Diurnal rhythm in ornithine decar☐ylase activity and noradrenergic and cholinergic markers in rat submaxillary lymph nodes

Diurnal variations in lymph node ornithine decar☐ylase activity were examined in submaxillary lymph nodes of rats injected with Freund's complete adjuvant or its vehicle. After immunization, lymph node ornithine decar☐ylase activity increased by about 10-fold. Both in immunized and non-immunized rats, a significant diurnal variation in ornithine decar☐ylase activity at early (i.e. 13.00 h, vehicle) or late afternoon (i.e. 17.00 h, Freund's adjuvant). Injection of Freund's adjuvant during daylight or at night resulted in similar day-night differences in submaxillary lymph node ornithine decar☐ylase activity. In rats subjected to the sympathetic postganglionic denervation (by ipsilateral superior cervical ganglionectomy) or the preganglionic parasympathetic decentralization (by chorda tympani section) of submaxillary lymph nodes, nyctohemeral variations in ornithine decar☐ylase were still present, showing a maximum at 17.00 h. Superior cervical ganglionectomy augmented lymph node ornithine decar☐ylase while chorda tympani section decreased it. When a unilateral superior cervical ganglionectomy plus chorda tympani section was performed, the diurnal changes in ornithine decar☐ylase were abolished. [³H]Norepinephrine uptake and tyrosine hydroxylase activity attained their maxima in submaxillary lymph nodes at early night. After immunization, these two presynaptic indicators of sympathetic activity in submaxillary lymph nodes augmented significantly. Neuronal [³H]choline uptake and [³H]choline conversion into acetylcholine (two indicators of cholinergic activity) also augmented in lymph nodes of rats injected with Freund's adjuvant. In immunized rats, maxima in [³H]choline uptake and [³H]acetylcholine synthesis were found at 13.00–17.00 h while in non-immunized rats, a maximum in acetylcholine synthesis was found at 17.00 h. The results are compatible with the view that the autonomic nervous system plays a role in circadian changes of immune responsiveness in lymphoid tissue and that a significant augmentation of presynaptic autonomic activity takes place during immunization in lymphoid tissue.     

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.     

Effects of ovariectomy and estradiol benzoate on high affinity choline uptake, ACh synthesis, and release from rat cerebral cortical synaptosomes

Several presynaptic processes were studied in cerebral cortical synaptosomes prepared from intact adult female rats or from ovariectomized animals that received 3 subcutaneous injections of either estradiol benzoate (10 μg/kg) or vehicle. Injections were given 1/day, every other day, and animals were sacrificed 1 h after the last injection. High affinity choline uptake and coupled acetylcholine (ACh) synthesis were reduced by ovariectomy, and restored to control levels by the estradiol benzoate injections. In contrast, low affinity choline uptake and depolarization-induced [³H]ACh release were unaffected by either ovariectomy or estradiol benzoate injections. These results suggest that changes in estradiol levels may alter the high affinity transport process regulating ACh synthesis in this tissue.     

Choline and acetylcholine metabolism in slice cultures of the newborn rat septum

The incorporation of [3H]choline into acetylcholine and other choline-containing compounds was investigated in slice cultures of the septal area of newborn rats. At choline concentrations in the range of the high affinity transport mechanism (0.1-1 microM) most of the labeled choline was incorporated into phosphorylcholine, followed by lipids, acetylcholine and the free choline pool. Hemicholinium-3 (1-10 microM) lead to a marked decrease of acetylcholine synthesis, whereas choline accumulation or phosphorylcholine synthesis were not decreased. Both basal and K+-induced release of acetylcholine were Ca2+ dependent. The efflux of choline was not stimulated by high K+. When choline was absent from the incubation medium, the slices were able to liberate significant amounts of the [3H]choline previously incorporated into phospholipids, and were also able to synthesize some acetylcholine. In choline-free medium, acetylcholine synthesis was greatly enhanced by depolarization. During the period in culture, there was a decrease of the incorporation rate of [3H]choline into phosphorylcholine and an increase of the incorporation rate into acetylcholine. The tissue structure was well preserved after several weeks in culture. After staining for acetylcholinesterase, the cholinergic neurons in the cultures showed a similar morphology to that seen in situ. The main conclusions of the present study are: cholinergic neurons in slice cultures develop and behave in a manner which is very similar to their in situ counterparts; the main divergence from previous studies of choline metabolism in tissue culture is the substantial incorporation rate of choline into acetylcholine at choline concentrations in the range of the high affinity uptake mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Differential long-term effect of AF64A on [3H]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 microliters/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]Ch 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.     

