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Blockade of voltage-gated calcium channels in rat inhibits repetitive cortical spreading depression
Neuroscience Letters 334 (2002) 123–126 www.elsevier.com/locate/neulet
Blockade of voltage-gated calcium channels in rat inhibits repetitive cortical spreading depression Frank Richter*, Andrea Ebersberger, Hans-Georg Schaible Department of Physiology, Friedrich Schiller University of Jena, Teichgraben 8, D-07740 Jena, Germany Received 29 July 2002; received in revised form 24 September 2002; accepted 24 September 2002
Abstract Blockers of L-, N-, and P/Q-type voltage-gated calcium channels (VGCCs) were topically applied to the cortical surface of anaesthetized adult rats to study their role in cortical spreading depression (CSD), a correlate of the migraine aura. By pricking the brain, single CSD could still be elicited after blockade of the three different types of VGCCs as in the untreated brain. Topical KCl application to the untreated cortex resulted in repetitive CSD. However, after application of blockers at either L-, or N-, or P/Q-type VGCCs to the cortical surface, application of KCl elicited only one or very few CSD, and their repetition rate was dramatically reduced. The results suggest that cortical excitability resulting in repetitive CSD is markedly influenced by N- and P/Q-type VGCCs and less by L-type VGCCs. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Spreading depression; Rat; Direct current potentials of the brain; Voltage-gated calcium channel; L-type calcium channel; Ntype calcium channel; P/Q-type calcium channel; Migraine model
Spreading depression in the cortical gray matter (cortical spreading depression, CSD) [15] is thought to be a putative neuronal mechanism underlying migraine aura [14]. CSD is a depolarization wave characterized by a transient negative DC shift that propagates slowly from a focus across the hemisphere. CSD is accompanied by a rise in extracellular potassium concentration ([K 1]e) to 60–70 mM, whereas sodium, calcium and chloride ions move into cells together with water [5,13,17,18]. Usually these transients recover within 90–120 s in adult animals. Whether CSD itself is initiating the pain phase of migraine is controversially disputed [3,6,10]. Calcium ions and voltage-gated calcium channels (VGCCs) play a role in CSD. In rat cerebral cortex addition of divalent cations such as calcium or magnesium (0.1 M) to the superfusing solution was known to block CSD initiation and propagation. CSD amplitudes declined to 20% when CSD reached a brain area pretreated with 0.15 M CaCl2 [5]. In hippocampal slices addition of Ni 21 or Co 21 ions to the bathing solutions blocked VGCCs and prevented propagation but not the initiation of SD [11]. On the other hand, CSD can also be facilitated when calcium ions are * Corresponding author. Tel.: 149-36-4193-8811; fax: 149-364193-8812 E-mail address: [email protected] (F. Richter).
removed from the extracellular environment [18]. To date, neither the precise role of calcium in CSD nor the type of VGCC(s) involved have been sufficiently explored. In the brain, at least three different VGCCs are present: L-type (Cav1.3(a1D)) channels are located at the cell somata and regulate cell excitability, P/Q-type (Cav2.1(a1A)) channels and N-type (Cav2.2(a1B)) channels are located on presynaptic endings (and on cell somata) and regulate neurotransmission, synaptic firing and shaping of action potentials [7]. Some evidence was provided for an involvement of P/Qtype channels in CSD. Leaner and tottering mice with mutations of the P/Q-calcium channels showed increased resistance to CSD and lack of self-regenerated repetitive CSDwaves [1]. In order to define which VGCCs are involved in CSD, we administered either blockers of L-, or N-, or P/Q-type channels topically to the cerebral cortex of anesthetized rats to study their influence on propagation of CSD. The experiments were approved by the animal protection committee and the regional government of Thueringen (reg.02-09/01). Twenty-one male Wistar rats (350–450 grams) were used for the experiments. The rats were anaesthetized with sodium pentobarbital (Trapanal w, Byk Gulden, Konstanz, Germany; initially 100 mg/kg i.p.). Absence of the corneal blink reflex was maintained by supplemental doses of 20 mg/kg i.p. The trachea, the left femoral vein and artery were
