Rolf Jackisch

Modulation by endogenous dopamine of the release of acetylcholine in the caudate nucleus of the rabbit

SummarySlices of the caudate nucleus of rabbits were preincubated with 3H-choline and then superfused. Stimulation by electrical pulses at 3 Hz or by 25 mmol/l potassium elicited an increase in tritium outflow which was calcium-dependent and, in the case of electrical stimulation, tetrodotoxin-sensitive.The dopamine receptor agonist apomorphine (0.01–1 μmol/l) decreased, whereas the antagonist haloperidol increased the electrically evoked overflow of tritium. Nomifensine and cocaine, used at concentrations known to inhibit the re-uptake of dopamine, also reduced the evoked overflow of tritium, and this reduction was antagonized by haloperidol. Combined pretreatment with reserpine and α-methyltyrosine methylester (α-MT), which lowered dopamine levels by 99.5%, increased the electrically evoked overflow, as did bretylium which is shown here to block action potential-induced release of dopamine. The facilitation by haloperidol and bretylium as well as the inhibition by nomifensine and cocaine were diminished or abolished after pretreatment with reserpine plus α-MT. Apomorphine decreased, and haloperidol increased, the potassium-evoked overflow of tritium; the effects were not changed by tetrodotoxin.The results indicate that the striatal dopamine receptors which, when activated, depress the release of acetylcholine, are akin to the D-2 type. Endogenous dopamine also acts on the receptors as shown by several manipulations with known effects on dopaminergic transmission. A large fraction of these dopamine receptors may be located on the cholinergic axon terminals.

Comparison of prostanoid forming capacity of neuronal and astroglial cells in primary cultures

Prostaglandin (PG) and thromboxane (TX) biosynthesis in primary neuronal and astroglial cell cultures was studied. Cultures obtained from fetal (15-16 days old) and neonatal rat brain hemispheres were characterized by chemical and immunocytochemical staining techniques as predominantly neurons or mature and immature astrocytes, respectively. Six-day old neuronal cell cultures grown in the presence of cytosine arabinoside (2 ?M) from the day 3 onwards were contaminated up to 10% with glioblasts. In astroglial cultures up to 3% of the cells were postively stained with a marker for oligodendroglial cells. Fibroblast contamination was below 1% in both cultures. Prostanoid formation (measured by specific radioimmunoassays) in 6-day old neuronal cell cultures was low (sum of the amount of PGs and TX formed: 1.16 +/- 0.17 (ng/mg protein/15 min) as compared to 14-day old cultured astroglial cells: 21.27 +/- 2.53 (ng/mg protein/15 min). Also the pattern of prostanoids formed was different in neuronal (PGD(2) ? PGF(2?) > TXB(2) ? PGE(2)) and astroglial cells (PGD(2) > TXB(2) ? PGF(2?) ? PGE(2) ? 6-ketoPGF(1?)). Preincubation with arachidonic acid (1 ?g/ml) did not affect prostanoid formation in both cultures, whereas it was stimulated 4-6-fold by addition of the calcium ionophore A23187 (1 ?M). These results, although found on cultured neuronal and glial cells of different stages of development, support the view that astroglial cells might play a crucial role in brain prostanoid synthesis.

Endogenous adenosine as a modulator of hippocampal acetylcholine release

SummaryModulation of acetylcholine release via adenosine receptors was studied in rabbit hippocampal slices, which were preincubated with 3H-choline and then continuously superfused. Electrical field stimulation of the slices elicited a release of acetylcholine, which was inhibited in a concentration-dependent manner by various adenosine receptor agonists. The effects of the agonists were antagonized by the methylxanthines. From the order of potency: cyclohexyladenosine > (−)phenylisopropyl-adenosine ((−)PIA) > 5′-N-ethylcarboxamideadenosine (NECA) > 2-chloradenosine > (+)phenylisopropyladenosine > adenosine, the inhibitory adenosine receptor may be classified as A1-(R1-)receptor. In experiments on rabbit caudate nucleus slices, adenosine receptor agonists only slightly decreased the evoked acetylcholine release.The presence of an inhibitory tone of endogenous adenosine on hippocampal acetylcholine release is supported by the following findings: 1) the methylxanthines theophylline, 8-phenyltheophylline and 3-isobutylmethyl-xanthine (IBMX) increased the evoked acetylcholine release in concentrations below those required for phosphodiesterase inhibition. 2) Adenosine uptake inhibitors, in contrast, decreased the evoked transmitter release. 3) Deamination of endogenous adenosine by addition of adenosine deaminase to the medium enhanced the acetylcholine release.In conclusion, acetylcholine release in the hippocampus is depressed at the level of the cholinergic nerve terminals by endogenous adenosine via A1-(Ri-)receptors.

