- Email: [email protected]
Involvement of adenosine triphosphate-sensitive K+ channels in glucose-sensing in the rat solitary tract nucleus
Neuroscience Letters 278 (2000) 77±80 www.elsevier.com/locate/neulet
Involvement of adenosine triphosphate-sensitive K 1 channels in glucose-sensing in the rat solitary tract nucleus Michel Dallaporta a, Jean Perrin a, Jean-Claude Orsini a, b,* a
Laboratoire de Neurobiologie, CNRS-Universite de Provence, 31 chemin Joseph Aiguier, F-13402 Marseille ceÂdex 20, France b Faculte des Sciences du Sport, Universite de la MeÂditerraneÂe, Marseille, France Received 15 September 1999; received in revised form 22 October 1999; accepted 8 November 1999
Abstract The presence of adenosine triphosphate-sensitive (ATP-sensitive) K 1 channels (KATP channels) in the caudal nucleus tractus solitarii (NTS), and their possible involvement in glucose-sensing, were assessed by extracellular recording of neuronal activity in rat hindbrain slices. In 21 out of 36 recorded cells, ®ring was increased by sulfonylureas and decreased by K 1 channel opener (KCO), indicating the existence of KATP channels in the caudal NTS. In seven out of the nine neurons activated by a 2 mM increase in the glucose level, the effects of sulfonylureas and KCO were consistent with the involvement of KATP channels in the glucose response. Conversely, the mechanism(s) underlying the response of glucose-depressed neurons remains to be clari®ed. Finally, the presence of KATP channels was also detected in some neurons that were unresponsive to a 2 mM change in the glucose level. Thus, KATP channels were pharmacologically identi®ed in the caudal NTS, where they may be partly involved in glucose sensing. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Single unit activity; Feeding; Glucose level; KATP channel; Rats; Hindbrain
In free-moving and free-eating rats maintained in a steady state condition, meal initiation is preceded and triggered by a slight decline in the blood glucose level, and mediated by glycemia-sensitive brain neurons responsive to preprandial hypoglycemia [13]. One of the possible localizations of the relevant glycemia-sensitive neurons is the caudal part of the nucleus tractus solitarii (NTS), a glucose-sensitive structure [1] where several neurons respond to moderate changes in blood glucose level through the local action of glucose molecules [17]. We recently investigated this glucose sensitivity in vitro [8]. Half of the neurons recorded in the area responded to a 2 mM increase in the extracellular glucose level by either the activation or the depression of ®ring, and most of them were catecholaminergic. Moreover, the effects of 2-deoxyglucose suggested that a compound generated by carbohydrate metabolism, such as adenosine triphosphate (ATP), was involved in the NTS glucose sensing. The glucose sensitivity of some peripheral cells, such as the pancreatic beta-cells, involves potassium channels inhibited by intracellular ATP (KATP channels), so that * Corresponding author. Tel.: 133-4-91-164-581; fax: 133-491-220-875. E-mail address: [email protected] (J.-C. Orsini)
they are depolarized by a high glucose level and hyperpolarized by hypoglycemia [15]. Similarly, the glucose level affects the neuronal activity of different brain regions through KATP channels. These channels, as in pancreatic beta-cells, are composed of two proteins, an inwardly rectifying pore-forming unit, Kir 6.2, and a high af®nity sulfonylurea receptor, SUR1 [11,12]. The beta cell-like KATP channels are closed by antidiabetic sulfonylureas such as tolbutamide or glibenclamide, and activated by K 1 channel openers (KCOs) such as diazoxide, and to a lesser degree cromakalim or pinacidil [3,18]. In the striatal dopaminergic terminals, however, KATP channels are composed of Kir 6.2 pore-forming units associated with another isoform of sulfonylurea receptor, probably SUR2A [19]. These KATP channels belong to another subtype similar to those observed in heart cells, in which the channel is also closed by sulfonylureas and activated by cromakalim or pinacidil, but unaffected by diazoxide [3,18]. Glucose-sensing in the caudal NTS might be mediated by KATP channels [8], as suggested by the presence of Kir 6.2 mRNA in most C2 and some A2 catecholaminergic neurons of the nucleus [9], despite the low density of high-af®nity binding sites for [ 3H]glibenclamide in this area [16]. The purpose of the present study was to apply the above
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 89 8- 8
78
M. Dallaporta et al. / Neuroscience Letters 278 (2000) 77±80
Table 1 Distribution of 11 NTS neurons as a function of their response to Sulfonylureas and KCOs a Response to sulfonyluraes Tolbutamide 3
(1)
2
(1)
2 1 2 1
Glibenclamide 1
(1)
1
(1) (2) (0) (0)
(0) a
Response to KCOs
Presence of KATP channels
Diazoxide
Pinacidil
(2) (0)
(2)
Yes (b) Yes (c)
(2) (0) (2) (0) (2)
Yes (b) Yes (b) No No No
(2) (2) (0) (0)
(1) Increase in ®ring; (2) decrease in ®ring; (0) no response; (b ): b-cell type KATP channels; (c): cardiac type KATP channels.