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 01 12 0- 5
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F. Richter et al. / Neuroscience Letters 334 (2002) 123–126
Fig. 1. Comparison of effects of calcium channel blockers applied to the cortical surface on the occurrence of repetitive CSDs elicited by KCl. Not more than one blocker was tested in any one animal, each sample represents data from a particular animal. (A) Schematic top view (not to scale) with exposed cortex to which the blocker was applied (encircled with shaded area) and openings for electrodes and KCl application (small circles in front of coronar suture). A reference electrode is placed on the nasal bone. (B) Single CSD recorded in a depth of 1200 mm elicited by pin prick (P) and repetitive CSDs after application of KCl to the cortex in control animals. (C) Single CSD and CSD after KCl, 60 min after application of v-agatoxin to the cortical area. The upper trace shows the untreated remote brain in a depth of 1200 mm, the lower trace the treated brain area in the same cortical depth. (D) Same presentation, but 60 min after application of v-conotoxin. (E) Same presentation, but 60 min after application of nimodipine. Scale bars refer to all traces.
cannulated. The mean arterial blood pressure was continuously monitored. Body temperature was kept at 37 8C by a feed-back controlled heating system. The head of the animal was fixed stereotactically and the skin above the skull was removed. The dura mater was exposed over the left hemisphere from bregma to lambda (from midline spanning 4–6 mm laterally) using a mini-drill under saline cooling and kept moist with Tyrode throughout the experiment. Two further openings (B 1.5–2 mm), one used for DC recording, the other for CSD elicitation were made at 2 mm anterior
and 2 mm and 4 mm lateral from bregma, respectively, on the left hemisphere. Underneath these openings the dura mater was incised. Dental acrylic was used to build a wall around the exposed dura to prevent accidental overflow of solutions (see also Fig. 1A). Prior to the DC-recordings the exposed dura mater was carefully removed. An Ag/AgCl reference electrode containing 2 M KCl was placed on the nasal bone. Intracortical DC potentials were recorded using glass micropipettes (tip diameter about 5 mm, resistance , 10 MV) filled with 150 mM NaCl. One electrode was introduced into the cortex through one of the small openings to a depth of 1200 mm. Another electrode consisted of three pipettes which were glued together, their tips staggered horizontally and vertically by 400 mm, respectively. This assembly was lowered into the exposed gray matter within the temporal trepanation site 0.8–1.2 cm apart from the frontal DC electrode. At this site DC shifts were recorded simultaneously at cortical depths of 400, 800, and 1200 mm. The signals were recorded using a four-channel high-impedance amplifier (Meier, Munich, Germany) and stored on a PC. According to widely used experimental procedures [5], single CSDs were elicited by a pin prick into the cortex with a needle (diameter 0.5 mm) at the frontal opening, and repetitive CSDs were elicited by a small KCl crystal (weighing 0.08 to 0.1 mg) placed onto the pia mater at the same site. CSDs were evaluated according to their amplitudes, CSD peak-time (time from beginning of depolarization to maximal amplitude or first peak), CSD-decline (time from beginning of repolarization to baseline), and CSD-duration (duration between start of depolarization and reaching the baseline). The following VGCC blockers were used: the N-type VGCC blocker v-conotoxin GVIA 10 26 M in 165 mM NaCl, pH 7.4 (Bachem, Heidelberg, Germany), the L-type VGCC blocker nimodipine 10 25 M in 165 mM NaCl, pH 7.4 (RBI, Natick, USA), and the P/Q-type VGCC blocker vagatoxin IVA 10 26 M in 165 mM NaCl, pH 