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Effects of pentobarbital on GABA-activated currents in acutely-isolated rat dentate gyrus granule neurons
Neuroscience Letters 353 (2003) 139–142 www.elsevier.com/locate/neulet
Effects of pentobarbital on GABA-activated currents in acutely-isolated rat dentate gyrus granule neurons M.S.F. Lima, C.E.L. Lindquistb, B. Birnira,b,* a
Membrane Biology Program, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, ACT 2601, Australia b Molecular and Cellular Physiology, Department of Physiological Sciences, Tornavagen 10 BMC F11, Lund University, 22184 Lund, Sweden Received 28 August 2003; received in revised form 17 September 2003; accepted 17 September 2003
Abstract Granule neurons from the rat dentate gyrus were acutely isolated and whole-cell currents recorded. Maximal enhancement of 7 mM gaminobutyric acid (GABA; EC30) evoked currents was obtained with 100 mM pentobarbital where the peak-current was 2.1 ^ 0.2 of control. One hundred mM pentobarbital alone evoked no current response whereas 1 mM pentobarbital elicited a current response that was 0.4 ^ 0.2 of the 100 mM GABA-activated peak current. In 100 mM pentobarbital, the GABA EC50 value shifted from 14 to 3 mM but the peaksaturating-current value was not altered. An off-current was recorded on removal of 100 mM and higher pentobarbital concentrations. Ten mM pentobarbital abolished the peak-current response to 7 and 100 mM GABA. The results show that in the granule neurons the drug potency differs for the different effects of pentobarbital at GABAA receptors with the modulatory and inhibitory effects requiring lower concentrations than the direct activation of the receptors. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: g-aminobutyric acidA; Hippocampus; Barbiturates; Inhibition; Receptor; Ion channel
g-aminobutyric acidA (GABAA) receptors are the most important neuronal inhibitory receptors in the mammalian brain. When GABA binds to the receptors it opens a chloride channel. These receptors are the target of therapeutic drugs such as the barbiturates, benzodiazepines and anaesthetics but the specific pharmacological profiles are dictated by the receptor subtype [2,9]. To date, 19 different mammalian subunits have been cloned and are grouped into different subfamilies (a1 – 6, b1 – 3, g1 – 3, r1 – 3, p, e, d and u). They assemble into hetero-pentameric receptors [2] that are functionally and pharmacologically distinct [2,9]. In the hippocampus the dentate gyrus normally functions as a filter and prevents propagation of synchronised activity into the hippocampus [4]. The hippocampal dentate gyrus granule neurons have been shown to express a variety of GABAA receptors [5,10]. In the epileptic hippocampus, the pharmacology of drugs that interact specifically with the GABAA receptors is altered. The GABA-activated current is less sensitive to modulation by benzodiazepines, more *
Corresponding author. Tel.: þ 46-46-222-0675; fax: þ46-46-222-7763. E-mail address: [email protected] (B. Birnir).
sensitive to inhibition by Zn2þ but the modulation by pentobarbital appears similar to what it was in healthy granule neurons [4,8]. The barbiturates are a group of drugs that have anaesthetic, anti-convulsant and anxiolytic properties. A commonly used barbiturate is pentobarbital. It has been demonstrated to have three major effects on whole-cell currents activated by GABA. GABA-activated currents may be potentiated or blocked by pentobarbital and at some GABAA receptors pentobarbital directly activates a current response [9]. The whole-cell current response in native neurons is set by the cumulative response of all the GABAA receptor subtypes that are expressed in the cell. In this study we examined in more detail the effects pentobarbital has on healthy, acutely-isolated dentate gyrus granule neurons. The results show that in a concentration-dependent manner pentobarbital modulated, activated and inhibited GABAA receptor currents. Experiments were performed on acutely-isolated granule neurons from the hippocampal dentate gyrus region from Wistar rats aged 16– 21 days. The method of isolating the neurons has been previously described [10]. Briefly, the rats were killed by decapitation, the brain removed and
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.09.025
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M.S.F. Lim et al. / Neuroscience Letters 353 (2003) 139–142
immersed in ice-cold artificial cerebrospinal fluid (ACSF) containing in mM: 126 NaCl, 26 NaHCO3, 20 glucose, 3 KCl, 2.6 CaCl2, 2.5 NaH2PO4 and 1.3 MgSO4. The solution was equilibrated with carbogen gas resulting in a pH of 7.4. 