pharmacological criteria while using conventional electrophysiological recording, to: (a) lend support to the presence of KATP channels in some caudal NTS neurons (neurons activated by tolbutamide and/or glibenclamide and depressed by diazoxide and/or pinacidil); (b) specify their subtype (beta cell-type in neurons depressed by diazoxide and cardiac type in neurons depressed by pinacidil and unaffected by diazoxide); (c) determine whether or not their modulation can mediate glucose sensitivity in this area (neurons responding in the same direction to glucose and tolbutamide or glibenclamide, and in the opposite direction to glucose and diazoxide or pinacidil). Male Wistar rats weighing 150±300 g were anesthetized by halothane inhalation (Fluothan, Coopers) and killed by decapitation. The brain was quickly removed and placed in chilled arti®cial cerebrospinal ¯uid (aCSF) saturated with O2 95%, CO2 5% containing (in mM) NaCl 125, KCl 5, KH2PO4 1.25, Mg SO4 2, CaCl2 3, NaHCO3 25.5, and glucose 5 (pH 7:4). The medulla oblongata was microdissected and 300-mm thick coronal slices were cut at the area postrema level with an oscillating tissue slicer (Camp-
den Instruments) and then transferred into a preincubation chamber ®lled with oxygenated aCSF at room temperature. More than 2 h after preincubation, the recording session started in a submerged type recording chamber (0.5 ml) perfused at a rate of 2.5 ml/min with oxygenated aCSF containing 3 or 4 mM glucose. The temperature of the chamber was kept at 348C. The perfusion medium entered the chamber close to the slice. Rapid switching to aCSF containing 5 or 6 mM glucose was achieved using an inlet ¯ow valve. The new solution reached the chamber in about 30 s (dead space time). The following substances were diluted in aCSF and bath applied: tolbutamide (Sigma) and diazoxide (RBI) dissolved in dimethylsulfoxide (DMSO) or NaOH 15 mM (®nal concentration in the chamber: 200 mM), glibenclamide (Sigma) dissolved in DMSO (®nal concentration in the chamber: 2 mM), pinacidil (Sigma) dissolved in NaOH 15 mM (®nal concentration in the chamber: 40 or 60 mM). Single unit activity was recorded extracellularly with glass microelectrodes (tip diameter: 2±3 mm) ®lled with 3 M NaCl. Electrodes were placed under microscopic obser-
Table 2 Distribution of 25 NTS neurons as a function of their response to Glucose, Sulfonylureas, and KCOs a Response to glucose
Response to sulfonylureas Tolbutamide
9 (1)
5 (2)
11 (0)
a
6 1 2 2 1 2
(2) (0)
2
(1)
2 1 2 4
(1) (2) (0) (0)
(1) (0)
Response to KCOs
Presence of KATP channels
Glibenclamide
Diazoxide
Pinacidil
(1) (1)
(0)
(2) (2)
(1) (2)
(2) (2)
(2)
1
(2)
(2)
1
(2) (2)
(0)
(1) (2) (0)
(1)
Yes * Yes * No Yes No No Yes Yes Yes Yes ** No No
(1) Increase in ®ring; (2) decrease in ®ring; (0) no response; *KATP channels possibly involved in glucose-sensing; **KATP channels possibly located in inhibitory afferents.
M. Dallaporta et al. / Neuroscience Letters 278 (2000) 77±80
Fig. 1. Example of a glucose-responsive neuron in which KATP channels may mediate glucose-sensing. It was activated by a bath application of the sulfonylurea glibenclamide and by a 2mM increase in the extracellular glucose level, and depressed by a bath application of the K 1 channel opener pinacidil.