7.4 (Bachem, Heidelberg, Germany). An amount of about 200 ml (supplemented if needed) of each blocker was applied to the exposed cortex area and remained there for 60 min before attempts were made to elicit CSD. Concentrations of VGCC blockers have been chosen that are specific for VGCC types [9], and the concentration in the tissue might even be lower than the applied concentration. Before application of a blocker to the surface of the cortex, a slight pin prick into the stimulation site evoked CSDrelated DC shifts lasting up to 90 s with amplitudes up to 25 mV both in the adjacent cortical area and in the remote cortex. The DC shifts migrated with a velocity of 2–3 mm/ min and affected usually all cortical depths. After application of either v-conotoxin, v-agatoxin or nimodipine to the cortical surface for 60 min, pin prick still evoked single CSDs. After v-conotoxin they were significantly lower in amplitude (in mean 24.3 ^ 0.3 mV before and 18.6 ^ 1.1 mV after vconotoxin). Significant changes in CSD-amplitude, -peaktime, -decline and -duration were observed neither upon
F. Richter et al. / Neuroscience Letters 334 (2002) 123–126
Fig. 2. Average numbers of CSD ^ SEM elicited by a single KCl application per 30 min in control animals and in animals treated with either v-conotoxin (con.), nimodipine (nim.) or v-agatoxin (aga.) administered to the cortical surface. Numbers below the columns give numbers of animals tested. In controls both brain areas were untreated, the bars show the recordings remote from (left) and close to the KCl application (right). *Significant reduction compared to control (Mann–Whitney U-test with Bonferroni’s post test, P , 0:05).
repetitive stimulation in control rats, nor after v-agatoxin and nimodipine. Propagation velocity of prick-induced CSD was unchanged after application of any blocker. The result was different when a series of CSD was elicited by application of a small KCl crystal to the stimulation site. While KCl usually elicits a series of CSD in untreated animals (Fig. 1B), only a few repetitions were seen after v-agatoxin or v-conotoxin in the treated brain area (Fig. 1C,D, upper trace) and in the untreated brain (Fig. 1C,D, lower trace; Fig. 2). Only after v-conotoxin the CSD parameters were changed. The first KCl-induced CSD were significantly smaller in amplitude (in mean 24.3 ^ 0.3 mV before and 17.5 ^ 0.9 mV after v-conotoxin), prolonged in peak-time (before: 15.0 ^ 1.6 s, after: 30.3 ^ 7.4 s), decline (before: 101.5 ^ 17.8 s, after: 132 ^ 13.9 s and duration (before: 141 ^ 4.0 s, after: 164.7 ^ 14.0 s). Nimodipine had a small effect on CSD-repetition rate (Figs. 1E and 2), amplitude (before: 15 ^ 1.4 s, after: 14.3 ^ 0.9 s) and peaktime (before: 24.8 ^ 3.9 s, after: 32.3 ^ 4.6 s) of the first KCl-induced CSD following KCl. The third CSD after KCl was not altered in these parameters. The present data show that all three types of VGCC play a role in the self-perpetuation of KCl-induced CSD. Striking effects were seen after blockade of N-type or P/Q-type VGCCs. With respect to P/Q-type VGCCs, the present data seem to be consistent with those of Ayata et al. [1] who have shown that mice with mutations in the CACNA1A gene encoding the a1A subunit of the P/Q-type calcium channel exhibit no repetitive CSDs after KCl challenge. The present data show that L-type VGCCs influence the generation of repetitive CSDs less. The precise role of Ca 21 in the generation and propagation of repetitive CSDs is not known. There are several
125