Synthesis of acetylcholine and γ-aminobutyric acid by dissociated cerebral cortical cells in vitro

Biosynthesis of acetylcholine and γ-aminobutyric acid was investigated in primary cultures derived from embryonic rat cerebral cortex. The cortical cultures contained a mixture of neurons and non-neuronal cells. Cortical neurons were viable for at least 2 months in vitro. The cultures accumulated [³H]choline from the medium and synthesized acetylcholine. Choline uptake was significantly reduced by hemicholinium-3 and acetylcholine synthesis by intact cultures was partially inhibited by naphthylvinylpyridine. In lysed cultures, greater than 90% of acetylcholine synthesizing activity was inhibited by naphthylvinylpyridine. Acetylcholine synthesis and storage by intact cells increased as the concentration of choline increased and approached saturation near 50 μM choline. Also, the synthesis and storage of γ-aminobutyric acid from [³H]glutamate increased as the concentration of glutamic acid increased and began to saturate near 25 μM glutamate. The rates of synthesis of acetylcholine from 5 μM choline and of γ-aminobutyric acid from 50 μM glutamate were linear for at least 30 min. The synthesis and accumulation of acetylcholine and γ-aminobutyric acid from their respective precursors showed a similar dependence on culture age; they increased constantly during the first 3 weeks in culture, thereafter they declined. Mixed cultures of cortical cells and either skeletal muscles or various other non-neuronal cells exhibited a 40–100% enhancement in both acetylcholine and γ-aminobutyric acid synthesis. Autoradiographic methods showed that a subpopulation of neurons in the cortical cultures accumulated γ-aminobutyric acid from the medium.     

Differential changes in presynaptic modulation of transmitter release during aging

The purpose of this study was to assess the functional role of presynaptic α2-autoreceptors in noradrenergic transmission in the hippocampus and dopamine-2 heteroreceptors in cholinergic transmission in the striatum in young, adult, and senescent rats. Male and female Wistar rats (4, 12, and 24 months old) were used and the release of radioactivity from striatal and hippocampal slices that had been loaded either with [³H]choline or with [³H]norepinephrine was measured at rest and in response to field stimulation (2 Hz, 360 shocks). The release was challenged by sulpiride, a selective dopamine-2 receptor antagonist, and CH-38083, a selective α2-adrenoceptor antagonist. The dissociation constant and the number of α2-adrenoceptors was also determined by binding studies using [³H]yohimbine as ligand in crude membrane preparations of frontal cortex. There were an age-related changes in α2-adrenoceptor-mediated negative feedback modulation of norepinephrine release and in the density and dissociation constant of α2-adrenoceptors. They were reduced in senescent rats. In contrast the presynaptic modulation of striatal cholinergic transmission by dopamine-2 receptors was not altered during aging, but the storage capacity of and the release of acetylcholine from cholinergic interneurons was significantly lower.     

Differential acetylcholine and GABA release from cultured chick retina cells

In the present work we investigated the mechanisms controlling the release of acetylcholine (ACh) and of γ-aminobutyric acid (GABA) from cultures of amacrine-like neurons, containing a subpopulation of cells which are simultaneously GABAergic and cholinergic. We found that 81.2 ± 2.8% of the cells present in the culture were stained immunocytochemically with an antibody against choline acetyltransferase, and 38.5 ± 4.8% of the cells were stained with an antibody against GABA. Most of the cells containing GABA (87.0 ± 2.9%) were cholinergic. The release of acetylcholine and GABA was mostly Ca²⁺-dependent, although a significant release of [³H]GABA occurred by reversal of its transporter. Potassium evoked the Ca²⁺-dependent release of [³H]GABA and [³H]acetylcholine, with EC₅₀ of 31.0 ± 1.0 mm and 21.6 ± 1.1 mm , respectively. The Ca²⁺-dependent release of [³H]acetylcholine was significantly inhibited by 1 μm tetrodotoxin and by low (30 nm ) ω-conotoxin GVIA (ω-CgTx GVIA) concentrations, or by high (300 nm ) nitrendipine (Nit) concentrations. On the contrary, the release of [¹⁴C]GABA was reduced by 30 nm nitrendipine, or by 500 nm ω-CgTx GVIA, but not by this toxin at 30 nm . The release of either transmitters was unaffected by 200 nm ω-Agatoxin IVA (ω-Aga IVA), a toxin that blocks P/Q-type voltage-sensitive Ca²⁺ channels (VSCC). The results show that Ca²⁺-influx through ω-CgTx GVIA-sensitive N-type VSCC and through Nit-sensitive L-type VSCC induce the release of ACh and GABA. However, the significant differences observed regarding the Ca²⁺ channels involved in the release of each neurotransmitter suggest that in amacrine-like neurons containing simultaneously GABA and acetylcholine the two neurotransmitters may be released in distinct regions of the cells, endowed with different populations of VSCC.     