500 mm thick hippocampal slices were cut and incubated in ACSF (35 8C) for 30 min. An enzyme solution was then added having a final concentration of 20 Units/ml Papain (Worthington), 1.1 mM cysteine, 0.22 mM EDTA and 13.4 mM mercaptoethanol and the incubation continued for an additional 30 min. The enzyme solution was then replaced with fresh carbogenated ACSF and the slices incubated for a further 30 min at room temperature (22 8C). The dentate gyri were dissected out of the hippocampal slices and triturated using a polished Pasteur pipette. Isolated neurons were placed in the recording chamber in the experimental bath solution and used for experiments. Whole-cell patch-clamp recordings were performed as described previously [10]. After establishing a whole-cell configuration, the cell was lifted up and positioned in front of a drug delivery tube. Drugs were applied using a fast perfusion system [10]. The currents were recorded with Axopatch amplifiers (1B or 200B) filtered, digitised and stored on a computer. The data were analysed by Channel 2 (M. Smith, ANU, Australia) or pClamp (Axon Instruments, USA) software analysis packages. Dose-response plots were constructed from the modulated GABA-activated peak currents and the peak offcurrents. Values are presented as mean ^ SEM for three to nine neurons and differences between groups are considered significant for P , 0:05 using Student’s t-test. We first examined what concentration range of pentobarbital was effective in modulating the current response to low concentration of GABA. Fig. 1A shows typical current responses to 7 mM GABA (GABA EC30 concentration [10]) and several (3, 10, 50 100 and 1000 mM) test concentrations of pentobarbital. The data are from different cells but normalised to the 7 mM GABA peakcurrent response in the same cell. Pentobarbital concentrations from 0.1 to 3 mM (n ¼ 15) did not significantly affect the amplitude of the peak-current response to the 7 mM GABA but in 10 mM pentobarbital (n ¼ 3) the peakcurrent amplitude was reduced to 0.6 ^ 0.1 of the 7 mM GABA response (Fig. 1Aa,c,B, P , 0:05). In the higher pentobarbital concentrations (50, 100 mM and 1 mM) the 7 mM GABA-activated peak current was enhanced. The greatest current enhancement was measured in 100 mM pentobarbital (n ¼ 8) where it was 2.1 ^ 0.2 of control (Figs. 1Aa,e,B). In 1 mM pentobarbital (n ¼ 3, Figs. 1Af, B) the peak-current enhancement was not significantly different from that of the 100 mM pentobarbital but there was an apparent increase in the initial rate of the current decay and an off-current was recorded when the drugs were removed (Fig. 1Af). Both the increased rate of the current decay and the presence of the off-current indicate a significant inhibitory effect of 1 mM pentobarbital in addition to the enhancing effect of the GABA-activated current response. Due to the clear evidence of inhibition at
Fig. 1. Pentobarbital modulation of the GABA-activated current. (A) Representative current responses to 7 mM GABA (a.) plus test concentrations of pentobarbital (b. 3 mM, c. 10 mM, d. 50 mM, e. 100 mM, f. 1 mM). The currents were normalised to the 7 mM GABA in the same cell. (B) Effects of pentobarbital concentrations on the 7 mM GABAactivated peak-current response. Currents were normalised to the 7 mM peak current in the same cell. Data points represent the averages from 3 to 9 neurons and SEM is shown if larger than the symbol. (C) Effects of 100 mM pentobarbital on GABA-activated currents. Data points represents the averages from three to six neurons and SEM is shown if larger than the symbol. The currents were normalised to the 1 mM GABA plus pentobarbital peak amplitude. The solid line shows the fit of a Hill-type equation to the data. The broken line is from Lim et al. [10] and shows the GABA-activation curve.
10 mM and again at 1 mM pentobarbital we did not attempt to fit the data with a concentration-response curve and the lines in Fig. 1B simply connect the data points. The effective concentration of pentobarbital for modulation of the GABA-activated current response in this study is similar to what has been reported for frog sensory neurons [1], chromaffin cells [12] and cultured rat hippocampal neurons [13] where it ranges between 10 and 300 mM pentobarbital. The concentration for the greatest enhancement of the peak-current amplitude ranged from 100 and 300 mM in these studies. The clinical half-saturation concentration (EC50) of pentobarbital for generating anaesthesia in mammals is 50 mM [7]. In this study the fractional enhancement of the GABA-activated current by 50 mM pentobarbital was 1.7 ^ 0.1 (n ¼ 3) of control. We then varied the GABA concentration in the presence of 100 mM pentobarbital. The results are shown in Fig. 1C and were fitted with a Hill-type equation: I00 ¼ Imax £ ½GABA^h =ð½EC50 ^h þ ½GABA^h Þ where Imax is the saturating peak-current amplitude, EC50 is the half-maximal concentration for the activation of the receptors and h is the Hill coefficient. All currents were normalised to the saturating 1 mM GABA plus pentobarbital currents. The data points are the averages of three or more experiments. The curve in the presence of GABA alone is taken from Lim et al. [10]. The apparent affinity for GABA increased about four times in the presence of 100 mM pentobarbital. The half-saturating concentration of
M.S.F. Lim et al. / Neuroscience Letters 353 (2003) 139–142