vation in the caudal part of the nucleus tractus solitarii, according to atlas plates [4]. Ampli®ed spikes were selected by a window discriminator. The number of spikes per 5 s was counted by the timer on an interface adapter board (National Instruments) in a computer (Macintosh, Apple) and the time course of the ®ring frequency was plotted on the screen. Once the spontaneous activity became stable, the glucose concentration of the bathing medium was changed or drugs were injected. Statistical analysis of data was performed as previously described [8]. Thirteen of 36 studied neurons were submitted to tolbutamide and glibenclamide. As both sulfonylureas induced always the same response, the other neurons were only tested with one of them. In a ®rst series (Table 1), the presence of KATP channels in the caudal NTS was indicated in seven of 11 tested neurons by their activation by tolbutamide and/or glibenclamide and their depression by diazoxide and/or pinacidil. The channels likely belong to the beta-cell type for six neurons depressed by diazoxide, and to the cardiac type for the other one which was depressed by pinacidil and unaffected by diazoxide. In a second series, to assess the possible mediation of glucose-sensing by KATP channels, 25 spontaneously active neurons were subjected to a 2 mM increase in glucose level and to a bath application of tolbutamide and/or glibenclamide. Neurons responsive to sulfonylurea were subsequently tested with diazoxide and/or pinacidil applications. Glucose increase activated nine neurons, depressed ®ve others, and failed to affect the last 11 (Table 2). Considering that the actual concentration of glucose might be lower in the central layers than in the peripheral layers of the slice, due to the delay required for the molecules to diffuse from the bath, recording was carried out at different depths. No correspondence was observed between the depth of the recorded neuron and the incidence of glucose responses. In seven out of the nine neurons activated by glucose, activity was increased by tolbutamide and/or glibenclamide and
79
decreased by pinacidil, suggesting the involvement of KATP channels in the glucose response (Fig. 1). The channel type was cardiac-like for one of the seven neurons, which was unaffected by diazoxide (and undetermined for the other six, for which diazoxide was not tested). Of the ®ve neurons depressed by glucose, two responded in the opposite direction to tolbutamide and/or glibenclamide and diazoxide and/ or pinacidil. As they were activated by sulfonylurea and depressed by KCO, they probably have KATP channels which do not mediate glucose-sensing. One glucosedepressed neuron was also depressed by both sulfonylureas and both KCOs, and the last two were unresponsive to tolbutamide (Fig. 2). Of the 11 glucose-unresponsive neurons, four were activated by sulfonylurea and depressed by KCO, suggesting the presence of KATP channels (Fig. 3), and one was depressed by sulfonylurea and activated by KCO, suggesting the presence of KATP channels in inhibitory afferents. Six other glucose-unresponsive neurons failed also to respond to sulfonylurea. Previous studies have reported the presence of beta-cell type KATP channels in the dorsal nucleus of the vagus nerve [11]. In the present study, a majority of the neurons recorded in the caudal NTS (7/11) were found to contain KATP channels which belong to the beta-cell type in all of them except one, which displayed the cardiac type. The second series suggested the involvement of KATP channels in the response to glucose of seven out of nine glucose-activated neurons. The open probability of these channels might be in equilibrium under normal glucose and oxygen supply, since they were opened by diazoxide and /or pinacidil and closed by sulfonylurea and glucose. For the two other glucose-activated neurons, a different mechanism of glucose-sensing is probably involved. For instance, a modulation of presynaptic purinergic receptors, as observed in the hippocampus [20] and the striatum [7] would be compatible with the presence of different subtypes of purinergic receptors in the NTS [5]. Concerning the glucose-depressed neurons, mediation of
Fig. 2. Example of a neuron depressed by a 2-mM increase in the extracellular glucose level. A bath application of the sulfonylurea tolbutamide had no effect, suggesting the lack of KATP channels.
80
M. Dallaporta et al. / Neuroscience Letters 278 (2000) 77±80
[3] [4] [5]
[6]
[7] Fig. 3. Example of a neuron unresponsive to 2-mM changes in the glucose level. However, this neuron met the pharmacological criteria for the presence of beta-cell type KATP channels: it was depressed and activated by bath applications of the K 1 channel opener diazoxide, and of the sulfonylurea tolbutamide, respectively.