Characterization of the excitatory amino acid receptor-mediated release of [H]acetylcholine from rat striatal slices

The pharmacological nature of the interaction of excitatory amino acids with striatal cholinergic neurons was investigated in vitro. Agonists of excitatory amino acid receptors evoked the release of [³H]acetylcholine from slices of rat striatum, in the presence of magnesium (1.2 mM). Removal of magnesium from the medium markedly increased the release of [³H]acetylcholine evoked by all excitatory amino acid receptor agonists tested, with the exception of kainate. In the absence but not the presence of magnesium, a clear rank order of potency was found: N-methyl-dl-aspartate = ibotenate >l-glutamate >l-aspartate cysteate > kainate = quisqualate.The excitatory amino acid receptor mediating [³H]acetylcholine release resembles the N-methyl-d-aspartate preferring (N-type) receptor, as previously characterized electrophysiologically, according to 3 criteria: (1) rank order of potency of agonists; (2) magnesium-sensitivity; and (3) antagonism by 2-amino-5-phosphonovalerate.The release of [³H]acetylcholine evoked by N-methyl-dl-aspartate was blocked by tetrodotoxin (0.5 μM). Moreover, N-methyl-dl-aspartate failed to evoke [³H]acetylcholine release from slices of hippocampus, where cholinergic afferents, rather than interneurons, are found. These results suggest that excitatory amino acids act at receptors on the dendrites of striatal cholinergic interneurons, giving rise to action potentials and release of acetylcholine from cholinergic nerve terminals.     

Lipid peroxidation and aluminium effects on the cholinergic system in nerve terminals

In the present study, we analyzed how aluminium and oxidative stress induced by ascorbate/Fe²⁺ affect the mechanisms related with the cholinergic system in a crude synaptosomal fraction isolated from rat brain. [³H]Choline uptake, [³H]acetylcholine release, membrane potential and Na⁺/K⁺-ATPase activity were determined in the presence or in the absence of aluminium in control conditions and in the presence of ascorbate (0.8 mM)/Fe²⁺ (2.5 μM). The extent of lipid peroxidation was measured by quantifying thiobarbituric acid reactive substances (TBARS). Under oxidizing conditions aluminium increased the formation of TBARS by about 30 %, but was without effect when the synaptosomal preparation was incubated in the absence of oxidants. Additionally, aluminium potentiated the inhibition of the high-affinity [³H]choline uptake observed following lipid peroxidation and had the same effect on the Na⁺/K⁺-ATPase activity. [³H]Acetylcholine release induced by 4-aminopyridine, and membrane potential were not significantly affected under oxidizing conditions, either in the absence or in the presence of aluminium. We can conclude that aluminium, by potentiating lipid peroxidation, affects the uptake of choline in nerve endings. This effect, occurring during brain oxidative injury, might contribute to the cholinergic dysfunction and neuronal cell degeneration known to occur in Alzheimer’s disease.     

Development of adrenergic nerve terminals: the effects of decentralization.

The effect of decentralization (deafferentation) on the ontogeny of adrenergic nerve terminals was studied in the rat iris. Transection of the cholinergic trunk, which innervates the superior cervial ganglion (SCG), in 2–3-day-old rats, inhibited the developmental increase in iris nerve terminal density, as indicated by fluorescence microscopy. However, terminal varicosities, containing the norepinephrine (NE) storage vesicles, appeared larger and more brightly fluorescent in decentralized irides. Tyrosine hydroxylase, localized to nerve terminal cytoplasm, developed to only 40% of normal in decentralized irides. In contrast, dopamine-ß-hydroxylase, which is localized to NE storage vesicles in varicosities, developed normally. The ability to accumulate and store [³H]NE was examined in control and decentralized terminals in the presence or absence of reserpine. This drug inhibits NE storage capacity without primarily affecting accumulation itself. Decentralization reduced thein vitro uptake of NE to 54%, measured after reserpine pretreatment; in contrast,apparent uptake of transmitter was reduced to only 76% in vehicle-treated rats. These results suggest that decentralization prevents the normal development of nerve terminal membrane without markedly interfering with vesicular storage capacity. This contention was supported by the observation that in vitro retention of [³H]NE over time was actually increased in decentralized irides. Our results suggest that the ontogeny of different subcellular structures is affected differently by deafferentation.The transsynaptic regulation of nerve terminal maturation appears to be most critical in younger rats, since decentralization exerts most marked effects when performed during the perinatal period.     

