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.     

Storage and release of acetylcholine in rat cortical synaptosomes: Effects ofd,l-2-(4-phenylpiperidino)cyclohexanol (AH5183)

A post-stimulation synthesis of acetylcholine (ACh), its incorporation into a 'stable-bound' (vesicular) compartment and subsequent release, were compared in K+-stimulated synaptosomes, in the absence and presence of 10 microM AH5183. The drug depressed by 16% the net intrasynaptosomal formation of ACh from 1 microM [3H]choline (Ch) in the medium, by competitively inhibiting (Ki approximately equal to 20 microM) the high-affinity Ch transport, but it had no direct effect on the intraterminal synthesis of ACh per se. The drug reduced incorporation of newly synthesized [3H]ACh into synaptic vesicles by 55% and subsequent K+-depolarization-induced release of [3H]ACh by 83%, although it had no effect on Ca2+ influx into synaptosomes. These results are consistent with the hypothesis that AH5183 blocks cholinergic neurotransmission presynaptically by interfering with recharging of synaptic vesicles with ACh. Since the reduction of ACh release in the presence of AH5183 had no direct effect on ACh synthesis, these results also suggest that the transmitter release is not prerequisite for enhancement of Ch uptake and ACh synthesis in stimulated nerve terminals.     

Pirenzepine-insensitive muscarinic autoreceptors regulate acetylcholine release in human neocortex

The release of [³H]acetylcholine ([³H]ACh) and its modulation mediated by autoreceptors were investigated in synaptosomes prepared from fresh human cerebral cortex prelabelled with [³H]choline ([³H]Ch) and depolarized in superfusion with 15 mM KCl. The K⁺-evoked release of tritium was almost totally accounted for by unmetabolized [³H]ACh and was largely calcium-dependent. Exogenous ACh decreased the depolarization-evoked release of [³H]ACh in a concentration-dependent manner (EC₅₀ = 1.5 μM). The inhibitory effect of ACh on [³H]ACh release was counteracted by the non-selective muscarinic antagonist atropine. In contrast, the selective M₁ receptor antagonist pirenzepine was ineffective. It is concluded that muscarinic autoreceptors regulating the release of ACh are present on cholinergic nerve terminals of human cerebral cortex and appear to belong to a pirenzepine-insensitive subtype.     

Compensatory elevation of acetylcholine synthesis in vivo by cholinergic neurons surviving partial lesions of the septohippocampal pathway

The present study characterized the effects of partial destruction of the cholinergic septohippocampal pathway on transmitter functions of surviving cholinergic neurons in the hippocampus. Partial and full fimbrial transections were performed, and 3 weeks after lesioning, cholinergic functions were assessed in vivo and in vitro. Hippocampal ChAT activity and the capacity of hippocampal slices to synthesize [3H]ACh in vitro decreased by 35% and 45%, respectively, following partial fimbrial lesions and by 68% and 85%, respectively, following full fimbrial lesions. [3H]ACh release from hippocampal slices in vitro was decreased by 57% and 87%, respectively, following partial and full fimbrial lesions. Partial lesions decreased high-affinity choline uptake into hippocampal synaptosomes by 52%. In contrast to the significant reductions in cholinergic parameters measured in vitro after partial fimbrial lesions, such partial lesions did not significantly alter in vivo measures of hippocampal cholinergic function. Levels of endogenous ACh and choline measured in the hippocampus following partial lesions were similar to that of control values. Also, the hippocampal content of newly synthesized [2H4]ACh and the [2H4]ACh synthesis rate were not significantly different from control values. However, following full fimbrial lesions, in vivo measures of hippocampal cholinergic function were decreased to a degree similar to that observed in vitro. Hippocampal levels of endogenous ACh and [2H4]ACh and the synthesis rate for [2H4]ACh were decreased by 73%, 72%, and 83%, respectively. These results suggest that, following partial destruction of afferent cholinergic fibers that innervate the hippocampal formation, residual cholinergic neurons are able to upregulate their capacity to synthesize and store ACh in vivo, thus compensating for lesion-induced losses of cholinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Galanin inhibits acetylcholine release from rat cerebral cortex via a pertussis toxin-sensitive G(i)protein

