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Effect of calcium channel antagonists on electrically-evoked release of 5-HT in rat hippocampus in vivo as studied by microdialysis
1923 P.th.311 [
Effect of calcium channel antagonists on electrically-evoked release of S-HT in mt
hippocmnpus in e/vo as studied by mieredialysis Sharp, T., Bramwell, S. and Grahame-Smith, D.G. MRC Clinical PImmm¢olog}, Unit, Radcliffe In~immry, Ox/ord OX2 6HF., U.K.
There is evidence that depolarization-evoked release of $-hydroxytryptamine (5-HT) from central nerve tegminah in vitro is mediated via an i~qux of extraceHular calcium through voltage-sensitive calcium channels (VSCC's) of the N-type. In particular, as well as being dependent on extracellular calcium, depolarization-evoked release of radiolabelled $-HT from slices of animal brain tissue can be inhibited by agents blocking N- but not L-type VSCC's (Middlemiss and Speddin& 1985; Dooley et al., 1987). Here we have attempted to study the pharmacological nature of the VSCC involved in depolarization-evoked release of endogenous $-HT in rat hippocampus in vivo utilizing brain microdialysis. Depolarization of 5-HT neurones innervating hippocampus (ventral) was achieved by electrical stimulation of their probable site of origin, the dorsal raphe nucleus (DRN). Adult male Sprague Dawley rats (280-320 8) were anaesthetized with chloral hydrate throughout the experimenL A microdialysis probe and a stimulating electrode were stereotaxically implanted into the ventral hippocampus and DRN, respectively. The microdialysis probe was peffused continuously with az-'~ticial ~,.~,Fc o n ~ ! pM cita.~rtopr-.--~-and perfusates were collected every 20 rain and then analysed for 5-HT using HPLC-EC (for further methodological details see Sharp et al., 1989). A control period of 2-3 h was allowed b~fore commencing nerve stimulations. In one group of experiments the DRN was electrically stimulated for 20 r ~ at 60 rain intervals with the p,,.dsc frequently being increased succ:~sive!y (2, 3, $ and 10 I ~ ) ~ith ~ c h stimulation pexio6, in another group of experiments an initial 20 rain period of electrical stimulation at 3 Hz was immediately followed by a change of perfusion medium (either calcium was removed or drugs were added) and then a second 20 rain period of stimulation at 3 ~ was applied 100-120 rain later. After a 2-3 h control period, basal levels of 5-HT in the hippocampal perfusates were readily detectable (approx. 0.05 pmol/sample) and stable. Electrical stimulation of the DRN indu~,~d a short-lasting and frequency-dependent (2-10 Hz) increase of $-HT in the hippocampal perfusates (n = 9 rats). Followin8 a control 3 Hz stimulation of the DRN, both removal of calcium from, or addition of tetrodotox~n (10 pM) to, the perfusion medium decreased basal 5-HT output by about 50~; and completely prevented a release 3f 5-HT in response to a second stimulation (n -- $ in each case). Similarly, local perfusion with the N/L-type VSCC antagonist cadmium (300 pM) reduced basal $-HT output to about 50~ of control levels and markedly attenuated the effect of stimulation of the DRN on $-HT output (n ==4). In contrast, local perfusion with the selective L-type VSCC antagonists, nifedipine (10 pM) and diltiazem (10 pM), failed to attenuate either basal or electrically-evoked ou~ut of 5-HT (n ==4 in each case). Preliminary experiments show that nickel (300 pit/), which has antagonist activity at the T-type VSCC, does not affect $-HT
output. These experiments show that, in the rat, low frequency electrical stimulation of the DRN induces a release of hippocampal $-HT through a depolarization-evoked/calcium-dependent mechanism. Furthermore, our data support '.he idea that in vivo, depolarization of hippocaml:' ' " .'TI"nerve terminals causes a release of $-HT via activation of an N-type VSCC.
Referenees Dool~,, DJ.: A. Lupp and G. Hertlting, 1987, Naunyn-Schmiedeberg'sArch. Pharmacol. 336, 467. Middlemiss, D.N. and M. Speddin& 1985, Nature 314, 94. Sharp, T., S. Bramwell, D. Clark and D.G. Grahame-Smith, 1989, J.NeurochenL 53, 234.