the response by presynaptic KATP channels, as observed in the substantia nigra [2] is not supported by the present pharmacological data. A possible glucose-sensing mechanism in these cells might involve modulation of sodium-potassium pumps by intracellular ATP, as suggested for the glucosedepressed neurons of the LHA [14]. An unexpected ®nding was the possible presence of KATP channels in four out of the 11 neurons unresponsive to a moderate glucose-level change, and also in the inhibitory afferents of another glucose-unresponsive neuron. Since the glucose-level modi®cations tested in the present study were only 2 mM, a plausible explanation of this discrepancy is that the response threshold of these neurons is too high to allow sensitivity to physiological glycemic changes. Instead, they may be involved in neuroprotective responses to drastic hypoxia or hypoglycemia, as in other brain regions [6,10,11]. In conclusion, KATP channels can be pharmacologically identi®ed in the caudal NTS, and may mediate most excitatory responses to moderate increases in the glucose level, while the origin of inhibitory-like responses remains to be investigated. It is also worth noting that a neuron endowed with KATP channels is not necessarily able to respond to moderate glucose-level ¯uctuations. This study was supported by the Institut Danone (Appel d'Offres Alimentation et Sante 1998). Mrs. HeÂleÁne Chagneux is acknowledged for expert technical assistance regarding the computer program. [1] Adachi, A., Kobashi, M. and Funahashi, M., Glucoseresponsive neurons in the brainstem. Obes. Res., 3 (1995) 7355±7405. [2] Amoroso, S., Schmid-Antomarchi, H., Fosset, M. and Ladzunski, M., Glucose, sulfonylureas and neurotransmit-
[8] [9] [10]
[11]
[12]
[13] [14]
[15] [16] [17]
[18]
[19] [20]
ter release: role of ATP-sensitive K 1 channels. Science, 247 (1990) 852±854. Babenko, A.P., Aguilar-Bryan, L. and Bryan, J., A view of SUR/K(IR)6.X, K-ATP channels. Annu. Rev. Physiol., 60 (1998) 667±687. Barraco, R., Elridi, M., Ergene, E., Parizon, M. and Bradley, D., An atlas of the rat subpostremal nucleus-tractus-solitarius. Brain Res. Bull., 29 (1992) 703±765. Barraco, R.A., O'Leary, D.S., Ergene, E. and Scislo, T.J., Activation of purinergic receptor subtypes in the nucleus tractus solitarius elicits speci®c regional vascular response patterns. J. Auton. Nerv. Syst., 59 (1996) 113±124. Ben Ari, Y., Krnjevic, K. and CreÂpel, V., Activators of the ATP-sensitive K 1 channels reduce anoxic depolarization in CA3 hippocampal neurons. Neuroscience, 37 (1990) 55± 60. Calabresi, P., Centonze, D., Pisani, A. and Bernardi, G., Endogenous adenosine mediates the presynaptic inhibition induced by aglycemia at corticostriatal synapses. J. Neurosci., 17 (1997) 4509±4516. Dallaporta, M., Himmi, T., Perrin, J. and Orsini, J.C., Solitary tract nucleus sensitivity to moderate changes in glucose level. NeuroReport, 10 (1999) 2657±2660. Dunn-Meynell, A.A., Rawson, N.E. and Levin, B.E., Distribution and phenotype of neurons containing the ATP-sensitive K 1 channel in rat brain. Brain Res., 814 (1998) 41±54. Guatteo, E., Federici, M., Siniscalchi, A., Knopfel, T., Mercuri, N.B. and Bernardi, G., Whole cell patch-clamp recordings of rat midbrain dopaminergic neurons isolate a sulphonylurea- and ATP-sensitive component of potassium currents activated by hypoxia. J. Neurophysiol., 79 (1998) 1239±1245. Karschin, A., Brockhaus, J. and Ballanyi, K., K-ATP channel formation by the sulphonylurea receptors SUR1 with Kir 6.2 subunits in rat dorsal vagal neurons in situ. J. Physiol. (Lond.), 509 (1998) 339±346. Karschin, C., Ecke, C., Ashcroft, F.M. and Karschin, A., Overlapping distribution of K-ATP channel-forming Kir 6.2 subunit and the sulfonylurea receptor SUR1 in rodent brain. FEBS Lett., 401 (1997) 59±64. Le Magnen, L., Neurobiology of Feeding and Nutrition, Academic Press, San Diego, CA, 1992, 385 pp. Oomura, Y., Ooyama, H., Sugimori, M., Nakamura, T. and Yamada, Y., Glucose inhibition of the glucose-sensitive neurones in the rat lateral hypothalamus. Nature, 247 (1974) 284±286. Rorsman, P., The pancreatic beta-cell as a fuel sensor: an electrophysiologist's viewpoint. Diabetologia, 40 (1997) 487±495. Treherne, J.M. and Ashford, M.L.J., The regional distribution of sulphonylurea binding sites in rat. Brain Neurosci., 40 (1991) 523±531. Yettefti, K., Orsini, J.C. and Perrin, J., Characteristics of glycemia-sensitive neurons in the nucleus tractus solitarii. Possible involvement in nutritional regulation. Physiol. Behav., 61 (1997) 93±100. Yokoshiki, H., Sunagawa, M., Seki, T. and Sperelakis, N., ATP-sensitive K 1 channels in pancreatic, cardiac, and vascular smooth muscle cells. Am. J. Physiol. Cell. Physiol., 43 (1998) C25±C37. Zhu, D.X.D., Sullivan, J.P. and Brioni, J.D., ATP-sensitive potassium channels regulate in vivo dopamine release in rat striatum. Jpn. J. Pharmacol., 79 (1999) 59±64. Zhu, P.J. and Krnjevic, K., Adenosine release is a major cause of failure of synaptic transmission during hypoglycaemia in rat hippocampal slices. Neurosci. Lett., 155 (1993) 128±131.