Islet-activating protein (pertussis toxin) diminishes ?2-adrenoceptor mediated effects on noradrenaline release

SummaryThe effect of islet-activating protein (IAP) on α2-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 with3H-noradrenaline and superfused continuously. IAP-pretreatment significantly enhanced the electrically evoked transmitter release and diminished the facilitatory effect of the α2-adrenoceptor antagonist yohimbine. In addition, the inhibitory effect of the α2-adrenoceptor agonist clonidine was reduced. These results provide circumstantial evidence that an inhibitory guanine-nucleotide-binding protein, most probably N1 of a presynaptically located adenylate cyclase, is involved in the α2-autoreceptor mediated modulation of noradrenaline release.

Characterization of Opioid Receptors Modulating Noradrenaline Release in the Hippocampus of the Rabbit

Noradrenaline (NA) release and its modulation via presynaptic opioid receptors were studied in rabbit hippocampal slices, which were preincubated with [3H]NA, continuously superfused in the presence of 30 μM cocaine and stimulated electrically. The evoked release of [3H]NA was strongly reduced by the preferential K‐agonists ethylketocyclazocine, dynorphin A1‐13, dynorphin A, trans‐3,4‐dichloro‐N‐methyl‐N‐[2‐(1‐pyrrolidinyl)‐cyclohexyl]‐benzeneacetamide (U‐50,488), and (–)‐5,9‐dimethyl‐2′‐OH‐2‐tetrahydrofurfuryl‐6,7‐benzomorphan [(–)‐MR 2034], whereas (+)‐MR 2035 [the (+)‐enantiomer of (–)‐MR 2034] was ineffective. In contrast, the preferential δ‐agonists Leu‐enkephalin, Met‐enkephalin, and D‐Ala2,‐D‐Leu5‐enkephalin (DADLE) as well as the μ‐agonists morphine, normorphine, D‐Ala2Gly‐ol5‐enkephalin (DAGO), and β‐casomorphin1‐4 amide (morphiceptin) were much less potent. However, in similar experiments on rat hippocampal slices DAGO (1 μM) was much more potent than ethylketocyclazocine (1 μM) or DADLE (1 μM). (–)‐N‐(3‐furylmethyl)‐α‐noretazocine [(–)‐MR 2266], 1 μM, a preferential K‐antagonist, antagonized the effect of ethylketocyclazocine more potently than (–)‐naloxone or (+)‐MR 2267 [the (+)‐enantiomer of (–)‐MR 2266]. Given alone, (–)‐MR 2266 slightly and (+)‐MR 2267 (1 μM each) greatly enhanced NA release, apparently due to α2‐adrenoceptor blockade since their effects were completely abolished in the presence of yohimbine (0.1 μM). The effects of DADLE (1 μM) and DAGO (1 μM) were also antagonized by (–)MR 2266 (0.1 μM) but not by the δ‐antagonist N,N‐diallyl‐Tyr‐Aib‐Phe‐Leu‐OH (ICI 174864), 0.3 μM. It is concluded that NA release in the rabbit hippocampus is inhibited via K‐receptors; our results do not support the presence of modulatory μ‐ and δ‐receptors in this tissue. However, in the rat hippocampus μ‐receptors may modulate NA release.

Effect of extracellular dopamine on the release of dopamine in the rabbit caudate nucleus: evidence for a dopaminergic feedback inhibition.