Galanin has been implicated in various physiological functions including memory, feeding and pain perception. Using rat cerebral cortical slices and synaptosome preparations incubated with [(3)H]choline in Kreb's-Ringer solution, galanin was shown to inhibit both spontaneous and K(+)-stimulated [(3)H]ACh release in a concentration-related manner [EC(50)= 35 nM]. The galanin-mediated inhibition on spontaneous and K(+)-stimulated [(3)H]ACh release was respectively regulated by pertussis toxin-sensitive G(alphai3)and G(alphai1). These suggest that galanin is a negative modulator of cortical cholinergic function and most probably acting on presynaptic cholinergic terminals. Although galantide blocked the galanin-mediated inhibitory effect on [(3)H]ACh release, it mimicked galanin in blocking K(+)-stimulated [(3)H]ACh release, indicating that galantide may have a more complicated pharmacology than being a galanin receptor antagonist. In addition, we demonstrate that galanin and beta-amyloid peptide(1-42)synergistically attenuated K(+)-evoked [(3)H]ACh release from synaptosomes prepared from rat cerebral cortex. Since galanin is increased in Alzheimer's disease brain, our results suggest that galanin may be involved in cholinergic dysfunctions that occur in Alzheimer's disease.     

Effects of 4-(2-Hydroxyethyl)-1-piperazine-ethanesulfonic acid (AH5183) on rat cortical synaptosome choline uptake, acetylcholine storage and release

The cholinergic vesicular uptake blocker, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (AH5183), had several effects on presynaptic cholinergic function that depended on the duration of treatment and dose. The synthesis, storage and release of newly synthesized [3H]ACh were monitored because the vesicular uptake of this pool of transmitter may be preferentially affected by the drug. Initially, high concentrations of AH5183 (over 10 microM) increased the spontaneous release but decreased the K+ depolarization-induced release of newly synthesized transmitter. [3H]Choline efflux was not altered by the drug. High affinity choline uptake was slightly (10-20%) inhibited by AH5183 in an apparently competitive but time-dependent manner. In contrast to its initial effects on [3H]ACh release, AH5183 (50nM-100 microM) very potently inhibited both the spontaneous and K+-induced release of [3H]ACh but not of [3H]choline after a 60 min preincubation. [3H]ACh levels in cytoplasmic (S3) and crude membrane (P3) fractions were not affected by a 2-min incubation with 10 microM AH5183. After a 60-min preincubation with this drug dose, however, the P3 and S3 levels of newly synthesized transmitter were decreased and increased, respectively. Subsequent fractionation of synaptosomes by sucrose-density gradient centrifugation revealed that these reductions in P3 [3H]ACh-levels were referable to reductions in two subfractions D and H that have been reported to contain low density vesicles and denser vesicles associated with plasma membranes, respectively.     

Presynaptic modulation of acetylcholine,noradrenaline, and serotonin release in the hippocampus of aged rats with various levels of memory impairments

Aged (25-27 months) Long-Evans female rats were distinguished according to whether they showed no significant impairment (AU), moderate impairment (AMI), or severe impairment (ASI) in a spatial reference-memory task. Young (3-5 months) rats served as controls. Electrically evoked overflow of tritium was assessed in hippocampal slices preloaded with [3H]choline or [3H]serotonin (5-HT). Nicotine-evoked overflow of tritium was measured after preloading with [3H]noradrenaline (NA). Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, and concentration of monoamines were assessed in homogenates. Aged rats exhibited reduced accumulation of [3H]choline and [3H]5-HT, increased accumulation of [3H]NA, and weaker electrically evoked overflow of [3H]acetylcholine ([3H]ACh) and [3H]5-HT. The overflow of [3H]NA was not altered consistently by aging. Roughly, drugs acting presynaptically had comparable effects in aged rats: oxotremorine and CP 93,129 inhibited the overflow of [3H]ACh, CP 93,129 and UK 14,304 reduced that of [3H]5-HT. ChAT or AChE activity, and 5-HT concentration were not changed by age; NA concentration was reduced. When significant, changes were comparable in AU, AMI, and ASI rats. Data show that aging alters cholinergic and serotonergic hippocampal innervations, release of ACh and 5-HT, but not presynaptic release-modulating mechanisms. These alterations do not account for variability in water-maze performance of aged rats.     