Involvement of adenosine triphosphate-sensitive K 1 channels in glucose-sensing in the rat solitary tract nucleus Michel Dallaporta a, Jean Perrin a, Jean-Claude Orsini a, b,* a
Laboratoire de Neurobiologie, CNRS-Universite de Provence, 31 chemin Joseph Aiguier, F-13402 Marseille ceÂdex 20, France b Faculte des Sciences du Sport, Universite de la MeÂditerraneÂe, Marseille, France Received 15 September 1999; received in revised form 22 October 1999; accepted 8 November 1999
Abstract The presence of adenosine triphosphate-sensitive (ATP-sensitive) K 1 channels (KATP channels) in the caudal nucleus tractus solitarii (NTS), and their possible involvement in glucose-sensing, were assessed by extracellular recording of neuronal activity in rat hindbrain slices. In 21 out of 36 recorded cells, ®ring was increased by sulfonylureas and decreased by K 1 channel opener (KCO), indicating the existence of KATP channels in the caudal NTS. In seven out of the nine neurons activated by a 2 mM increase in the glucose level, the effects of sulfonylureas and KCO were consistent with the involvement of KATP channels in the glucose response. Conversely, the mechanism(s) underlying the response of glucose-depressed neurons remains to be clari®ed. Finally, the presence of KATP channels was also detected in some neurons that were unresponsive to a 2 mM change in the glucose level. Thus, KATP channels were pharmacologically identi®ed in the caudal NTS, where they may be partly involved in glucose sensing. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Single unit activity; Feeding; Glucose level; KATP channel; Rats; Hindbrain
In free-moving and free-eating rats maintained in a steady state condition, meal initiation is preceded and triggered by a slight decline in the blood glucose level, and mediated by glycemia-sensitive brain neurons responsive to preprandial hypoglycemia [13]. One of the possible localizations of the relevant glycemia-sensitive neurons is the caudal part of the nucleus tractus solitarii (NTS), a glucose-sensitive structure [1] where several neurons respond to moderate changes in blood glucose level through the local action of glucose molecules [17]. We recently investigated this glucose sensitivity in vitro [8]. Half of the neurons recorded in the area responded to a 2 mM increase in the extracellular glucose level by either the activation or the depression of ®ring, and most of them were catecholaminergic. Moreover, the effects of 2-deoxyglucose suggested that a compound generated by carbohydrate metabolism, such as adenosine triphosphate (ATP), was involved in the NTS glucose sensing. The glucose sensitivity of some peripheral cells, such as the pancreatic beta-cells, involves potassium channels inhibited by intracellular ATP (KATP channels), so that * Corresponding author. Tel.: 133-4-91-164-581; fax: 133-491-220-875. E-mail address: [email protected] (J.-C. Orsini)
they are depolarized by a high glucose level and hyperpolarized by hypoglycemia [15]. Similarly, the glucose level affects the neuronal activity of different brain regions through KATP channels. These channels, as in pancreatic beta-cells, are composed of two proteins, an inwardly rectifying pore-forming unit, Kir 6.2, and a high af®nity sulfonylurea receptor, SUR1 [11,12]. The beta cell-like KATP channels are closed by antidiabetic sulfonylureas such as tolbutamide or glibenclamide, and activated by K 1 channel openers (KCOs) such as diazoxide, and to a lesser degree cromakalim or pinacidil [3,18]. In the striatal dopaminergic terminals, however, KATP channels are composed of Kir 6.2 pore-forming units associated with another isoform of sulfonylurea receptor, probably SUR2A [19]. These KATP channels belong to another subtype similar to those observed in heart cells, in which the channel is also closed by sulfonylureas and activated by cromakalim or pinacidil, but unaffected by diazoxide [3,18]. Glucose-sensing in the caudal NTS might be mediated by KATP channels [8], as suggested by the presence of Kir 6.2 mRNA in most C2 and some A2 catecholaminergic neurons of the nucleus [9], despite the low density of high-af®nity binding sites for [ 3H]glibenclamide in this area [16]. The purpose of the present study was to apply the above
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 89 8- 8
78
M. Dallaporta et al. / Neuroscience Letters 278 (2000) 77±80
Table 1 Distribution of 11 NTS neurons as a function of their response to Sulfonylureas and KCOs a Response to sulfonyluraes Tolbutamide 3
(1)
2
(1)
2 1 2 1
Glibenclamide 1
(1)
1
(1) (2) (0) (0)
(0) a
Response to KCOs
Presence of KATP channels
Diazoxide
Pinacidil
(2) (0)
(2)
Yes (b) Yes (c)
(2) (0) (2) (0) (2)
Yes (b) Yes (b) No No No
(2) (2) (0) (0)
(1) Increase in ®ring; (2) decrease in ®ring; (0) no response; (b ): b-cell type KATP channels; (c): cardiac type KATP channels.