SummarySlices of the head of the rabbit caudate nucleus were preincubated with 10−7 M 3H-dopamine and then superfused, and the effect of unlabeled dopamine on the outflow of tritium was investigated. In most experiments, nomifensine was added throughout superfusion in order to block uptake of the unlabeled amine. Nomifensine was a potent inhibitor of the uptake of 3H-dopamine into rabbit caudate synaptosomes, with an IC50 of 5·10−8 M at a 3H-dopamine concentration of 4·10−8 M.In the absence of nomifensine, unlabeled dopamine (10−7 M and higher concentrations) accelerated the basal outflow of tritium from preincubated slices. 10−5 M nomifensine strongly counteracted the acceleration. In the presence of nomifensine, unlabeled dopamine (10−7 to 10−6 M) caused a concentrationdependent decrease of the overflow of tritium evoked by electrical stimulation at 0.1 Hz. Chlorpromazine and haloperidol (in the presence of nomifensine) increased the stimulation evoked overflow and antagonized the inhibitory effect of dopamine.It is concluded that extracellular dopamine shares with other dopaminergic agonists the ability to inhibit action potential-evoked release of intraneuronal dopamine. The inhibition is mediated by specific receptors. The results support the hypothesis that previously released dopamine, by an action on these receptors, can inhibit further release of dopamine.

Effects of septal and/or raphe cell suspension grafts on hippocampal choline acetyltransferase activity, high affinity synaptosomal uptake of choline and serotonin, and behavior in rats with extensive septohippocampal lesions

At 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 [3H]choline and [3H]serotonin. The remaining rats were used for histological verifications on brain sections stained for acetylcholinesterase (AChE). The lesions increased locomotor activity, 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 effects on hippocampal AChE positivity, increased both the ChAT activity and the choline uptake to 70% ad 38% of the 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 rich 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Downregulation of muscarinic- and 5-HT1B-mediated modulation of [3H]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 microM), physostigmine (acetylcholinesterase inhibitor, 0.1 microM), oxotremorine (muscarinic agonist, 0.01 microM-10 microM), mecamylamine (nicotinic antagonist, 10 microM), methiothepin (mixed 5-HT1/5-HT2 antagonist, 10 microM), 8-OH-DPAT (5-HT1A agonist, 1 microM), 2-methyl-serotonin (5-HT3 agonist, 1 microM) and CP 93129 (5-HT1B agonist, 0.1 microM-100 microM), 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-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 exert at the level of the cholinergic terminal.

Presynaptic opioid receptors modulating acetylcholine release in the hippocampus of the rabbit

SummarySlices of the rabbit hippocampus were pre-incubated with 3H-choline, rinsed and superfused continuously. The release of 3H-acetylcholine in these slices, evoked by electrical field stimulation, was strongly reduced by the preferential κ-agonists ethylketocyclazocine, dynorphin A (1–13) and dynorphin A (1–17). Dynorphin A (1–9) and (−)MR 2034 [(−)5,9-dimethyl-2′-OH-2-tetrahydrofurfuryl-6,7-benzomorphan] were less potent, the (+)enantiomer of (−)MR 2034 was ineffective. Whereas the μ-agonist DAGO (d-Ala2-Gly-ol5-enkephalin) showed significant depressant effects, two other μ-agonists morphine and morphiceptine, as well as the δ-agonists DADLE (d-Ala2-d-Leu5-enkephalin) and Leu-enkephalin were much less inhibitory. The preferential μ-antagonist (−)naloxone as well as (−)MR 2266 [(−)N-(3-furylmethyl)-α-noretazocine], a preferential κ-antagonist, did not increase acetylcholine release when given alone, but antagonized the effect of ethyl-ketocyclazocine; (−)MR 2266 (Ke: 1.6 nmol/l) was about 4 times more potent than (−)naloxone (Ke: 6.3 nmol/l). The inhibitory effects of DAGO and DADLE were abolished by (−)MR 2266 (0.1 μmol/l) but not by the δ-antagonist ICI 174864, (N,N-diallyl-Tyr-Aib-Phe-Leu-OH, 0.3 μmol/l). It is concluded that the release of acetylcholine in the hippocampus of the rabbit is inhibited at the level of the axon terminals via κ-receptors; in addition, μ-receptors may be present. An inhibitory tone of endogenous opioid peptides on hippocampal acetylcholine release could not be demonstrated. Experiments on rat hippocampal slices showed that in this species μ- rather than κ-receptors may modulate acetylcholine release.

Graft-derived cholinergic reinnervation of the hippocampus prevents a lasting increase of hippocampal noradrenaline concentration induced by septohippocampal damage in rats

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 [3H]choline (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.