MKC-231, a choline uptake enhancer: (2) Effect on synthesis and release of acetylcholine in AF64A-treated rats

The effect of MKC-231 on acetylcholine (ACh) synthesis and release was studied in the hippocampus of normal and AF64A-treated rats. AF64A (3 nmol/brain, i.c.v.) produced significant reduction of high-affinity choline uptake (HACU) and high K+-induced ACh release in hippocampal synaptosomes. Treatments with MKC-231 (10(-8) and 10(-7) M) showed significant reverse of the decrease in both HACU and ACh release. In hippocampal slices superfused with choline-containing artificial cerebro-spinal fluid (ACSF), high K+-induced ACh release was gradually decreased by repeated alteration of resting and high K+ stimulations in AF64A-treated rats. However, addition of MKC-231 (10(-8) to 10(-7) M) in the superfusate reduces this decrease. In vivo microdialysis studies indicate MKC-231 (10 mg/kg, p.o.) significantly reversed reduction of basal ACh concentrations in AF64A-treated rats, measured by radioimmunoassay without a cholinesterase inhibitor in the perfusate. These results indicate MKC-231 improves AF64A-induced cholinergic hypofunction by enhancing HACU, subsequently facilitating ACh synthesis and release in vitro and in vivo.     

Downregulation of muscarinic- and 5-HT1B-mediated modulation of [H]acetylcholine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts of septal origin

Adult Long-Evans female rats sustained electrolytic fimbria-fornix lesions and, two weeks later, received intrahippocampal suspension grafts of fetal septal tissue. Sham-operated and lesion-only rats served as controls. Between 6.5 and 8 months after grafting, both the [³H]choline accumulation and the electrically evoked [³H]acetylcholine ([³H]ACh) release were assessed in hippocampal slices. The release of [³H]ACh was measured in presence of atropine (muscarinic antagonist, 1 μM), physostigmine (acetylcholinesterase inhibitor, 0.1 μM), oxotremorine (muscarinic agonist, 0.01 μM–10 μM), mecamylamine (nicotinic antagonist, 10 μM), methiothepin (mixed 5-HT₁/5-HT₂ antagonist, 10 μM), 8-OH-DPAT (5-HT_1A agonist, 1 μM), 2-methyl-serotonin (5-HT₃ agonist, 1 μM) and CP 93129 (5-HT_1B agonist, 0.1 μM–100 μM), or without any drug application as a control. In lesion-only rats, the specific accumulation of [³H]choline was reduced to 46% of normal and the release of [³H]ACh to 32% (nCi) and 43% (% of tissue tritium content). In the grafted rats, these parameters were significantly increased to 63%, 98% and 116% of control, respectively. Physostigmine reduced the evoked [³H]ACh release and was significantly more effective in grafted (−70%) than in sham-operated (−56%) or lesion-only (−54%) rats. When physostigmine was superfused throughout, mecamylamine had no effect. Conversely, atropine induced a significant increase of [³H]ACh release in all groups, but this increase was significantly larger in sham-operated rats (+209%) than in the other groups (lesioned: +80%; grafted: +117%). Oxotremorine dose-dependently decreased the ([³H]ACh) release, but in lesion-only rats, this effect was significantly lower than in sham-operated rats. Whatever group was considered, 8-OH-DPAT, methiothepin and 2-methyl-serotonin failed to induce any significant effect on [³H]ACh release. In contrast, CP 93129 dose-dependently decreased [³H]ACh release. This effect was significantly weaker in grafted rats than in the rats of the two other groups. Our data confirm that cholinergic terminals in the intact hippocampus possess inhibitory muscarinic autoreceptors and serotonin heteroreceptors of the 5-HT_1B subtype. They also show that both types of receptors are still operative in the cholinergic terminals which survived the lesions and in the grafted cholinergic neurons. However, the muscarinic receptors in both lesioned and grafted rats, as well as the 5-HT_1B receptors in grafted rats show a sensitivity which seems to be downregulated in comparison to that found in sham-operated rats. In the grafted rats, both types of downregulations might contribute to (or reflect) an increased cholinergic function that results from a reduction of the inhibitory tonus which ACh and serotonin exert at the level of the cholinergic terminal.     