possibilities. The role of VGCCs in the release of transmitters could be important. Ca 21 plays a role in the release of glutamate that is involved in the propagation of CSD [18]. In the study of Ayata et al. [1] the mice with P/Q-type mutations showed less glutamate release than normal mice when KCl was administered. Because P/Q- as well as Ntype VGCCs are located presynaptically [19] and control exocytosis, the similar effects of v-agatoxin and v-conotoxin on repetitive CSDs may have a common cause, namely reduction of glutamate. This reduction could weaken the ‘positive feedback’ cycle in which both increased [K 1]o and release of excitatory neurotransmitters (e.g. glutamate and aspartate) are needed for self-regeneration of repetitive CSD [18]. This assumption is supported by the inhibition of potassium-evoked glutamate and serotonin release after treatment with v-conotoxin or v-agatoxin [12]. However, at present it is not precisely known whether P/Qand N-type channels have a similar localization or influence release of transmitters from the same population of neurons. Since the CSD parameters were only changed after N-type channel blockers it is also possible that further transmitters and other N-type dependent processes are involved in the time course of CSDs. We cannot exclude that the reduction of CSD repetition rate in the remote and untreated brain area could be due to diffusion of the blockers, since the wall built around the exposed brain area only prevented the overflow but not migration below the skull bone. The mechanism of nimodipine could be different because L-type channels are mainly located on the postsynaptic site. However, L-type channels control excitability, and changes of excitability may affect the release of transmitters [2]. Other functions of Ca 21 are conceivable. Migrating waves of intracellular Ca 21 across glial cells were observed during propagation and repetition of CSDs [8,16] raising the possibility that glia cells are important in the generation of repetitive CSDs. With respect to migraine, particular importance has been ascribed to the P/Q-type VGCC because patients with hemiplegic migraine exhibited a mutation of this channel. However, this may be a special case. In a recent study on familial migraine with aura such genetic changes could not be identified [4]. Thus it does not appear that only P/Q-type channels should be related to neuronal events of migraine. Rather several VGCCs could be involved in generation of repetitive CSDs and migraine aura. This study was supported by Interdisciplinary Center for Clinical Research Jena (IZKF B378-10102).
[1] Ayata, C., Shimizu-Sasamata, M., Lo, E.H., Noebels, J.L. and Moskowitz, M.A., Impaired neurotransmitter release and elevated threshold for cortical spreading depression in mice with mutations in the a1A subunit of P/Q type calcium channels, Neuroscience, 95 (2000) 639–645. [2] Barral, J., Poblette, F., Mendoza, E., Pineda, J.C., Galarragga, E. and Bargas, J., High-affinity inhibition of gluta-
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F. Richter et al. / Neuroscience Letters 334 (2002) 123–126 mate release from corticostriatal synapses by omegaagatoxin TK, Eur. J. Pharmacol., 430 (2001) 167–173. Bolay, H., Reuter, U., Dunn, A.K., Huang, Z.H., Boas, D.A. and Moskowitz, M.A., Intrinsic brain activity triggers trigeminal meningeal afferents in a migraine model, Nat. Med., 8 (2002) 136–142. Brugnoni, R., Leone, M., Rigamonti, A., Moranduzzo, E., Cornelio, F., Mantegazza, R. and Bussone, G., Is the CACNA1A gene involved in familial migraine with aura? Neurol. Sci., 23 (2002) 1–5. Buresˇ , J., Buresˇ ova´ , O. and Kriva´ nek, J., The Mechanism and Application of Lea˜ o’s Spreading Depression of Electroencephalographic Activity, Academic Press, New York, 1974 410 pp.. Ebersberger, A., Schaible, H.G., Averbeck, B. and Richter, F., Is there a correlation between spreading depression, neurogenic inflammation and nociception that might cause migraine headache? Ann. Neurol., 49 (2001) 7–13. Ertel, E.A., Campbell, K.P., Harpold, M.M., Hofmann, F., Mori, Y., Perez-Reyes, E., Schwartz, A., Snutch, T.P., Tanabe, T., Birnbaumer, L., Tsien, R.W. and Catterall, W.A., Nomenclature of voltage-gated calcium channels, Neuron, 25 (2000) 533–535. Glaum, S.R., Holzwarth, J.A. and Miller, R.J., Glutamate receptors activate Ca 21 mobilization and Ca 21 influx into astrocytes, Proc. Natl. Acad. Sci. USA, 87 (1990) 3454–3458. Hoffmann, F., Lacinova, L. and Klugbauer, N., Voltagedependent calcium channels: from structure to function, Rev. Physiol. Biochem. Pharmacol., 139 (1999) 35–87. Iadecola, C., From C.S.D. to headache: a long and winding road, Nat. Med., 8 (2002) 110–112.