Enhancement of [H]dopamine release and its [H]metabolites in rat striatum by nicotinic drugs

Recent evidence has identified directly muscarinic acetylcholine receptor (m-ACh R) and nicotinic acetylcholine receptor (n-ACh R) in the brain utilizing receptor binding assay. Several studies suggest that release of dopamine (DA) in the striatum is regulated by presynaptic receptors present on dopaminergic terminals. In the present study, the effects of cholinergic drugs on [³H]DA release were examined using micropunched tissue and synaptosomes obtained from rat striatum. ACh (5 × 10⁻⁴M) significantly increased spontaneous [³H]DA release, and the overflow was partially inhibited by d-tubocurarine (1 mM) but not atropine. Nicotine, lobeline, coniine and spartein, nicotinic agonists, significantly increased spontaneous and 25 mM K⁺ evoked [³H]DA release almost in a dose-dependent manner. In contrast, oxotremorine (2 × 10⁻⁴M), muscarinic agonist, did not any change in [³H]DA efflux. Furthermore, the metabolites of [³H]DA were separated by column chromatography. The main metabolite of [³H]DA in the spontaneous release from rat striatal synaptosomes was [³H]DOPAC (3,4-dihydroxyphenylacetic acid). Lobeline (5 × 10⁻⁵M) accelerated the outflow of [³H]DOPAC and [³H]OMDA metabolites (O-methylated and deaminated metabolites).These results could give rise to the suggestion that there was n-ACh R on the dopaminergic nerve terminals in the striatum and n-ACh R might have related to a directly excitatory effect on the DA release.     

Blockade of N- and Q-type Ca channels inhibit K-evoked [H]acetylcholine release in rat hippocampal slices

In the present study, we examined the contribution of specific Ca²⁺ channels to K⁺-evoked hippocampal acetylcholine (ACh) release using [³H]choline loaded hippocampal slices. [³H]ACh release was Ca²⁺-dependent, blocked by the nonspecific Ca²⁺ channel blocker verapamil, but not by blockade of L-type Ca²⁺ channels. The N-type Ca²⁺ channel blocker, ω-conotoxin GVIA (ω-CgTx GVIA; 250 nM) inhibited [³H]ACh release by 44% and the P/Q-type Ca²⁺ channel blocker ω-agatoxin IVA (ω-Aga IVA; 400 nM) inhibited [³H]ACh release by 27%, with the combination resulting in a nearly additive 79% inhibition. Four hundred or one thousand nM ω-Aga IVa was necessary to inhibit [³H]ACh release, ω-Conotoxin MVIIC (ω-CTx-MVIIC) was used after first blocking N-type Ca²⁺ channels with ω-CgTx GVIA (1 μM). Under these conditions, 500 nM ω-CTx-MVIIC led to a nearly maximal inhibition of the ω-CgTx GVIA-insensitive [³H]ACh release. Based on earlier reports about the relative sensitivity of cloned and native Ca²⁺ channels to these toxins, this study indicates that N- and Q-type Ca²⁺ channels primarily mediate K⁺-evoked hippocampal [³H]ACh release.     

Neurotransmitter synthesis, storage and release by aggregating cell cultures of rat brain

Rotation-mediated aggregating cell cultures of mechanically dissociated fetal (15–16 days gestation) rat brains between 25 and 35 days in vitro were examined for their ability to synthesize neurotransmitters and putative neurotransmitters from radioactively labeled precursors added to the culture medium. Cultures derived from whole brain synthesized [³H]acetylcholine from [³H]choline, [³H]γ-aminobutyric acid from l-[³H]glutamic acid, [³H]dopamine from l-[³H]tyrosine, [³H]dopamine and [³H]norepinephrine from l-[³H]dihydroxyphenylalanine, and [³H]serotonin from l-[³H]tryptophan. Veratridine increased and tetrodotoxin decreased the rate of [³H]-dopamine synthesized by aggregates derived from midbrain plus hindbrain. In chase experiments in which aggregates were incubated for 4 h with radioactively labeled precursors and then for 4 h with non-radioactively labeled precursors, addition of veratridine (50 μM) during the second 4 h incubation significantly decreased the amounts of radioactively labeled acetylcholine, l-glutamic acid, dopamine and serotonin recovered from aggregates. Tetrodotoxin (5 μM) present during the chase significantly increased the amounts of [³H]acetylcholine and [³H]dopamine recovered from the aggregates. In addition, reserpine (4 μM) markedly depleted [³H]dopamine from aggregates in these experiments. These results indicate that these cultured cells synthesized neurotransmitters and in addition suggest that some of these compounds are stored by and released from electrically active cells within the aggregates.     