Neurotransmitter release and its presynaptic modulation in the rat hippocampus after selective damage to cholinergic or/and serotonergic afferents

Male Long-Evans rats sustained injections of 5,7-dihydroxytryptamine (5,7-DHT) into the fimbria-fornix and the cingular bundle or/and intraseptal injections of 192 IgG-saporin to induce serotonergic or/and cholinergic hippocampal denervations; Sham-operated rats served as controls. Four to ten weeks after lesioning, we measured (i). the electrically evoked release of acetylcholine ([3H]ACh), noradrenaline ([3H]NA) and serotonin ([3H]5-HT) in hippocampal slices in the presence of drugs acting on auto- or heteroreceptors, (ii). the nicotine-evoked release of NA and (iii). the choline acetyltransferase (ChAT) activity and the concentration of monoamines in homogenates. Saporin lesions reduced the accumulation of [3H]choline, the release of [3H]ACh and the ChAT activity, but increased the concentration of NA and facilitated the release of [3H]NA evoked by nicotine. 5,7-DHT lesions reduced the accumulation and the release of [3H]5-HT, the concentration of 5-HT, and also facilitated the release of [3H]NA evoked by nicotine. Accumulation and electrically evoked release of [3H]NA were not altered by either lesion. The combination of both toxins resulted in an addition of their particular effects. The 5-HT(1B) receptor agonist, CP 93129, and the muscarinic agonist, oxotremorine, reduced the release of [3H]ACh in control and 5,7-DHT-lesioned rats; in rats injected with saporin, their effects could not be measured reliably. CP 93129 and the alpha(2)-adrenoceptor agonist, UK 14304, reduced the release of [3H]5-HT in all groups by about 65%. In conclusion: (i). selective neurotoxins can be combined to enable controlled and selective damage of hippocampal transmitter systems; (ii). 5-HT exerts an inhibitory influence on the nicotine-evoked release of NA, but partial serotonergic lesions do not influence the release of ACh at a presynaptic level and (iii). presynaptic modulatory mechanisms involving auto- and heteroreceptors may be conserved on fibres spared by the lesions.     

MKC-231, a choline uptake enhancer: (3) mode of action of MKC-231 in the enhancement of high-affinity choline uptake

MKC-231, a putative cholinergic activity, is reported to improve learning and memory impaired in AF64A-treated animals. MKC-231 enhances high-affinity choline uptake (HACU) known as the rate-limiting step of acetylcholine (ACh) synthesis. We investigated the mode of action (MOA) of HACU enhancement by MKC-231. Intracerebroventricular (i.c.v.) injections of AF64A (3 nmol/brain) resulted in significant HACU reduction in hippocampal synaptosomes. Treatment with MKC-231 increased Vmax of HACU and Bmax of [3H]-HC-3 binding 1.6 and 1.7-fold, respectively. In studies of [3H]-MKC-231 binding and Biacore analysis, MKC-231 showed noticeable affinity for cloned high-affinity choline transporters (CHT1). The present study suggests that MKC-231 directly affects trafficking of CHT1 and increases the numbers of transporter, working for HACU, at the synaptic membrane.     

Effects of AF64A on [3H]acetylcholine synthesis in neuron-enriched primary brain cell cultures

[3H]Acetylcholine (ACh) synthesis was measured in primary neuronal cultures from neonatal rat brains. Neuronal [3H]ACh synthesis was blocked by hemicholinium-3 and depended on the age of the cultures, increasing for ca. 10 days, and eventually declining. The irreversible inhibitor AF64A (10 or 30 microM) inhibited [3H]ACh synthesis from [3H]choline at concentrations (10 or 30 microM) with affecting choline acetyltransferase activity. Nine-day-old cultures recovered 90% of their [3H]ACh synthesis within 7 days after AF64A, while 13-day-old cultures never recovered. These results suggest that the turnover of neuronal choline transporters is age-related.     