[11] Jing, J., Aitken, P.G. and Somjen, G.G., Role of calcium channels in spreading depression in rat hippocampal slices, Brain Res., 604 (1993) 251–259. [12] Keith, R.A., Mangano, T.J., Lampe, R.A., DeFeo, P.A., Hyde, M.J. and Donzanti, B.A., Comparative actions of synthetic omega-grammotoxin SIA and synthetic omega-Aga-IVA on neuronal calcium entry and evoked release of neurotransmitters in vitro and in vivo, Neuropharmacology, 34 (1995) 1515–1528. [13] Kraig, P.R. and Nicholson, C., Extracellular ionic variations during spreading depression, Neuroscience, 3 (1978) 1045– 1059. [14] Lauritzen, M., Pathophysiology of the migraine aura: the spreading depression theory, Brain, 117 (1994) 199– 210. [15] Lea˜ o, A.A.P., Spreading depression of activity in the cerebral cortex, J. Neurophysiol., 7 (1944) 359–390. [16] Needergaard, M., Direct signalling from astrocytes to neurons in cultures of mammalian brain cells, Science, 263 (1994) 1768–1771. [17] Phillips, J.M. and Nicholson, C., Anion permeability in spreading depression investigated with ion sensitive microelectrodes, Brain Res., 173 (1979) 567–571. [18] Somjen, G.G., Mechanisms of spreading depression and hypoxic spreading depression-like depolarization, Physiol. Rev., 81 (2001) 1065–1096. [19] Timmermann, D.B., Westenbroek, R.E., Schousboe, A. and Catterall, W.A., Distribution of high-voltage-activated calcium channels in cultured gamma-aminobutyric acidergic neurons from mouse cerebral cortex, J. Neurosci. Res., 67 (2002) 48–61.
Blockade of voltage-gated calcium channels in rat inhibits repetitive cortical spreading depression Frank Richter*, Andrea Ebersberger, Hans-Georg Schaible Department of Physiology, Friedrich Schiller University of Jena, Teichgraben 8, D-07740 Jena, Germany Received 29 July 2002; received in revised form 24 September 2002; accepted 24 September 2002
Abstract Blockers of L-, N-, and P/Q-type voltage-gated calcium channels (VGCCs) were topically applied to the cortical surface of anaesthetized adult rats to study their role in cortical spreading depression (CSD), a correlate of the migraine aura. By pricking the brain, single CSD could still be elicited after blockade of the three different types of VGCCs as in the untreated brain. Topical KCl application to the untreated cortex resulted in repetitive CSD. However, after application of blockers at either L-, or N-, or P/Q-type VGCCs to the cortical surface, application of KCl elicited only one or very few CSD, and their repetition rate was dramatically reduced. The results suggest that cortical excitability resulting in repetitive CSD is markedly influenced by N- and P/Q-type VGCCs and less by L-type VGCCs. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Spreading depression; Rat; Direct current potentials of the brain; Voltage-gated calcium channel; L-type calcium channel; Ntype calcium channel; P/Q-type calcium channel; Migraine model
Spreading depression in the cortical gray matter (cortical spreading depression, CSD) [15] is thought to be a putative neuronal mechanism underlying migraine aura [14]. CSD is a depolarization wave characterized by a transient negative DC shift that propagates slowly from a focus across the hemisphere. CSD is accompanied by a rise in extracellular potassium concentration ([K 1]e) to 60–70 mM, whereas sodium, calcium and chloride ions move into cells together with water [5,13,17,18]. Usually these transients recover within 90–120 s in adult animals. Whether CSD itself is initiating the pain phase of migraine is controversially disputed [3,6,10]. Calcium ions and voltage-gated calcium channels (VGCCs) play a role in CSD. In rat cerebral cortex addition of divalent cations such as calcium or magnesium (0.1 M) to the superfusing solution was known to block CSD initiation and propagation. CSD amplitudes declined to 20% when CSD reached a brain area pretreated with 0.15 M CaCl2 [5]. In hippocampal slices addition of Ni 21 or Co 21 ions to the bathing solutions blocked VGCCs and prevented propagation but not the initiation of SD [11]. On the other hand, CSD can also be facilitated when calcium ions are * Corresponding author. Tel.: 149-36-4193-8811; fax: 149-364193-8812 E-mail address: [email protected] (F. Richter).