Effect of calcium channel antagonists on electrically-evoked release of S-HT in mt
hippocmnpus in e/vo as studied by mieredialysis Sharp, T., Bramwell, S. and Grahame-Smith, D.G. MRC Clinical PImmm¢olog}, Unit, Radcliffe In~immry, Ox/ord OX2 6HF., U.K.
There is evidence that depolarization-evoked release of $-hydroxytryptamine (5-HT) from central nerve tegminah in vitro is mediated via an i~qux of extraceHular calcium through voltage-sensitive calcium channels (VSCC's) of the N-type. In particular, as well as being dependent on extracellular calcium, depolarization-evoked release of radiolabelled $-HT from slices of animal brain tissue can be inhibited by agents blocking N- but not L-type VSCC's (Middlemiss and Speddin& 1985; Dooley et al., 1987). Here we have attempted to study the pharmacological nature of the VSCC involved in depolarization-evoked release of endogenous $-HT in rat hippocampus in vivo utilizing brain microdialysis. Depolarization of 5-HT neurones innervating hippocampus (ventral) was achieved by electrical stimulation of their probable site of origin, the dorsal raphe nucleus (DRN). Adult male Sprague Dawley rats (280-320 8) were anaesthetized with chloral hydrate throughout the experimenL A microdialysis probe and a stimulating electrode were stereotaxically implanted into the ventral hippocampus and DRN, respectively. The microdialysis probe was peffused continuously with az-'~ticial ~,.~,Fc o n ~ ! pM cita.~rtopr-.--~-and perfusates were collected every 20 rain and then analysed for 5-HT using HPLC-EC (for further methodological details see Sharp et al., 1989). A control period of 2-3 h was allowed b~fore commencing nerve stimulations. In one group of experiments the DRN was electrically stimulated for 20 r ~ at 60 rain intervals with the p,,.dsc frequently being increased succ:~sive!y (2, 3, $ and 10 I ~ ) ~ith ~ c h stimulation pexio6, in another group of experiments an initial 20 rain period of electrical stimulation at 3 Hz was immediately followed by a change of perfusion medium (either calcium was removed or drugs were added) and then a second 20 rain period of stimulation at 3 ~ was applied 100-120 rain later. After a 2-3 h control period, basal levels of 5-HT in the hippocampal perfusates were readily detectable (approx. 0.05 pmol/sample) and stable. Electrical stimulation of the DRN indu~,~d a short-lasting and frequency-dependent (2-10 Hz) increase of $-HT in the hippocampal perfusates (n = 9 rats). Followin8 a control 3 Hz stimulation of the DRN, both removal of calcium from, or addition of tetrodotox~n (10 pM) to, the perfusion medium decreased basal 5-HT output by about 50~; and completely prevented a release 3f 5-HT in response to a second stimulation (n -- $ in each case). Similarly, local perfusion with the N/L-type VSCC antagonist cadmium (300 pM) reduced basal $-HT output to about 50~ of control levels and markedly attenuated the effect of stimulation of the DRN on $-HT output (n ==4). In contrast, local perfusion with the selective L-type VSCC antagonists, nifedipine (10 pM) and diltiazem (10 pM), failed to attenuate either basal or electrically-evoked ou~ut of 5-HT (n ==4 in each case). Preliminary experiments show that nickel (300 pit/), which has antagonist activity at the T-type VSCC, does not affect $-HT
output. These experiments show that, in the rat, low frequency electrical stimulation of the DRN induces a release of hippocampal $-HT through a depolarization-evoked/calcium-dependent mechanism. Furthermore, our data support '.he idea that in vivo, depolarization of hippocaml:' ' " .'TI"nerve terminals causes a release of $-HT via activation of an N-type VSCC.
Referenees Dool~,, DJ.: A. Lupp and G. Hertlting, 1987, Naunyn-Schmiedeberg'sArch. Pharmacol. 336, 467. Middlemiss, D.N. and M. Speddin& 1985, Nature 314, 94. Sharp, T., S. Bramwell, D. Clark and D.G. Grahame-Smith, 1989, J.NeurochenL 53, 234.