effect. To examine the effect of narcotic gases at pressure on the rat striatal dopamine release, we have used two gases, with different narcotic potency, at sublethargic pressure, nitrogen at 3 MPa and argon at 2 MPa. In addition, to dissociate the effect of the pressure, we have used nitrous oxide at 0.1 MPa to induce narcosis at very low pressure, and helium at 8 MPa to study the effect of pressure per se. In all the narcotic conditions we have recorded a decrease of the striatal dopamine release. In contrast, helium pressure induced an increase of DA release. For the pressures used, the results suggest that the decrease of dopamine release was independent of such an effect of the pressure. However, for the same narcotic gas, the measurements of the extracellular DA performed in the striatum seem to reflect an opposing effect of pressure, since the decrease in DA release is lower with increasing pressure.     

Differential modulation of NMDA-stimulated [H]dopamine release from rat striatum by neuropeptide Y and σ receptor ligands

Although the identity of the endogenous ligands for sigma (σ) receptors is unknown, neuropeptide Y (NPY) has been named as a possible candidate for a natural transmitter at these receptors. Using a superfusion system, we compared the effect of NPY on NMDA-stimulated [³H]dopamine release in rat striatum to that of the σ agonists (+)-pentazocine and BD737. In contrast to (+)-pentazocine- or BD737-mediated inhibition of release, NPY enhanced release. However, the same σ antagonists (BD1008, DuP734, haloperidol and DTG) that reverse (+)-pentazocine- or BD737-mediated inhibition, as well as a Y receptor antagonist, PYX-1, all reversed the enhancement. PYX-1 also reversed the (+)-pentazocine- and BD737-mediated inhibition of release. Peptide YY (PYY) and [Leu³¹,Pro³⁴]NPY did not mimic the effect of NPY. NPY_13–36 enhanced release to the same extent as NPY but the effect was not reversed by σ antagonists. Our findings are consistent with the potential role of NPY as an endogenous ligand for a subtype of σ receptor with characteristics different from Y₁, Y₂ and Y₃ receptors but sensitive to PYX-1.     

Regulation of NMDA-stimulated [14C]GABA and [3H]acetylcholine release by striatal glutamate and dopamine receptors.

Striatal function is heavily influenced by glutamatergic and dopaminergic afferent input. To ultimately better understand how the N-methyl-D-aspartate (NMDA) antagonist, phencyclidine (PCP), alters striatal function, we sought to determine how NMDA receptor function is influenced by activation of other glutamatergic receptors and by dopaminergic receptors. To this end, we used NMDA-stimulated efflux of [14C]GABA and [3H]acetylcholine (ACh) from striatal slices to assess the influence of these receptors on NMDA function. NMDA-stimulated [14C]GABA release was more sensitive to NMDA and glycine antagonists than was [3H]ACh release, suggesting that different NMDA receptors regulate the release of these neurotransmitters. Furthermore, NMDA-stimulated [3H]ACh release was inhibited by a D2 receptor mechanism whereas NMDA-stimulated [14C]GABA release was enhanced by D1 receptor activation. NMDA and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) interact additively to evoke [3H]ACh release, and synergistically to evoke [14C]GABA release. An additive effect of NMDA and kainate (KA) was found on [14C]GABA release, but NMDA and KA acted in a less than additive manner in evoking [3H]ACh release. KA-stimulated [3H]ACh release was largely blocked by NMDA antagonists, suggesting mediation through activation of NMDA receptors, probably secondary to KA-induced glutamate release. A selective group II metabotropic receptor agonist inhibited NMDA-stimulated [14C]GABA and [3H]ACh release. On the other hand, NMDA-stimulated [14C]GABA release was potentiated by activation of group I metabotropic receptors. Thus, in addition to the differential modulation by D1- and D2-like receptors, the release of striatal neurotransmitters by NMDA receptor activation depends on the extent to which the other glutamate receptors, both ionotropic and metabotropic, are activated.     

Lateralized changes in behavior and striatal dopamine release following unilateral tactile stimulation of the perioral region: a microdialysis study

Intracranial microdialysis was used to measure dopamine (DA) release in the ventrolateral neostriatum of freely moving rats before and after unilateral tactile stimulation was applied to the orofacial region. Several behavioral parameters which have been linked to changes in nigrostriatal DA transmission (scanning, or snout contact with the walls of the observation chamber, turning and locomotion) were measured as well. Orofacial stimulation was followed by an asymmetrical increase in DA release with concentrations of transmitter higher in the neostriatum ipsilateral to the side of stimulation. Asymmetrical scanning behavior was observed during the time period when DA release was asymmetric, with rats favoring use of the side of the face contralateral to increased DA release. Increase in the DA metabolites DOPAC and HVA were found in the striatum ipsilateral to stimulation, but were delayed 40 min following the increase in DA.     