removed from the extracellular environment [18]. To date, neither the precise role of calcium in CSD nor the type of VGCC(s) involved have been sufficiently explored. In the brain, at least three different VGCCs are present: L-type (Cav1.3(a1D)) channels are located at the cell somata and regulate cell excitability, P/Q-type (Cav2.1(a1A)) channels and N-type (Cav2.2(a1B)) channels are located on presynaptic endings (and on cell somata) and regulate neurotransmission, synaptic firing and shaping of action potentials [7]. Some evidence was provided for an involvement of P/Qtype channels in CSD. Leaner and tottering mice with mutations of the P/Q-calcium channels showed increased resistance to CSD and lack of self-regenerated repetitive CSDwaves [1]. In order to define which VGCCs are involved in CSD, we administered either blockers of L-, or N-, or P/Q-type channels topically to the cerebral cortex of anesthetized rats to study their influence on propagation of CSD. The experiments were approved by the animal protection committee and the regional government of Thueringen (reg.02-09/01). Twenty-one male Wistar rats (350–450 grams) were used for the experiments. The rats were anaesthetized with sodium pentobarbital (Trapanal w, Byk Gulden, Konstanz, Germany; initially 100 mg/kg i.p.). Absence of the corneal blink reflex was maintained by supplemental doses of 20 mg/kg i.p. The trachea, the left femoral vein and artery were
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 01 12 0- 5
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F. Richter et al. / Neuroscience Letters 334 (2002) 123–126
Fig. 1. Comparison of effects of calcium channel blockers applied to the cortical surface on the occurrence of repetitive CSDs elicited by KCl. Not more than one blocker was tested in any one animal, each sample represents data from a particular animal. (A) Schematic top view (not to scale) with exposed cortex to which the blocker was applied (encircled with shaded area) and openings for electrodes and KCl application (small circles in front of coronar suture). A reference electrode is placed on the nasal bone. (B) Single CSD recorded in a depth of 1200 mm elicited by pin prick (P) and repetitive CSDs after application of KCl to the cortex in control animals. (C) Single CSD and CSD after KCl, 60 min after application of v-agatoxin to the cortical area. The upper trace shows the untreated remote brain in a depth of 1200 mm, the lower trace the treated brain area in the same cortical depth. (D) Same presentation, but 60 min after application of v-conotoxin. (E) Same presentation, but 60 min after application of nimodipine. Scale bars refer to all traces.
