Characterization of muscarinic autoreceptors in the rabbit hippocampus and caudate nucleus.

Oxotremorine-induced inhibition of electrically evoked release of 3H-acetylcholine from brain slices preincubated with 3H-choline was used to characterize muscarinic autoreceptors in rabbit hippocampus and caudate nucleus. From the shifts to the right of the concentration-response curves of oxotremorine in the presence of muscarinic receptor antagonists, the following pKB values [95% C.I.] were determined in the hippocampus: tripinamide: 8.7 [8.5, 8.8]; himbacine: 8.4 [8.3, 8.5]; AQ-RA 741: 8.3 [8.2, 8.5]; 4-DAMP: 8.2 [8.0, 8.3]; hexahydrosiladifenidol: 7.4 [7.2, 7.5]; AF-DX 116: 7.3 [7.1, 7.4]; pirenzepine: 6.8 [6.6, 7.0]; and PD102807: 6.3 [6.0, 6.5]. In the caudate nucleus: tripinamide: 9.1 [8.9, 9.2]; 4-DAMP: 8.3 [8.2, 8.5]; himbacine: 8.1 [8.0, 8.2]; AQ-RA 741: 8.1 [8.0, 8.3]; hexahydrosiladifenidol: 7.3 [7.2, 7.4]; AF-DX 116: 7.1 [7.0, 7.2]; pirenzepine: 6.7 [6.6, 6.8]; and PD102807: 6.5 [6.2, 6.8]. These pKB values fit best to literature values for M2 receptors, suggesting that the muscarinic autoreceptor of the rabbit hippocampus and caudate nucleus is the m2 gene product.

Properties of 3,4-diaminopyridine-evoked dopamine and acetylcholine release in rabbit caudate nucleus slices: involvement of facilitatory adenosine A2 receptors or nitric oxide?

The 3H-overflow from slices of the rabbit caudate nucleus preincubated with tritiated dopamine (DA), or choline, and then superfused and stimulated twice with 3,4-diaminopyridine (3,4-DAP; 25 microM, 1 min), was explored as an in vitro model for evoked release of DA, or acetylcholine (ACh), respectively. In both cases the 3,4-DAP-evoked 3H-overflow was tetrodotoxin-sensitive and Ca(2+)-dependent and hence most probably represents action potential-induced exocytotic release of DA or ACh, respectively. Using pairs of preferential agonists/antagonists it was shown, that evoked DA release was inhibited via presynaptic D2 autoreceptors (quinpirole/domperidone) and kappa-opioid receptors (U-50488H/norbinaltorphimine). No evidence was found for the presence of presynaptic adenosine A1 or A2 receptors on dopaminergic terminals. Moreover, 3,4-DAP-evoked DA release was unaffected by increased intracellular cyclic AMP levels or by drugs affecting the NO/guanylate cyclase pathway. In a similar manner it was shown that 3,4-DAP-evoked ACh release was inhibited via presynaptic muscarine autoreceptors (oxotremorine/atropine) and dopamine D2 heteroreceptors (quinpirole/domperidone). Again, no evidence for the involvement of the NO/guanylate cyclase system in the modulation of ACh release was found, whereas the presence of inhibitory adenosine A1 receptors, but not of facilitatory A2 receptors, could be clearly established. It is concluded, that 3,4-DAP-evoked 3H-overflow from rabbit caudate nucleus slices preincubated with [3H]DA or [3H]choline, represents a simple and useful in vitro model for action potential-induced DA or ACh release, respectively. Moreover, at least in this model or rabbit brain region, facilitatory adenosine A2 receptors and the NO/guanylate cyclase system seem not to be involved in the release of these transmitters.

The influx of Ca2+ and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels.

We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca2+ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca2+]i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker omega-conotoxin (0.1 mumol/l) diminished the release of noradrenaline and the increase of intraterminal Ca2+ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 mumol/l) diminished the release of noradrenaline by 25% and the increase of [Ca2+]i by 39%. The P-type channel blocker omega-agatoxin (0.3 mumol/l) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca2+ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca2+]i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca2+ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.

3,4-Diaminopyridine masks the inhibition of noradrenaline release from chick sympathetic neurons via presynaptic alpha 2-adrenoceptors: insights into the role of N- and L-type calcium channels.