pharmacological criteria while using conventional electrophysiological recording, to: (a) lend support to the presence of KATP channels in some caudal NTS neurons (neurons activated by tolbutamide and/or glibenclamide and depressed by diazoxide and/or pinacidil); (b) specify their subtype (beta cell-type in neurons depressed by diazoxide and cardiac type in neurons depressed by pinacidil and unaffected by diazoxide); (c) determine whether or not their modulation can mediate glucose sensitivity in this area (neurons responding in the same direction to glucose and tolbutamide or glibenclamide, and in the opposite direction to glucose and diazoxide or pinacidil). Male Wistar rats weighing 150±300 g were anesthetized by halothane inhalation (Fluothan, Coopers) and killed by decapitation. The brain was quickly removed and placed in chilled arti®cial cerebrospinal ¯uid (aCSF) saturated with O2 95%, CO2 5% containing (in mM) NaCl 125, KCl 5, KH2PO4 1.25, Mg SO4 2, CaCl2 3, NaHCO3 25.5, and glucose 5 (pH 7:4). The medulla oblongata was microdissected and 300-mm thick coronal slices were cut at the area postrema level with an oscillating tissue slicer (Camp-
den Instruments) and then transferred into a preincubation chamber ®lled with oxygenated aCSF at room temperature. More than 2 h after preincubation, the recording session started in a submerged type recording chamber (0.5 ml) perfused at a rate of 2.5 ml/min with oxygenated aCSF containing 3 or 4 mM glucose. The temperature of the chamber was kept at 348C. The perfusion medium entered the chamber close to the slice. Rapid switching to aCSF containing 5 or 6 mM glucose was achieved using an inlet ¯ow valve. The new solution reached the chamber in about 30 s (dead space time). The following substances were diluted in aCSF and bath applied: tolbutamide (Sigma) and diazoxide (RBI) dissolved in dimethylsulfoxide (DMSO) or NaOH 15 mM (®nal concentration in the chamber: 200 mM), glibenclamide (Sigma) dissolved in DMSO (®nal concentration in the chamber: 2 mM), pinacidil (Sigma) dissolved in NaOH 15 mM (®nal concentration in the chamber: 40 or 60 mM). Single unit activity was recorded extracellularly with glass microelectrodes (tip diameter: 2±3 mm) ®lled with 3 M NaCl. Electrodes were placed under microscopic obser-
Table 2 Distribution of 25 NTS neurons as a function of their response to Glucose, Sulfonylureas, and KCOs a Response to glucose
Response to sulfonylureas Tolbutamide
9 (1)
5 (2)
11 (0)
a
6 1 2 2 1 2
(2) (0)
2
(1)
2 1 2 4
(1) (2) (0) (0)
(1) (0)
Response to KCOs
Presence of KATP channels
Glibenclamide
Diazoxide
Pinacidil
(1) (1)
(0)
(2) (2)
(1) (2)
(2) (2)
(2)
1
(2)
(2)
1
(2) (2)
(0)
(1) (2) (0)
(1)
Yes * Yes * No Yes No No Yes Yes Yes Yes ** No No
(1) Increase in ®ring; (2) decrease in ®ring; (0) no response; *KATP channels possibly involved in glucose-sensing; **KATP channels possibly located in inhibitory afferents.
M. Dallaporta et al. / Neuroscience Letters 278 (2000) 77±80
Fig. 1. Example of a glucose-responsive neuron in which KATP channels may mediate glucose-sensing. It was activated by a bath application of the sulfonylurea glibenclamide and by a 2mM increase in the extracellular glucose level, and depressed by a bath application of the K 1 channel opener pinacidil.