cannulated. The mean arterial blood pressure was continuously monitored. Body temperature was kept at 37 8C by a feed-back controlled heating system. The head of the animal was fixed stereotactically and the skin above the skull was removed. The dura mater was exposed over the left hemisphere from bregma to lambda (from midline spanning 4–6 mm laterally) using a mini-drill under saline cooling and kept moist with Tyrode throughout the experiment. Two further openings (B 1.5–2 mm), one used for DC recording, the other for CSD elicitation were made at 2 mm anterior
and 2 mm and 4 mm lateral from bregma, respectively, on the left hemisphere. Underneath these openings the dura mater was incised. Dental acrylic was used to build a wall around the exposed dura to prevent accidental overflow of solutions (see also Fig. 1A). Prior to the DC-recordings the exposed dura mater was carefully removed. An Ag/AgCl reference electrode containing 2 M KCl was placed on the nasal bone. Intracortical DC potentials were recorded using glass micropipettes (tip diameter about 5 mm, resistance , 10 MV) filled with 150 mM NaCl. One electrode was introduced into the cortex through one of the small openings to a depth of 1200 mm. Another electrode consisted of three pipettes which were glued together, their tips staggered horizontally and vertically by 400 mm, respectively. This assembly was lowered into the exposed gray matter within the temporal trepanation site 0.8–1.2 cm apart from the frontal DC electrode. At this site DC shifts were recorded simultaneously at cortical depths of 400, 800, and 1200 mm. The signals were recorded using a four-channel high-impedance amplifier (Meier, Munich, Germany) and stored on a PC. According to widely used experimental procedures [5], single CSDs were elicited by a pin prick into the cortex with a needle (diameter 0.5 mm) at the frontal opening, and repetitive CSDs were elicited by a small KCl crystal (weighing 0.08 to 0.1 mg) placed onto the pia mater at the same site. CSDs were evaluated according to their amplitudes, CSD peak-time (time from beginning of depolarization to maximal amplitude or first peak), CSD-decline (time from beginning of repolarization to baseline), and CSD-duration (duration between start of depolarization and reaching the baseline). The following VGCC blockers were used: the N-type VGCC blocker v-conotoxin GVIA 10 26 M in 165 mM NaCl, pH 7.4 (Bachem, Heidelberg, Germany), the L-type VGCC blocker nimodipine 10 25 M in 165 mM NaCl, pH 7.4 (RBI, Natick, USA), and the P/Q-type VGCC blocker vagatoxin IVA 10 26 M in 165 mM NaCl, pH 7.4 (Bachem, Heidelberg, Germany). An amount of about 200 ml (supplemented if needed) of each blocker was applied to the exposed cortex area and remained there for 60 min before attempts were made to elicit CSD. Concentrations of VGCC blockers have been chosen that are specific for VGCC types [9], and the concentration in the tissue might even be lower than the applied concentration. Before application of a blocker to the surface of the cortex, a slight pin prick into the stimulation site evoked CSDrelated DC shifts lasting up to 90 s with amplitudes up to 25 mV both in the adjacent cortical area and in the remote cortex. The DC shifts migrated with a velocity of 2–3 mm/ min and affected usually all cortical depths. After application of either v-conotoxin, v-agatoxin or nimodipine to the cortical surface for 60 min, pin prick still evoked single CSDs. After v-conotoxin they were significantly lower in amplitude (in mean 24.3 ^ 0.3 mV before and 18.6 ^ 1.1 mV after vconotoxin). Significant changes in CSD-amplitude, -peaktime, -decline and -duration were observed neither upon
F. Richter et al. / Neuroscience Letters 334 (2002) 123–126
Fig. 2. Average numbers of CSD ^ SEM elicited by a single KCl application per 30 min in control animals and in animals treated with either v-conotoxin (con.), nimodipine (nim.) or v-agatoxin (aga.) administered to the cortical surface. Numbers below the columns give numbers of animals tested. In controls both brain areas were untreated, the bars show the recordings remote from (left) and close to the KCl application (right). *Significant reduction compared to control (Mann–Whitney U-test with Bonferroni’s post test, P , 0:05).