Alteration of acetylcholine synthesis in mouse brain cortex in mild hypoxie hypoxia

Summary Acetylcholine synthesis in four brain regions (cerebral neocortex, hippocampus, septum and striatum) of the mouse during mild hypoxic hypoxia was measured by using [U-¹⁴C]glucose and [²H₄] choline. At the same time, concentrations of norepinephrine and dopamine in four brain regions (cerebral neocortex, hippocampus, striatum and hypothalamus) were also estimated.During 12% O₂ hypoxia, concentrations of acetylcholine in the striatum were significantly decreased (P<0.05), whereas [²H₄] acetylcholine, lactate and glucose did not alter in any regions studied. During 12% O₂ hypoxia, concentrations of choline and [²H₄]choline were significantly increased in all regions examined (P<0.05), except the [²H₄]choline in the striatum. Radioactivity (dpm/100 mg protein) and specific activity (dpm/nmol) of acetylcholine were significantly decreased in the cerebral neocortex, hippocampus and septum (P<0.01) during 12% O₂ hypoxia. A particularly marked decrease was found in the hippocampus, strongly suggesting that cholinergic terminals are particularly sensitive to hypoxia. In addition, these data also suggest that the acetylcholine synthesis from glucose might be more sensitive to hypoxia than that from choline.During 12% O₂g hypoxia, concentrations of catecholamine did not alter in any regions examined, whereas during 9% O₂ hypoxia dopamine was significantly decreased in the cerebral neocortex and hippocampus (P<0.05).     

Muscarinic receptors on dopamine terminals in the cat caudate nucleus: Neuromodulation of [H]dopamine release in vitro by endogenous acetylcholine

The directly acting muscarinic receptor agonist oxotremorine (1.8–10 μM) produced an increase in electrically evoked [³H]dopamine release from slices of the cat caudate. The maximal increase caused by oxotremorine was 40%, and was antagonized by the muscarinic receptor blocking agent atropine (0.1 μM). Exposure to the acetylcholinesterase (AChE) inhibitor physostigmine (1 μM) resulted in a 50% increase in electrically evoked [³H]dopamine release. The increase caused by physostigmine was also antagonized by atropine (0.1 μM).Atropine did not, however, alter the modulations in [³H]dopamine release mediated by the dopamine autoreceptor: the increase in electrically evoked [³H]dopamine release caused by the dopamine receptor antagonist S-sulpiride (0.1 μM) and the decrease caused by the dopamine receptor agonist pergolide (30 nM) were unaffected by atropine (0.1 μM). These results indicate that the muscarinic receptor-mediated and dopamine autoreceptor-mediated presynaptic effects on [³H]dopamine release are independent.The present results suggest that in the electrically depolarized caudate slice in vitro, released endogenous acetylcholine may interact with muscarinic receptors faciliting depolarization-evoked [³H]dopamine release,ifAChE is inhibited. These muscarinic receptors may be located on dopamine nerve terminals. In the context of present neuroanatomical knowledge, the action of released endogenous acetylcholine on dopamine terminals may be a non-synaptic neuromodulation.     

Rapid desensitization of presynaptic dopamine autoreceptors during exposure to exogenous dopamine

When nomifensine is employed to inhibit neuronal uptake, exposure to dopamine (DA) (0.1–0.3 μM) or apomorphine (0.01–0.1 μM) inhibited, in a concentration-dependent manner, the electrically evoked release of [³H]dopamine from slices of the rabbit caudate nucleus. Apomorphine inhibited transmitter release independently of the time of exposure to the drug (6–32 min). On the other hand, the inhibitory effect of exogenous dopamine occurred only if a short period (4–12 min) of exposure was employed. In studies on the electrically evoked release of [³H]acetylcholine in slices of the rabbit caudate nucleus there was no evidence for desensitization to apomorphine or exogenous dopamine at the level of the dopamine receptors that inhibit [³H]acetylcholine release. These results indicate that the dopamine autoreceptors modulating [³H]dopamine release in the caudate nucleus become subsensitive after a few minutes of exposure to exogenous dopamine. This effect does not occur at the level of the dopamine receptors which inhibit the release of [³H]acetylcholine.