Inhibition of high affinity choline transport attenuates both cholinergic and non-cholinergic effects of ethylcholine aziridinium (AF64A)

Ethylcholine aziridinium (AF64A) has been proposed as a specific cholinergic neurotoxin. In earlier studies, using AF64A, we reported that slow infusion of 1-2 nmol of this compound into each lateral ventricle of Sprague-Dawley rats resulted in small, and transient decreases in noradrenaline (NA) and serotonin (5-HT) levels in the hippocampus, while inducing a permanent and significant cholinergic hypofunction in the same brain region. The experiments described in this paper were designed to test the hypothesis that such noradrenergic and serotonergic changes after small doses of AF64A are secondary to the changes observed in cholinergic neurons. Levels of NA, and of 5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) were measured concurrently with levels of acetylcholine (ACh), in various brain regions of rats in which the effect of AF64A was attenuated, and in respective control animals. The effect of AF64A was diminished by inhibiting the interaction of AF64A with the high affinity transport site for choline (HAChT). This was achieved using hemicholinium-3 (HC-3), which does not cross the blood-brain barrier, and A-4 (a bis 4-methylpiperidine analog of HC-3), which is centrally active following its peripheral administration. A-4 (20 or 40 mg/kg i.p.) or HC-3 (10 micrograms/ventricle) had no effect on ACh, NA, 5-HT or 5-HIAA levels in saline-treated rats. However, all treatments significantly attenuated the decrease in ACh content produced by AF64A pretreatment. Transient decreases in NA, 5-HT and 5-HIAA contents after AF64A treatment were prevented or reduced by prior treatment with A-4 or HC-3. These results indicate that changes in noradrenergic and serotonergic neurons following AF64A administration are not due to non-specific toxicity of AF64A, but may be the result of adaptation of these neurons to withdrawal of cholinergic input, which would normally inhibit the release of NA and 5-HT. These results also indicate that AF64A can be used to produce specific lesions of hippocampal cholinergic nerve terminals.     

Septal grafts and evoked acetylcholine release in the rat hippocampus after 192 IgG-saporin lesions

Adult rats received intraseptal injections of 192 IgG-saporin and intrahippocampal grafts of septal cells. Between 6 and 10 months later, we assessed baseline and electrically-evoked release of tritium in hippocampal slices preloaded with [(3)H]choline, and the uptake of [(3)H]choline, [(3)H]noradrenaline and [(3)H]serotonin by hippocampal synaptosomes. The lesions reduced the accumulation of [(3)H]choline by approximately 40%, the evoked release of [(3)H]acetylcholine by approximately 90%, and the uptake of [(3)H]choline by synaptosomes by 90% in the dorsal hippocampus, but increased the relative baseline release of [(3)H]choline by +43%, and the synaptosomal uptake of [(3)H]noradrenaline (66%) and [(3)H]serotonin (58%) in the ventral hippocampus. The increased noradrenaline uptake may account for sympathetic ingrowth. Although the grafts of fetal septal neurons produced modest cholinergic effects, these effects were positive and significant.     

Veratridine-induced breakdown of cytosolic acetylcholine in rat hippocampal minces: An intraterminal form of acetylcholinesterase or choline O-acetyltransferase?

