Long-Evans female rats sustained aspirative lesions of the septohippocampal pathways and, 2 weeks later, received into the dorsal hippocampus grafts prepared from the septal area (rich in cholinergic ...neurons; Group Sep) or from the mesencephalic raphe (poor in cholinergic neurons; Group Rap) of rat fetuses. Lesion-only (Group Les) and virtually intact (Group Sham) rats served as controls. Between 9.5 and 10.5 months after grafting surgery, we found the lesions to decrease choline acetyltransferase activity (ChAT), high affinity synaptosomal uptake of 3Hcholine (HACU) and serotonin concentration (5-HT), as well as to increase the noradrenaline concentration (NA) in the dorsal hippocampus. Raphe grafts increased 5-HT to 456% of normal, but had only weak or no effects on the other lesion-induced modifications in brain neurochemistry. Septal grafts dramatically increased ChAT activity and HACU, enhanced 5-HT, and reduced NA to near-normal levels. We also found a significant negative correlation between HACU and NA in rats with lesions, whether grafted or not. These data show that grafts providing the denervated hippocampus with a new cholinergic innervation might be able to exert inhibitory effects on the lesion-induced increase of NA. Since such an increase is indicative of sympathetic sprouting, the finding of reduced NA in rats with graft-derived cholinergic reinnervation of the hippocampus is in line with the hypothesis that hippocampal cholinergic denervation plays a crucial role in the induction of sympathetic sprouting. However, our data do not allow to distinguish whether grafts rich in cholinergic neurons inhibited the sympathetic sprouting itself, or rather reduced the NA content of sprouted fibers.
The effect of the alpha 2-adrenoceptor agonist clonidine on 3,4-diaminopyridine (3,4-DAP)-evoked 3Hnoradrenaline (32HNA) release in rat hippocampus slices was studied in the presence or absence (+1 ...mM EGTA) of extracellular Ca2+. 3H overflow (consisting mainly of unmetabolized 3HNA) was evoked by addition of 100 microM 3,4-DAP for 10 min to the medium, which always contained 1 microM desipramine. Ligands for L-type voltage-sensitive Ca2+ channels (VSCC) did not affect the evoked 3HNA release, whereas the preferential N-type VSCC antagonist omega-conotoxin was inhibitory, both in the presence and even more potently in the absence of Ca2+, suggesting an involvement of N-type VSCC in the mechanism of 3,4-DAP-evoked 3HNA release. In the absence of extracellular Ca2+ the initial Na+ influx, which has been previously proposed to liberate Ca2+ from intracellular stores for the exocytotic process, most probably occurs via N-type VSCC. Clonidine inhibited the 3,4-DAP-evoked 3HNA release in a concentration-dependent manner, both in the presence and even more potently in the absence of Ca2+; its effects were antagonized by yohimbine. In the presence of extracellular Ca2+ the clonidine effect was not changed by addition of omega-conotoxin. Similar effects of clonidine were found in slices from the rabbit hippocampus. Since the availability of Ca2+ from intracellular stores seems to predominate in the present model, our results lend some support to the suggestion that alpha 2-adrenoceptor activation might affect intracellular mechanisms of Ca2+ homeostasis.
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 3Hcholine accumulation and the electrically evoked 3Hacetylcholine (3HACh) release were assessed in hippocampal slices. The release of 3HACh was measured in presence of atropine (muscarinic antagonist, 1 mu M), physostigmine (acetylcholinesterase inhibitor, 0.1 mu M), oxotremorine (muscarinic agonist, 0.01 mu M-10 mu M), mecamylamine (nicotinic antagonist, 10 mu M), methiothepin (mixed 5-HT1/5-HT2 antagonist, 10 mu M), 8-OH-DPAT (5-HT1A agonist, 1 mu M), 2-methyl-serotonin (5-HT3 agonist, 1 mu M) and CP 93129 (5-HT1B agonist, 0.1 mu M-100 mu M), or without any drug application as a control. In lesion-only rats, the specific accumulation of 3Hcholine was reduced to 46% of normal and the release of 3HACh 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 3HACh release and was significantly more effective in grafted (-70%) than in sham-operated (-56%) or lesion-only (-54%) rats. When physostigminewas superfused throughout, mecamylamine had no effect. Conversely, atropine induced a significant increase of 3HACh 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 3HACh 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 3HACh release. In contrast, CP 93129 dose-dependently decreased 3HACh 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-HT1B 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-HT1B 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 exertat the level of the cholinergic terminal.