repetitive stimulation in control rats, nor after v-agatoxin and nimodipine. Propagation velocity of prick-induced CSD was unchanged after application of any blocker. The result was different when a series of CSD was elicited by application of a small KCl crystal to the stimulation site. While KCl usually elicits a series of CSD in untreated animals (Fig. 1B), only a few repetitions were seen after v-agatoxin or v-conotoxin in the treated brain area (Fig. 1C,D, upper trace) and in the untreated brain (Fig. 1C,D, lower trace; Fig. 2). Only after v-conotoxin the CSD parameters were changed. The first KCl-induced CSD were significantly smaller in amplitude (in mean 24.3 ^ 0.3 mV before and 17.5 ^ 0.9 mV after v-conotoxin), prolonged in peak-time (before: 15.0 ^ 1.6 s, after: 30.3 ^ 7.4 s), decline (before: 101.5 ^ 17.8 s, after: 132 ^ 13.9 s and duration (before: 141 ^ 4.0 s, after: 164.7 ^ 14.0 s). Nimodipine had a small effect on CSD-repetition rate (Figs. 1E and 2), amplitude (before: 15 ^ 1.4 s, after: 14.3 ^ 0.9 s) and peaktime (before: 24.8 ^ 3.9 s, after: 32.3 ^ 4.6 s) of the first KCl-induced CSD following KCl. The third CSD after KCl was not altered in these parameters. The present data show that all three types of VGCC play a role in the self-perpetuation of KCl-induced CSD. Striking effects were seen after blockade of N-type or P/Q-type VGCCs. With respect to P/Q-type VGCCs, the present data seem to be consistent with those of Ayata et al. [1] who have shown that mice with mutations in the CACNA1A gene encoding the a1A subunit of the P/Q-type calcium channel exhibit no repetitive CSDs after KCl challenge. The present data show that L-type VGCCs influence the generation of repetitive CSDs less. The precise role of Ca 21 in the generation and propagation of repetitive CSDs is not known. There are several
125
possibilities. The role of VGCCs in the release of transmitters could be important. Ca 21 plays a role in the release of glutamate that is involved in the propagation of CSD [18]. In the study of Ayata et al. [1] the mice with P/Q-type mutations showed less glutamate release than normal mice when KCl was administered. Because P/Q- as well as Ntype VGCCs are located presynaptically [19] and control exocytosis, the similar effects of v-agatoxin and v-conotoxin on repetitive CSDs may have a common cause, namely reduction of glutamate. This reduction could weaken the ‘positive feedback’ cycle in which both increased [K 1]o and release of excitatory neurotransmitters (e.g. glutamate and aspartate) are needed for self-regeneration of repetitive CSD [18]. This assumption is supported by the inhibition of potassium-evoked glutamate and serotonin release after treatment with v-conotoxin or v-agatoxin [12]. However, at present it is not precisely known whether P/Qand N-type channels have a similar localization or influence release of transmitters from the same population of neurons. Since the CSD parameters were only changed after N-type channel blockers it is also possible that further transmitters and other N-type dependent processes are involved in the time course of CSDs. We cannot exclude that the reduction of CSD repetition rate in the remote and untreated brain area could be due to diffusion of the blockers, since the wall built around the exposed brain area only prevented the overflow but not migration below the skull bone. The mechanism of nimodipine could be different because L-type channels are mainly located on the postsynaptic site. However, L-type channels control excitability, and changes of excitability may affect the release of transmitters [2]. Other functions of Ca 21 are conceivable. Migrating waves of intracellular Ca 21 across glial cells were observed during propagation and repetition of CSDs [8,16] raising the possibility that glia cells are important in the generation of repetitive CSDs. With respect to migraine, particular importance has been ascribed to the P/Q-type VGCC because patients with hemiplegic migraine exhibited a mutation of this channel. However, this may be a special case. In a recent study on familial migraine with aura such genetic changes could not be identified [4]. Thus it does not appear that only P/Q-type channels should be related to neuronal events of migraine. Rather several VGCCs could be involved in generation of repetitive CSDs and migraine aura. This study was supported by Interdisciplinary Center for Clinical Research Jena (IZKF B378-10102).
[1] Ayata, C., Shimizu-Sasamata, M., Lo, E.H., Noebels, J.L. and Moskowitz, M.A., Impaired neurotransmitter release and elevated threshold for cortical spreading depression in mice with mutations in the a1A subunit of P/Q type calcium channels, Neuroscience, 95 (2000) 639–645. [2] Barral, J., Poblette, F., Mendoza, E., Pineda, J.C., Galarragga, E. and Bargas, J., High-affinity inhibition of gluta-
126
[3]
[4]
[5]
[6]
[7]
[8]
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