Rat hippocampal minces were loaded with N-methyl-[3H]acetylcholine ([3H]ACh) in the presence of the 'poorly penetrating' acetylcholinesterase (EC 3.1.1.7, AChE) inhibitor echothiophate and the effect of the depolarizing agent veratridine determined on the subcellular storage and release of [3H]ACh and [3H]choline. Results indicated that veratridine stimulated the release of [3H]ACh from a crude vesicular fraction (P3) by a Ca2+-dependent process, while simultaneously accelerating the breakdown of cytosolic (S3) [3H]ACh. A portion of the [3H]choline derived from the hydrolyzed S3 [3H]ACh was donated to the P3 fraction for [3H]ACh formation and release. When the identical experiment was done using hippocampal minces from septal lesioned rats, veratridine did not stimulate either the Ca2+-dependent release of [3H]ACh or the hydrolysis of cytosolic [3H]ACh. Incubation of control hippocampal minces with paraoxon, an AChE inhibitor which can penetrate cholinergic nerve terminals more rapidly than echothiophate, prevented veratridine from stimulating the Ca2+-dependent release of [3H]ACh from the P3 fraction. Instead, it then stimulated the Ca2+-independent release of [3H]ACh from the S3 fraction. When minces were incubated with the choline O-acetyltransferase (EC 2.3.1.6, ChAT) inhibitor 4-(1-naphthyl)vinyl pyridine (NVP), veratridine was no longer able to stimulate the Ca2+-dependent release of labelled ACh either. Instead, veratridine stimulated the Ca2+-independent release of labelled ACh from the S3 fraction. NVP also abolished the veratridine-induced, Ca2+-dependent release of total ACh. Both paraoxon and NVP inhibited the reversible reaction of ionically bound ChAT prepared from rat brain when tested in vitro, yet paraoxon was much less potent than NVP, and was unable to inhibit this reaction at the low concentration which prevented the veratridine induced breakdown of S3 [3H]ACh during mince incubation. Veratridine depolarization of hippocampal minces stimulated the activity of a membrane-bound fraction of ChAT associated with the P3 fraction, but this fraction of ChAT did not become more sensitive to inhibition by paraoxon during tissue incubation. Veratridine depolarization of minces also increased the activity of membrane-bound AChE, but this enzyme was not inhibited by the low NVP concentration which prevented the veratridine-induced breakdown of S3 [3H]ACh. The veratridine-induced increase in membrane-bound ChAT activity was dependent on the presence of extracellular Ca2+ in the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Central interactions between dihydropyridines and cholinergic systems in the control of blood pressure in rat

Intracerebroventricular (i.c.v.) injection of the 1,4-dihydropyridine (DHP) calcium channel agonist, Bay K8644 (30 micrograms/kg) increased mean blood pressure and the K+-evoked release of [3H]acetylcholine ([3H]ACh) from hippocampal slices in spontaneously hypertensive rats (SHR). The Bay K8644-induced hypertension was inhibited by a pretreatment with methylatropine (80 micrograms/kg i.c.v.). In SHR, nicardipine, a DHP calcium channel antagonist, reduced mean blood pressure when i.c.v. injected (10 micrograms/kg). The nicardipine-induced hypotension was reduced by a pretreatment with hemicholinium-3 (20 micrograms, i.c.v.). Nicardipine (1 microM) did not modify, in SHR, the K+-evoked release of [3H]ACh, but inhibited the Bay K8644-induced increase in the ACh release. In normotensive rats, neither Bay K8644 nor nicardipine modify blood pressure, when centrally injected, or the stimulated release of [3H]ACh from hippocampal slices. The participation of central DHP sites in the cholinergic transmission in genetic hypertension is discussed.     

Modulation of acetylcholine and 5-hydroxytryptamine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts containing cholinergic and/or serotonergic neurons

Three-month-old Long-Evans female rats sustained aspirative lesions of the dorsal septohippocampal pathways and, 2 weeks later, received intrahippocampal suspension grafts containing fetal cells from the mesencephalic raphe (rich in serotonergic neurons; RAPHE), the medial septum and the diagonal band of Broca (rich in cholinergic neurons; SEPT), or a mixture of both (COTR). Lesion-only (LES) and sham-operated rats (SHAM) were used as controls. Hippocampal slices of these rats (5-9 month after surgery) were preincubated with [3H]choline or [3H]5-HT, superfused continuously (in the presence of hemicholinium-3 or fluvoxamine) and stimulated electrically (360 pulses, 2 ms, 3 Hz, 26-28 mA) in order to study the presynaptic modulation of acetylcholine (ACh) and serotonin (5-HT) release. The accumulation of [3H]choline and the evoked overflow of [3H]ACh were significantly reduced in slices from LES and RAPHE rats, but reached a close-to-normal level in SEPT and COTR rats. As to accumulation and overflow of [3H]5-HT, the lesion-induced reduction was compensated for only in RAPHE and COTR rats. The relative amount of evoked [3H]5-HT release (in % of tissue-3H) was significantly increased in LES and SEPT rats. Only slight differences (group LES) were found in the sensitivity of muscarinic and serotonergic autoreceptors towards oxotremorine and CP 93,129, respectively. Moreover, CP 93,129 induced a significantly weaker inhibition of ACh release in slices of COTR rats than in all other groups. Using the 5-HT1A receptor agonist 8-OH-DPAT and antagonist Way 100,635, no evidence for a modulatory influence of 5-HT1A receptors was found in RAPHE and COTR rats. It is concluded that despite substantial lesion- and graft-induced changes in the amount of ACh and 5-HT released by hippocampal slices of lesion-only or grafted rats, the presynaptic modulation of these transmitters is only slightly affected by changes in the neuronal environment.     

