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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 Ca 2+ 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 [Ca 2+ ] i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker ω-conotoxin (0.1 μmol/1) diminished the release of noradrenaline and the increase of intraterminal Ca 2+ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 μmol/1) diminished the release of noradrenaline by 25% and the increase of [Ca 2+ ] i by 39%. The P-type channel blocker ω-agatoxin (0.3 μmol/1) 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 Ca 2+ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca 2+ ] 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 Ca 2+ 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.

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.

3t 31 days of age, Long-Evans female rats sustained aspirative lesions of the septohippocampal pathways and, 14 days later, received intrahippocampal suspension grafts prepared from the region including the medial septum and the diagonal band of Broca (Group S, n = 11), from the region including the mesencephalic raphe (Group R, n = 11) or from both regions together (Group S + R, n = 11). Sham-operated (Group Sham, n = 9) and lesion-only (Group Les, n = 11) rats served as non-grafted controls. Seven Sham, 7 Les and 8 rats from each transplant group were tested for home cage activity (6 months after grafting) and radial maze performance (between 7.5 and 8.5 months post-grafting). One month after completion of behavioral testing, the dorsal hippocampi of these rats were prepared for measuring choline acetyltransferase (ChAT) activity and high affinity synaptosomal uptake of both [ 3 H]choline and [ 3 H]serotonin. The remaining rats were used for histological verifications on brain sections stained for acetylcholinesterase (AChE). The lesions increased locomotor activitu, impaired radial maze learning and, in the dorsal hippocampus, reduced AChE positive staining, decreased ChAT activity (−73%) as well as high affinity uptake of both choline (−81%) and serotonin (−82%). Neither type of transplant produced any significant behavioral recovery. However, septal transplants increased hippocampal AChE positivity, restored ChAT activity and enhanced choline uptake to 116% and 70% of the values found in sham-operated rats, respectively; they had no significant effect on uptake of serotonin. Transplants from the raphe region had weak affects on hippocampal AChE positivity, increased both the ChAT activity and the choline uptake to 70% ad 38% of sham-operated rats, respectively, and produced an (over)compensation of the serotonin uptake which reached 324% of the values found in sham-operated rats. The co-transplantation of both regions resulted in restoration of ChAT activity (113% of sham-operated rats values), choline uptake (83% of sham-operated rats) and serotonin uptake (129% of sham-operated rats). Our neurochemical data show that after extensive denervation of the hippocampus, intrahippocampal grafts of fetal neurons may foster a neurotransmitter-specific recovery which depends upon the anatomical origin of the grafted cells; a graft rich in serotonergic neurons overcompensates the serotonergic deficit, a graft irich in cholinergic neurons attenuates the cholinergic deficit, whereas a mixture of both types of grafts produces recovery from both types of deficits. Thereby, both the feasibility and the interest of the co-grafting technique are confirmed. However, our behavioral data also suggest that separate or combined attenuation of the lesion-induced cholinergic and serotonergic deficits in the dorsal hippocampus does not seem to be a sufficient condition to induce attenuation of the lesion-induced behavioral deficits (hyperactivity, impaired memory).

Effects of aspirative fimbria-fornix lesions and intrahippocampal grafts of fetal septal-diagional band or hippocampal tissue were examined, in Long Evans female rats, on spontaneous alternation, radial maze learning, hippocampal acetylcholine concentrations and [3H]choline accumulation by hippocampal slices. Septohippocampal damage decreased all of these variables. Septal-diagonal band grafts increased hippocampal acetylcholine levels as well as [3H]choline accumulation of tissue (when incubated for 45 min), but they had no effect on alternation rates and further impaired radial maze performances. No such behavioral and neurochemical effects were observed in rats with hippocampal grafts. Our data suggest that factors other than graft-induced improvement of cholinergic functions in the denervated hippocampus may be involved in the expression of behavioral effects by intrahippocampal acetylcholine-rich grafts.

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 μM, 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 Ca2+-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 κ-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 involvement of nitric oxide (NO) in the evoked release of noradrenaline (NA) was studied in rat hippocampal slices preincubated with [ 3 H]NA and stimulated with 3,4-diaminopyridine (3,4-DAP; 200 μM) for 2 min. The 3,4-DAP-evoked [ 3 H]overflow was enhanced by the NO synthase substrate l -arginine, but not by d -arginine; it was reduced by the NO synthase inhibitor N G -mitro- l -arginine, which 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 μM) from rat hippocampus synaptosomes was not affected by l -arginine or N G -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. From these findings we conclude that endogenously formed NO facilitates 3,4-DAP-evoked NA release in rat hippocampus, possibly by a cyclic GMP dependent mechanism; NMDA receptor stimulation and glutamate release seem not to be involved in this phenomenon.