We have investigated the participation of the N-type (omega-conotoxin GVIA-sensitive) and L-type (nifedipine-sensitive) calcium channels in the alpha 2-adrenoceptor mediated autoinhibition of the release of [3H]noradrenaline from chick sympathetic neurons in culture. Blockade of 3,4-diaminopyridine-sensitive potassium channels resulted in tetrodotoxin-sensitive and calcium-dependent increase of the release of [3H]noradrenaline evoked by electrical stimulation. Nifedipine attenuated the evoked release under control conditions by 20%, but in the presence of 3,4-diaminopyridine by 51%, while omega-conotoxin decreased the release under control conditions by 87% and in the presence of 3,4-diaminopyridine by only 43%. The L-type calcium channel activator Bay k 8644 increased the evoked release of the transmitter both in the absence and in the presence of 3,4-diaminopyridine. Under control conditions, the alpha 2-adrenoceptor agonist UK 14304 decreased the evoked release by 57% and the alpha 2-adrenoceptor antagonist rauwolscine increased it by 14%. Nifedipine did not prevent this modulation. In the presence of 3,4-diaminopyridine, UK 14304 lost its effect on the release of noradrenaline, but its inhibitory action was restored when nifedipine, but not omega-conotoxin, was added. Changes in the increase of intracellular calcium concentration ([Ca2+]i) evoked by electrical stimulation, measured in the cell processes by microfluorimetry, paralleled the changes in the release of [3H]noradrenaline. Under control conditions, nifedipine attenuated the rise of intracellular calcium by only 16%, while omega-conotoxin did so by 66%. 3,4-Diaminopyridine enhanced the evoked rise of [Ca2+]i; in its presence the rise of intracellular calcium was about equally reduced by nifedipine and omega-conotoxin (by 46 and 36%, respectively). These effects were additive. UK 14304 diminished the peak concentration of [Ca2+]i elicited by the standard electrical stimulation by 31% and rauwolscine antagonised this effect. UK 14304 did not measurably inhibit the stimulation-evoked rise of intraterminal [Ca2+]i in the presence of 3,4-diaminopyridine but it produced an inhibition by 26% if nifedipine had been applied together with 3,4-diaminopyridine. Our observations show that, under control conditions, the stimulated release of [3H]noradrenaline is mainly associated with the opening of N-type channels, while in the presence of 3,4-diaminopyridine the contribution of L-type channels becomes more important. The alpha 2-adrenoceptor stimulation by UK 14304 inhibits the release of [3H]noradrenaline but, in the presence of 3,4-diaminopyridine, the inhibition of release can only be observed if the massive influx through L-type calcium channels is prevented. These data suggest that presynaptic alpha 2-adrenoceptors of chick sympathetic neurons preferentially influence the N-type calcium channels.

Presynaptic nicotinic receptors stimulate increases in intraterminal calcium of chick sympathetic neurons in culture.

Stimulation of chick sympathetic neurons in culture by the cholinergic agonists acetylcholine, nicotine, and 1,1-dimethyl-4-phenylpiperazinium (all at 10-1,000 mumol/L) induced concentration-dependent increases of free calcium levels measured by fura 2 fluorescence in neuronal processes. The response evoked by acetylcholine had both nicotinic and muscarinic components, whereas that induced by 1,1-dimethyl-4-phenylpiperazinium was purely nicotinic. Tetrodotoxin (0.3 mumol/L) blocked completely the increase of intraterminal free calcium level evoked by electrical stimulation. On the other hand, stimulation with 1,1-dimethyl-4-phenylpiperazinium still evoked 20-25% of the control response in the presence of tetrodotoxin. The concentration-response relationship of 1,1-dimethyl-4-phenylpiperazinium stimulation did not differ in the absence and in the presence of tetrodotoxin. The nicotinic antagonists d-tubocurarine (10 mumol/L) and mecamylamine (10 mumol/L), but not alpha-bungarotoxin (125 nmol/L), prevented the increase of intraterminal free calcium level evoked by 1,1-dimethyl-4-phenylpiperazinium (100 mumol/L) in the presence of tetrodotoxin. These observations indicate the presence of nicotinic receptors on neuronal processes that increase the intraterminal concentration of free calcium and probably modulate transmitter release. Their pharmacological properties are similar to those of nicotinic receptors located on neuronal cell bodies.

3,4-Diaminopyridine-evoked noradrenaline release in rat hippocampal slices: facilitation by endogenous or exogenous nitric oxide.