Muscarinic and dopaminergic receptor subtypes on striatal cholinergic interneurons

Unilateral stereotaxic injection of small amounts of the cholinotoxin, AF64A, caused minimal nonselective tissue damage and resulted in a significant loss of the presynaptic cholinergic markers [3H]hemicholinium-3 (45% reduction) and choline acetyltransferase (27% reduction). No significant change from control was observed in tyrosine hydroxylase or tryptophan hydroxylase activity; presynaptic neuronal markers for dopamine- and serotonin-containing neurons, respectively. The AF64A lesion resulted in a significant reduction of dopamine D2 receptors as evidenced by a decrease in [3H]sulpiride binding (42% reduction) and decrease of muscarinic non-M1 receptors as shown by a reduction in [3H]QNB binding in the presence of 100 nM pirenzepine (36% reduction). Saturation studies revealed that the change in [3H]sulpiride and [3H]QNB binding was due to a change in Bmax not Kd. Intrastriatal injection of AF64A failed to alter dopamine D1 or muscarinic M1 receptors labeled with [3H]SCH23390 and [3H]pirenzepine, respectively. In addition, no change in [3H]forskolin-labeled adenylate cyclase was observed. These results demonstrate that a subpopulation of muscarinic receptors (non-M1) are presynaptic on cholinergic interneurons (hence, autoreceptors), and a subpopulation of dopamine D2 receptors are postsynaptic on cholinergic interneurons. Furthermore, dopamine D1, muscarinic M1 and [3H]forskolin-labeled adenylate cyclase are not localized to striatal cholinergic interneurons.     

AF64A(ethylcholine aziridinium ion)-induced basal forebrain lesion impairs maze performance

Rats were given bilateral injections of ethylcholine aziridinium ion, AF64A (1 nmol/side) into the basal forebrain (BF). One month later, choline acetyltransferase activity was reduced by 25% in the frontal cortex (FC). There was a marked decrease in cortical uptake of [3H]choline, but [3H]GABA and [3H]dopamine uptake was not affected by the injection. Histological analysis confirmed that this dose of AF64A caused acetylcholinesterase staining in the FC to disappear. Acquisition and retention of a T-maze task were impaired in the rats with BF lesions one month after the injection. Acquisition of the water-filled multiple T-maze task was also impaired by AF64A. These observations suggest that the cholinergic component in the BF is involved in spatial memory.     

Modulation of hippocampal norepinephrine release by cholinergic agonists is altered by AF64A lesion

The effect of lesioning hippocampal cholinergic neurons with the neurotoxin AF64A on the ability of cholinergic agonists to modulate stimulation-induced release of ³H-norepinephrine (NE) from rat hippocampal slices was studied. Rats received intracerebroventricular injections of either AF64A (ethylcholine mustard aziridinium, 2 nmol) or vehicle (sham operated). Six weeks after treatment, release of ³H-NE evoked by electrical stimulation (2 Hz, 2 min) in the presence or absence of cholinergic agonists and/or antagonists was measured. Activation of M₂ receptors with oxotremorine (in the presence of the M₁ antagonist pirenzepine) caused a small inhibition of NE release, which was abolished in hippocampi from AF64A-treated rats. The K_d for high-affinity binding of the selective M₂ ligand [³H] AF-DX 384 was increased 10-fold in lesioned tissues. The M₁ selective agonist McN-A-343 produced a significant enhancement of NE release, which was unchanged by AF64A lesion. Binding studies with [³H] pirenzepine showed no change in the affinity or number of M₁ receptors. Nicotine also caused a significant enhancement of evoked NE release, but this effect was markedly reduced in tissues from AF64A-treated rats. AF64A treatment caused a twofold decrease in the number of [³H] nicotine binding sites. This study suggests that long-term lesion of hippocampal cholinergic neurons with AF64A alters the function of postsynaptic muscarinic M₂ and nicotinic cholinergic receptors that modulate the release of NE in the hippocampus.