The arachidonic acid (AA) metabolites prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha) and thromboxane B2 (TXB2) were measured in the dorsal hippocampus, amygdala/pyriform cortex and parietal cortex of the rat brain following the application of kainic acid (KA, s.c. 10 mg/kg). The first significant increases in the prostanoids were seen 10 min following the KA injection, at this time the first behavioral change 'staring' was observed. In the hippocampus the highest concentrations of the PGs were reached 30 min after the injection of the neurotoxin. At this time frequent wet dog shakes (WDS) and rare focal convulsions effecting head and extremities occurred. In the amygdala/pyriform cortex and parietal cortex the maximal prostanoid formation was seen after 120 min. At this time tonic clonic seizures were registered. In contrast to other seizure models, where PGD2 was the predominant prostanoid formed, PGF2 alpha was the major AA-metabolite found in KA-treated animals. From the time-course of the seizure-related behavior and the increased prostanoid synthesis we conclude that the initiation of the prostanoid synthesis was triggered by the increased neuronal activity rather than by cell damage.

Basal levels of 5 cerebral prostanoids (PGD 2 , PGF 2α , PGE 2 , 6-keto-PGF 1α and throm☐ane/TX/B 2 ) were measured radioimmunologically in normal and convulsion-prone gerbils. Significantly less PGD 2 , PGE 2 and 6-keto-PGF 1α was found in the brain of seizure-sensitive animals. After treatment with indomethacin, which reduced the amount of brain cyclo-oxygenase products, also normal gerbils exhibited convulsions following environmental stress. The results are in accordance with the hypothesis that endogenous prostanoids play a role in the regulation of seizure susceptibility.

Prostanoid forming capacity in vitro and convulsion-induced prostanoid formation in vivo were studied in the developing rat brain. For comparison, prostanoid synthesis in homogenates of primary astrocyte cultures of different ages was also examined. There was no significant prostanoid production in homogenates from primary astrocyte cultures prepared one week after cultivation. Two-week-old astrocyte cultures possessed a prostanoid synthesizing system of high specific activity. The relative proportions of the products were similar to those obtained in brain homogenates of adult rats, prostaglandin D2 (PGD2) being the major product. Prostanoid forming capacity of brain homogenates was low at birth, increased during development and nearly reached adult values by day 21. Generalized convulsions could be evoked by pentylenetetrazol (PTZ) irrespective of age, but convulsion-induced prostanoid formation characteristic of adult rodents did not take place before the third week of postnatal life. The close similarities between the characteristic features of prostanoid synthesis in both brain and astroglial homogenates, together with the coincidence during brain development of the expression of cerebral prostanoid synthesis with the appearance of mature astrocytes suggest that astrocytes are an important source of brain prostanoids.

The levels of 5 different prostanoids (PGD2, PGF2 alpha, PGE2, TXB2 and 6-keto-PGF1 alpha) formed in whole mouse brain in vivo were measured by specific radioimmunoassays. Basal concentrations were found to be very low (few ng/g wet weight). A marKed increase occurred during convulsions induced by either pentylenetetrazole or by electroconvulsive shock. Under both conditions the major cyclooxygenase product detected was PGD2, followed by PGF2 alpha and lower concentrations of the other prostanoids. The non-steroidal anti-inflammatory drugs flurbiprofen, indomethacin, and diclofenac dose-dependently inhibited the pentylenetetrazole-induced formation of prostaglandins. Concomitantly these 3 compounds dose-dependently increased the acute toxicity of pentylenetetrazole (decrease in LD50). Conversely, if levels of cerebral prostaglandins were enhanced by a preceding electroshock, the toxicity of pentylenetetrazole was significantly reduced (increase in LD50), and the time of onset of clonic seizures was markedly prolonged. Both the effect on the latency time and the LD50 could be reversed if the cerebral prostaglandin synthesis was prevented by indomethacin, or if the time interval between the electroshock and pentylenetetrazole administration was extended, so that the electroshock-stimulated prostaglandin concentrations had declined to basal levels again. These findings indicate that endogenous prostanoids formed in mouse brain during convulsions might possess anticonvulsive properties.

The distribution of the two major cycloxygenase products prostaglandin D2 (PGD2) and prostaglandin F2α (PGF2α) in 7 different regions of the brain (medulla, cerebellum, hypothalamus, striatum, midbrain, hippocampus and cerebral cortex) was studied. Basal levels were highest in hypothalamus and cortex. Following convulsions elicited by environmental stress prostaglandin concentrations increased in all areas, with largest increases (10–20-fold) in hippocampus and cortex, reaching 70 ng/g PGD2 in hippocampus and 115 ng/g PGD2 in cortex. These results demonstrate that, during spontaneous seizures, there is a greater increase in prostanoid production in those areas involved in the convulsive process.