vation in the caudal part of the nucleus tractus solitarii, according to atlas plates [4]. Ampli®ed spikes were selected by a window discriminator. The number of spikes per 5 s was counted by the timer on an interface adapter board (National Instruments) in a computer (Macintosh, Apple) and the time course of the ®ring frequency was plotted on the screen. Once the spontaneous activity became stable, the glucose concentration of the bathing medium was changed or drugs were injected. Statistical analysis of data was performed as previously described [8]. Thirteen of 36 studied neurons were submitted to tolbutamide and glibenclamide. As both sulfonylureas induced always the same response, the other neurons were only tested with one of them. In a ®rst series (Table 1), the presence of KATP channels in the caudal NTS was indicated in seven of 11 tested neurons by their activation by tolbutamide and/or glibenclamide and their depression by diazoxide and/or pinacidil. The channels likely belong to the beta-cell type for six neurons depressed by diazoxide, and to the cardiac type for the other one which was depressed by pinacidil and unaffected by diazoxide. In a second series, to assess the possible mediation of glucose-sensing by KATP channels, 25 spontaneously active neurons were subjected to a 2 mM increase in glucose level and to a bath application of tolbutamide and/or glibenclamide. Neurons responsive to sulfonylurea were subsequently tested with diazoxide and/or pinacidil applications. Glucose increase activated nine neurons, depressed ®ve others, and failed to affect the last 11 (Table 2). Considering that the actual concentration of glucose might be lower in the central layers than in the peripheral layers of the slice, due to the delay required for the molecules to diffuse from the bath, recording was carried out at different depths. No correspondence was observed between the depth of the recorded neuron and the incidence of glucose responses. In seven out of the nine neurons activated by glucose, activity was increased by tolbutamide and/or glibenclamide and
79
decreased by pinacidil, suggesting the involvement of KATP channels in the glucose response (Fig. 1). The channel type was cardiac-like for one of the seven neurons, which was unaffected by diazoxide (and undetermined for the other six, for which diazoxide was not tested). Of the ®ve neurons depressed by glucose, two responded in the opposite direction to tolbutamide and/or glibenclamide and diazoxide and/ or pinacidil. As they were activated by sulfonylurea and depressed by KCO, they probably have KATP channels which do not mediate glucose-sensing. One glucosedepressed neuron was also depressed by both sulfonylureas and both KCOs, and the last two were unresponsive to tolbutamide (Fig. 2). Of the 11 glucose-unresponsive neurons, four were activated by sulfonylurea and depressed by KCO, suggesting the presence of KATP channels (Fig. 3), and one was depressed by sulfonylurea and activated by KCO, suggesting the presence of KATP channels in inhibitory afferents. Six other glucose-unresponsive neurons failed also to respond to sulfonylurea. Previous studies have reported the presence of beta-cell type KATP channels in the dorsal nucleus of the vagus nerve [11]. In the present study, a majority of the neurons recorded in the caudal NTS (7/11) were found to contain KATP channels which belong to the beta-cell type in all of them except one, which displayed the cardiac type. The second series suggested the involvement of KATP channels in the response to glucose of seven out of nine glucose-activated neurons. The open probability of these channels might be in equilibrium under normal glucose and oxygen supply, since they were opened by diazoxide and /or pinacidil and closed by sulfonylurea and glucose. For the two other glucose-activated neurons, a different mechanism of glucose-sensing is probably involved. For instance, a modulation of presynaptic purinergic receptors, as observed in the hippocampus [20] and the striatum [7] would be compatible with the presence of different subtypes of purinergic receptors in the NTS [5]. Concerning the glucose-depressed neurons, mediation of
Fig. 2. Example of a neuron depressed by a 2-mM increase in the extracellular glucose level. A bath application of the sulfonylurea tolbutamide had no effect, suggesting the lack of KATP channels.
80
M. Dallaporta et al. / Neuroscience Letters 278 (2000) 77±80
[3] [4] [5]
[6]
[7] Fig. 3. Example of a neuron unresponsive to 2-mM changes in the glucose level. However, this neuron met the pharmacological criteria for the presence of beta-cell type KATP channels: it was depressed and activated by bath applications of the K 1 channel opener diazoxide, and of the sulfonylurea tolbutamide, respectively.