Extracellular calcium alters frequency modulation of [H]acetylcholine release from rat hippocampal slices

The concentration of extracellular Ca²⁺ has been shown to enhance or attenuate [³H]acetylcholine (ACh) release subsequent to a conditioning stimulus in rat brain hippocampal slices. Slices were incubated in vitro in [³H]choline solution. Subsequently the slices were subjected to two consecutive electrical stimulations separated by 15 or 30 min at 0.25, 1, 4 and 16 Hz and [³H]ACh release was assessed. It was found that a conditioning stimulus may reduce [³H]ACh release during a second stimulation. This phenomenon is frequency related and disappears when the two stimulations are 30 min apart. High extracellular Ca²⁺ (4.0 mM) further attenuated [³H]ACh release during the second stimulation whereas low Ca²⁺ (0.32 mM) abolished the decrease in [³H]ACh release following the second stimulation in all frequencies tested.     

Daily cocaine treatment produces a persistent reduction of [H]dopamine uptake in vitro in rat nucleus accumbens but not in striatum

Daily administration of cocaine (15 mg/kg i.p. × 3 days) led to a decrease in the total amount of 15 nM [³H]dopamine uptake in rat nucleus accumbens with no changes in uptake in the striatum when tested 24 h after the last injection. There was an increase in theK_m for dopamine uptake in the nucleus accumbens of cocaine-treated rats, with no change in theV_max. Furthermore, cocaine was a more potent inhibitor of [³H]dopamine uptake in vitro in the nucleus accumbens of rats treated with cocaine than in those which had received saline.     

Modulatory action of dopamine on acetylcholine-responsive striatal and accumbal neurons in awake, unrestrained rats

In ambulant rats, iontophoresis of low concentrations of dopamine (DA) enhances the response of neurons in striatum and nucleus accumbens to iontophoretic glutamate. In an extension of this line of investigation, we tested the effects of acetylcholine (ACh), a presumed modulator of neuronal function in these same brain regions, and assessed possible DA-ACh interactions. Data were obtained from spontaneously active neurons known to respond to ACh (5–30 nA) when the animals rested quietly with no overt movement. ACh iontophoresis either excited or inhibited striatal and accumbal activity but excitatory effects predominated in both areas. With multiple applications of ACh, especially at the lowest currents tested, either response often was interspersed with instances of no change in firing rate. Responsiveness to ACh also diminished during periods of spontaneous movement when basal firing showed phasic increases in activity. In fact, neurons with the highest rates of basal activity showed the smallest magnitude response to ACh. Prolonged applications (120–180 s) of DA attenuated basal firing as well as the iontophoretic effects of ACh both during the DA application itself and for up to 1 min after DA ejection offset. The result of these inhibitory effects was no net change in the relative magnitude of the ACh response. Thus, although ACh can modulate striatal and accumbal neuronal activity, DA does not regulate this effect in the same way that it regulates the neuronal responsiveness to glutamate.     

Differential long-term effect of AF64A on [H]ACh synthesis and release in rat hippocampal synaptosomes

The activities of various presynaptic cholinergic parameters were determined in hippocampal synaptosomes of rats 29 weeks after intracerebroventricular injection of ethylcholine aziridinium (AF64A) (3 nmol/2 μl/side) or vehicle (saline). Synaptosomes were preloaded with [³H]choline ([³H]Ch), treated with diisopropyl fluorophosphate to inhibit cholinesterase activity and then were assayed for their content of [³H]Ch and [³H]acetylcholine ([³H]ACh) and for their ability to synthesize and release [³H]ACh. In synaptosomes from AF64A-treated rats compared with synaptosomes from vehicle-treated rats we observed that: (i) specific uptake of [³H]ACh was reduced to 60% of control; (ii) residing [³H]ACh levels were 43% of control while residing [³H]Ch levels were 72% of control; (iii) basal and K⁺-induced [³H]ACh release were 77% and 73% of control, respectively; (iv) high K⁺-induced synthesis of [³H]ACh was only 9% of control; (v) but, choline acetyltransferase activity remained relatively high, being 80% of control. These results suggest that AF64A-induced cholinergic hypofunction is expressed by both loss of some cholinergic neurons and impairment in the functioning of the spared neurons.