The involvement of nitric oxide (NO) in the evoked release of noradrenaline (NA) was studied in rat hippocampal slices preincubated with [3H]NA and stimulated with 3,4-diaminopyridine (3,4-DAP; 200 microM) for 2 min. The 3,4-DAP-evoked [3H]overflow was enhanced by the NO synthase substrate L-arginine, but not by D-arginine; it was reduced by the NO synthase inhibitor NG-nitro-L-arginine, which also antagonized the effects of L-arginine. The corresponding nitro derivative of D-arginine was inactive and unable to block the effects of L-arginine. Also drugs known to produce NO in-vitro, like sodium nitroprusside (SNP), 3-morpholino-sydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) enhanced the 3,4-DAP-evoked NA release. The NO scavenger hemoglobin showed no significant effects when given alone, but reduced or abolished, respectively, the facilitatory effects of SNP, or SNAP and L-arginine. The cyclic GMP derivatives 8-Br-cGMP and Sp-8-p-chlorophenylthioguanosine-3',5'-cyclic monophosphorothioate (Sp-8-pCPT-cGMPS) also acted facilitatory, whereas the corresponding Rp-enantiomer of the latter compound was inactive, but antagonized the effect of Sp-8-pCPT-cGMPS. NA release evoked by 3,4-DAP (10 microM) from rat hippocampus synaptosomes was not affected by L-arginine or NG-nitro-L-arginine but slightly increased by SNAP and Sp-8-pCPT-cGMPS. Antagonists at NMDA, non-NMDA and metabotropic glutamate receptors neither affected the 3,4-DAP-evoked NA release nor the facilitatory effect of L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)

Sympathetic sprouting: time course of changes of noradrenergic, cholinergic, and serotonergic markers in the denervated rat hippocampus.

As a first step for experiments investigating the presynaptic characteristics of sympathetic fibers grown into the denervated hippocampus, we studied the time course of changes of neurochemical markers in the rat hippocampus, subsequent to aspiration lesions of the fimbria-fornix and the overlying callosal and cortical structures. At various postsurgical delays (1, 2, 8, 24, and 40 weeks), the activity of choline acetyltransferase, the high-affinity synaptosomal uptake of choline and noradrenaline, and the concentrations of noradrenaline, serotonin, and 5-hydroxyindoleacetic acid were measured in a dorsal, an intermediate, and a ventral part of the hippocampus. Levels of all markers were significantly reduced shortly (1-2 weeks) after the lesions. However, whereas the cholinergic (choline uptake and choline acetyltransferase activity) and the serotonergic (concentrations of serotonin and 5-hydroxyindoleacetic acid) markers remained significantly reduced for up to 40 weeks, both noradrenergic markers recovered to near-normal (noradrenaline uptake) or even supranormal (noradrenaline concentration) levels, although with clear-cut differences in the time course and the regional characteristics. The noradrenaline content reached control levels already 8 weeks after lesion surgery and was about two to three times higher 40 weeks later, with the most dramatic effects in the ventral hippocampus. In contrast, high-affinity noradrenaline uptake reached control values only 24 weeks after lesion and exceeded them only in the ventral hippocampus 40 weeks after surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences of the electrical and nicotinic receptor stimulation-evoked liberation of norepinephrine from chicken sympathetic neurons in culture: possible involvement of different pools of the transmitter.

We studied the release of [3H]norepinephrine from chicken sympathetic neurons in culture evoked by nicotinic and electrical stimulation with an intention to establish functional identity or nonidentity of the two stimuli in investigations of neurotransmitter release. Nicotinic stimulation evoked extracellular calcium dependent release of [3H]norepinephrine and the rise of intracellular calcium concentration. The release was completely blocked by nicotinic antagonists hexamethonium (100 mumol/l) and mecamylamine (10 mumol/l), and decreased by tetrodotoxin (0.3 mumol/l) and omega-conotoxin (0.1 mumol/l) to 17% and 27%, resp. The intracellular calcium response was decreased by nicotinic antagonists and tetrodotoxin, but not changed by omega-conotoxin. The electrical stimulation-evoked release was blocked by both tetrodotoxin and omega-conotoxin, and decreased by previous electrical, but not nicotinic, stimulation. The differential sensitivity to omega-conotoxin and tetrodotoxin,and the inability of nicotinic stimulation to decrease the liberation by following electrical stimulation may suggest the mobilization of different pools of the transmitter.