the response by presynaptic KATP channels, as observed in the substantia nigra [2] is not supported by the present pharmacological data. A possible glucose-sensing mechanism in these cells might involve modulation of sodium-potassium pumps by intracellular ATP, as suggested for the glucosedepressed neurons of the LHA [14]. An unexpected ®nding was the possible presence of KATP channels in four out of the 11 neurons unresponsive to a moderate glucose-level change, and also in the inhibitory afferents of another glucose-unresponsive neuron. Since the glucose-level modi®cations tested in the present study were only 2 mM, a plausible explanation of this discrepancy is that the response threshold of these neurons is too high to allow sensitivity to physiological glycemic changes. Instead, they may be involved in neuroprotective responses to drastic hypoxia or hypoglycemia, as in other brain regions [6,10,11]. In conclusion, KATP channels can be pharmacologically identi®ed in the caudal NTS, and may mediate most excitatory responses to moderate increases in the glucose level, while the origin of inhibitory-like responses remains to be investigated. It is also worth noting that a neuron endowed with KATP channels is not necessarily able to respond to moderate glucose-level ¯uctuations. This study was supported by the Institut Danone (Appel d'Offres Alimentation et Sante 1998). Mrs. HeÂleÁne Chagneux is acknowledged for expert technical assistance regarding the computer program. [1] Adachi, A., Kobashi, M. and Funahashi, M., Glucoseresponsive neurons in the brainstem. Obes. Res., 3 (1995) 7355±7405. [2] Amoroso, S., Schmid-Antomarchi, H., Fosset, M. and Ladzunski, M., Glucose, sulfonylureas and neurotransmit-
[8] [9] [10]
[11]
[12]
[13] [14]
[15] [16] [17]
[18]
[19] [20]
ter release: role of ATP-sensitive K 1 channels. Science, 247 (1990) 852±854. Babenko, A.P., Aguilar-Bryan, L. and Bryan, J., A view of SUR/K(IR)6.X, K-ATP channels. Annu. Rev. Physiol., 60 (1998) 667±687. Barraco, R., Elridi, M., Ergene, E., Parizon, M. and Bradley, D., An atlas of the rat subpostremal nucleus-tractus-solitarius. Brain Res. Bull., 29 (1992) 703±765. Barraco, R.A., O'Leary, D.S., Ergene, E. and Scislo, T.J., Activation of purinergic receptor subtypes in the nucleus tractus solitarius elicits speci®c regional vascular response patterns. J. Auton. Nerv. Syst., 59 (1996) 113±124. Ben Ari, Y., Krnjevic, K. and CreÂpel, V., Activators of the ATP-sensitive K 1 channels reduce anoxic depolarization in CA3 hippocampal neurons. Neuroscience, 37 (1990) 55± 60. Calabresi, P., Centonze, D., Pisani, A. and Bernardi, G., Endogenous adenosine mediates the presynaptic inhibition induced by aglycemia at corticostriatal synapses. J. Neurosci., 17 (1997) 4509±4516. Dallaporta, M., Himmi, T., Perrin, J. and Orsini, J.C., Solitary tract nucleus sensitivity to moderate changes in glucose level. NeuroReport, 10 (1999) 2657±2660. Dunn-Meynell, A.A., Rawson, N.E. and Levin, B.E., Distribution and phenotype of neurons containing the ATP-sensitive K 1 channel in rat brain. Brain Res., 814 (1998) 41±54. Guatteo, E., Federici, M., Siniscalchi, A., Knopfel, T., Mercuri, N.B. and Bernardi, G., Whole cell patch-clamp recordings of rat midbrain dopaminergic neurons isolate a sulphonylurea- and ATP-sensitive component of potassium currents activated by hypoxia. J. Neurophysiol., 79 (1998) 1239±1245. Karschin, A., Brockhaus, J. and Ballanyi, K., K-ATP channel formation by the sulphonylurea receptors SUR1 with Kir 6.2 subunits in rat dorsal vagal neurons in situ. J. Physiol. (Lond.), 509 (1998) 339±346. Karschin, C., Ecke, C., Ashcroft, F.M. and Karschin, A., Overlapping distribution of K-ATP channel-forming Kir 6.2 subunit and the sulfonylurea receptor SUR1 in rodent brain. FEBS Lett., 401 (1997) 59±64. Le Magnen, L., Neurobiology of Feeding and Nutrition, Academic Press, San Diego, CA, 1992, 385 pp. Oomura, Y., Ooyama, H., Sugimori, M., Nakamura, T. and Yamada, Y., Glucose inhibition of the glucose-sensitive neurones in the rat lateral hypothalamus. Nature, 247 (1974) 284±286. Rorsman, P., The pancreatic beta-cell as a fuel sensor: an electrophysiologist's viewpoint. Diabetologia, 40 (1997) 487±495. Treherne, J.M. and Ashford, M.L.J., The regional distribution of sulphonylurea binding sites in rat. Brain Neurosci., 40 (1991) 523±531. Yettefti, K., Orsini, J.C. and Perrin, J., Characteristics of glycemia-sensitive neurons in the nucleus tractus solitarii. Possible involvement in nutritional regulation. Physiol. Behav., 61 (1997) 93±100. Yokoshiki, H., Sunagawa, M., Seki, T. and Sperelakis, N., ATP-sensitive K 1 channels in pancreatic, cardiac, and vascular smooth muscle cells. Am. J. Physiol. Cell. Physiol., 43 (1998) C25±C37. Zhu, D.X.D., Sullivan, J.P. and Brioni, J.D., ATP-sensitive potassium channels regulate in vivo dopamine release in rat striatum. Jpn. J. Pharmacol., 79 (1999) 59±64. Zhu, P.J. and Krnjevic, K., Adenosine release is a major cause of failure of synaptic transmission during hypoglycaemia in rat hippocampal slices. Neurosci. Lett., 155 (1993) 128±131.