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
3Hcholine accumulation and the electrically evoked
3Hacetylcholine (
3HACh) release were assessed in hippocampal slices. The release of
3HACh 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-HT
1/5-HT
2 antagonist, 10 μM), 8-OH-DPAT (5-HT
1A agonist, 1 μM), 2-methyl-serotonin (5-HT
3 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
3Hcholine was reduced to 46% of normal and the release of
3HACh 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
3HACh 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
3HACh 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 (
3HACh) 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
3HACh release. In contrast, CP 93129 dose-dependently decreased
3HACh 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.
Effects of aspirative fimbria-fornix lesions and intrahippocampal grafts of fetal septal-diagonal band or hippocampal tissue were examined, in Long Evans female rats, on spontaneous alternation, ...radial maze learning, hippocampal acetylcholine concentrations and 3Hcholine accumulation by hippocampal slices. Septohippocampal damage decreased all of these variables. Septal-diagonal band grafts increased hippocampal acetylcholine levels as well as 3Hcholine 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 effect of islet-activating protein (IAP) on alpha 2-adrenoceptor mediated modulation of noradrenaline release in the rabbit hippocampus was studied. Slices of the hippocampus were incubated for 6 ...h with IAP, subsequently loaded with 3H-noradrenaline and superfused continuously. IAP-pretreatment significantly enhanced the electrically evoked transmitter release and diminished the facilitatory effect of the alpha 2-adrenoceptor antagonist yohimbine. In addition, the inhibitory effect of the alpha 2-adrenoceptor agonist clonidine was reduced. These results provide circumstantial evidence that an inhibitory guanine-nucleotide-binding protein, most probably Ni of a presynaptically located adenylate cyclase, is involved in the alpha 2-autoreceptor mediated modulation of noradrenaline release.
Long Evans female rats sustained aspirative lesions of the septohippocampal pathways; subsequently, they received intrahippocampal suspension grafts of fetal septal-diagonal band or hippocampal ...tissue. The long term (8-10 months post-surgery) effects of these treatments were examined in the hippocampus for the following variables: concentration of hippocampal acetylcholine (ACh), muscarinic-stimulated (carbachol) formation of inositol monophosphate, accumulation of tritiated choline, noradrenaline (3H-NA) and serotonin (3H-5-HT), electrically evoked release of 3H-acetylcholine (3H-ACh), 3H-NA and 3H-5-HT, and choline acetyltransferase (ChAT) activity. The lesions decreased the levels of endogenous ACh, the accumulation of 3H-choline and 3H-5-HT and the evoked release of both 3H-ACh and 3H-5-HT as well as the ChAT activity, but they failed to significantly affect the muscarinic-stimulated formation of inositol monophosphate and the accumulation and release of 3H-NA. Grafts of hippocampal cells were found to be ineffective on all lesion-induced effects. In contrast, grafts of septal-diagonal band origin attenuated the deficit of hippocampal concentrations of ACh and accumulation of 3H-choline without, however, improving release of 3H-ACh, accumulation and release of 3H-5-HT, and ChAT activity. These observations suggest that: (i) denervation-induced hippocampal muscarinic supersensitivity might not be long-lasting or the lesions, which in some cases spared the lateral edges of the fimbria, failed to induce any muscarinic supersensitivity, (ii) intrahippocampal grafts rich in cholinergic neurons do not foster recovery from the lesion-induced noncholinergic deficits we assessed, (iii) recovery of function may be expressed by some but not all biochemical or pharmacological cholinergic variables and (iv) graft-derived hippocampal reinnervation may be less efficient than the endogenous innervation of intact rats as indicated by the restoration of only some of the variables related to cholinergic function by intrahippocampal septal-diagonal band grafts.
45Ca2+ uptake measurements were performed on intact and osmotically lysed synaptosomes from rat brain to study the possible influence of prostaglandins (PGs) on Ca2+ movements into and within the ...nerve endings. The K+-induced 45Ca2+ uptake of intact synaptosomes was not influenced by several inhibitors of PG synthesis. 45Ca2+ uptake in lysed synaptosomal preparations was promoted by ATP and seemed to be largely attributable to mitochondria, as it was inhibited by mitochondrial poisons. This Ca2+ uptake was strongly reduced by PG synthesis inhibitors but also by PG precursor fatty acids. Both PG synthesis inhibitors and precursors, according to their relative efficacy in blocking Ca2+ uptake, were able to induce Ca2+ efflux from preloaded intrasynaptosomal organelles. The PGs E2, F2 alpha, D2, and thromboxane B2 were without effect on 45Ca2+ uptake in lysed synaptosomal preparations. On the basis of our results it does not seem likely that PGs influence Ca2+ availability by modulating Ca2+ fluxes into or within the nerve endings. The observed inhibitory effects of PG synthesis inhibitors and precursors on the intrasynaptosomal Ca2+ uptake might be due to unspecific impairment of mitochondrial functions.