Behavioural and neurochemical effects of superior cervical ganglionectomy in rats with septo-hippocampal lesions.

This longitudinal study, extending over 12 months, assessed the behavioural and biochemical effects of hippocampal sympathetic ingrowth (HSI) into the partially denervated hippocampus. Male Long-Evans rats received fimbria-fornix lesions (FIFO) or sham operations at 90 days of age. At the same time half of the rats from each group sustained bilateral ablation of the superior cervical ganglia (SCGX). A battery of behavioural tests, measuring spontaneous alternation, activity in the open field and home cage, and radial-maze performance, were employed, starting after one very short (16 days) and one extended (216 days) post-operative delay. Neurochemical analyses measuring choline acetyltransferase (ChAT) activity, high-affinity choline (HACU) and noradrenaline uptake by hippocampal synaptosomes (HANU), hippocampal noradrenaline ([NA]), serotonin ([5-HT]) and 5-hydroxyindoleacetic acid ([5-HIAA]) concentrations were carried out in a dorsal, a "middle" and a ventral region of the hippocampus. Lesion of the FIFO induced a significant and enduring deficit in radial-maze performance, in addition to a persistent locomotor hyperactivity. ChAT and HACU were significantly depleted in all three regions of the hippocampus at 12 months, and these deficits were negatively correlated with maze performance. SCGX in the presence of the FIFO lesion significantly reduced [NA] in the middle region of the hippocampus, as compared to SCGX rats, and contributed to a restoration of lesion-induced depletions in [5-HT] and [5-HIAA] in the middle and ventral hippocampal regions, whilst failing to elicit any behavioural changes at either time point. It is concluded that if lesion-induced HSI indeed occurred, as is suggested by neurochemical evidence, it had no effect upon the observed behavioural deficits elicited by transection of the FIFO in the rat.

The effects of intrahippocampal raphe and/or septal grafts in rats with fimbria-fornix lesions depend on the origin of the grafted tissue and the behavioural task used.

Long-Evans female rats sustained electrolytic lesions of the fimbria and the dorsal fornix and, two weeks later, received intrahippocampal suspension grafts of fetal tissue. The grafts were prepared from regions including either the medial septum and the diagonal band of Broca (septal grafts), or the mesencephalic raphe (raphe grafts), or from both these regions together (co-grafts). All rats were submitted to a series of behavioural tests (home cage and open-field locomotion, spontaneous alternation, radial-arm maze and Morris water maze performance) run over two periods after grafting (one to nine weeks and 20-35 weeks). Two weeks after completion of behavioural testing, histological (acetylcholinesterase and Cresyl Violet staining) and/or neurochemical (choline acetyltransferase activity, high-affinity synaptosomal uptake of choline and serotonin, noradrenaline, serotonin and 5-hydroxyindolacetic acid concentrations) verifications were performed on the hippocampus. Compared to sham-operated rats, lesion-only rats exhibited hyperactivity which was transient in a familiar environment (home cage) and lasting in an unfamiliar one (open field), decreased rates of spontaneous T-maze alternation, and impaired memory performance in both the radial-arm maze and the Morris water maze. These rats also showed decreased cholinergic and serotonergic markers with a maximal depletion in the septal two-thirds of the hippocampus. Noradrenaline concentration tended to be increased in the dorsal third of the hippocampus, but was not modified in the other two-thirds. While septal grafts specifically increased the cholinergic markers and raphe grafts the serotonergic ones, neither of these grafts produced a lasting effect on any behavioural variable. Conversely, the co-grafts, which increased both the cholinergic and serotonergic markers in the septal two-thirds of the hippocampus, completely normalized the Morris water maze probe trial performance, but failed to affect any of the other behavioural variables. Our present results confirm that grafts of fetal neurons injected into the denervated hippocampus may induce a neurochemical recovery that depends on the anatomical origin of the grafted cells, and that co-grafting two fetal brain regions allows the combination of their individual neurochemical properties. Furthermore, our results show that these neurochemical effects of the co-grafts may be involved in the recovery of behavioural function observed in the water maze. However, somewhat paradoxically, those effects appear inefficient for inducing any recovery in other behavioural tasks, even in the radial-arm maze; which is assumed to measure similar spatial functions.(ABSTRACT TRUNCATED AT 400 WORDS)