GABA (EC50) was shifted from 14 to 3 mM in the presence of 100 mM pentobarbital but the Hill coefficients were similar, 1.3 and 1.2, respectively. Pentobarbital directly activates some GABAA receptor subtypes [1,2,11,13 –15]. We examined if pentobarbital directly activated GABAA receptors expressed in the dentate gyrus granule neurons. In 12 cells 100 mM pentobarbital failed to evoke a current response whereas all the cells responded to 100 mM GABA. We applied 1 mM pentobarbital to another six cells and in all cases an inward current was evoked. The 1 mM pentobarbital evoked current was on the average 0.4 ^ 0.2 (n ¼ 4) of the 100 mM GABA evoked current in the same cell. The concentration required for direct activation appears to vary widely in native cells. In chromaffin cells and mammalian dorsal root ganglion neurons pentobarbital concentrations as low as 30 and 60 mM, respectively, were effective in evoking a current response [12,14] whereas in cultured hippocampal neurons concentrations greater than 100 mM were required with an EC50 for activation of 330 mM [13]. The sensitivity of cells to pentobarbital depends on the pentobarbital EC50 of the receptors expressed in the cell but also on the relative fraction of receptors expressed that are not activated by pentobarbital. In dentate gyrus granule neurons the a4 subunit is prominent [4,9]. a4 containing GABAA receptors are not directly activated by pentobarbital and the relative insensitivity to pentobarbital in this study suggests a significant population of a4 containing GABAA receptors in these cells. The results are in accordance with our previous study on dentate gyrus granule neurons where we showed that the GABA-activated whole-cell currents were only slightly sensitive to the benzodiazepine, diazepam and a significant part of the whole-cell plateau current was inhibited by 3 mM zinc, both indicative of receptors containing the a4 subunit [10]. In epileptic dentate gyrus neurons the altered pharmacology correlates with an even higher level of a4 containing receptors in the cells [4]. Our results with the 10 mM pentobarbital plus GABA indicated that an inhibition by pentobarbital was present in low mM pentobarbital plus GABA but was masked as the pentobarbital concentration was increased to 50 or 100 mM by the greater current-enhancement effects of higher pentobarbital concentrations. However, a transient offcurrent response was produced when 1 mM pentobarbital plus GABA was washed off the cells (see Fig. 1A) and is indicative of channel block [14]. We examined if we could use the off-current response as a tool to detect channel block that was not revealed due to the greater potentiation of the whole-cell current by pentobarbital. An off-current may not be recorded and therefore overlooked if the drugs are not rapidly removed off the cell. The isolated neurons are particularly well suited to test for the off-current response of the granule neurons as the drugs can be applied and removed rapidly with a fast application system [10]. The results are shown in Fig. 2. The lowest concentration that evoked an off-current when washed rapidly off the cells was 100 mM
141
Fig. 2. Pentobarbital inhibition of the GABA-activated current. (A) Offcurrents were recorded when 7 mM GABA plus 100 mM (a.), 1 mM (b.) or 10 mM (c.) pentobarbital or 100 mM GABA plus 10 mM pentobarbital (d.) were washed rapidly off the cells. (B) Peak amplitude of the off-current as a fraction of the 7 mM GABA-activated peak-current. The data are averages from three to eight neurons and SEM is shown if larger than the symbol.
pentobarbital (Fig. 2Aa). The average off-current amplitude as a fraction of the peak 7 mM GABA-activated current is shown in Fig. 2B. The off-current amplitude increased as the pentobarbital concentration was raised from 100 mM to 10 mM pentobarbital where it was on average 1.0 ^ 0.1 (n ¼ 8) of the 7 mM GABA-activated peak-current amplitude. In addition to producing a large off-current response, 10 mM pentobarbital also abolished the GABAactivated current response (Fig. 2Ac). We examined if the inhibition of the GABA-activated current was reduced if a higher concentration of GABA (100 mM) plus 10 mM pentobarbital was applied to the cells (Fig. 2Ad, n ¼ 3) but the level of the peak GABA-activated current inhibition was similar to that recorded in 7 mM GABA plus 10 mM pentobarbital (Fig. 2Ac and d). Transient rebound-currents (off-currents) have been recorded in frog sensory neurons (3 mM pentobarbital [1]), chromaffin cells (5 mM pentobarbital [12]), mammalian dorsal root ganglion neurons (600 mM pentobarbital [14]) and in cultured hippocampal neurons (1 mM pentobarbital ^ 1 mM GABA [13]) when the drugs were washed off the cells. The degree of block and current enhancement by pentobarbital has been shown to be influenced by the nature of the amino acid located at the 120 position in the second transmembrane region in a1b1 GABAA receptors [3,6]. Whether the inhibition represented by the reduced peak-current at 10 mM pentobarbital and the inhibition represented by the off-current are due to the same or different mechanism cannot be determined from our studies. If the different subtypes of GABAA receptors
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expressed in a neuron differ in their susceptibility to inhibition by pentobarbital this will influence the effective pentobarbital concentration range for potentiation and the level of current enhancement of the respective GABAactivated current. We have shown that GABAA receptors expressed in the dentate gyrus granule neuron are potentiated, directly activated and blocked by pentobarbital. The drug sensitivity of the three effects of pentobarbital differs with the modulatory and inhibitory effects requiring lower concentration (10 – 100 mM) than direct activation of the receptors.
[4]
[5]
[6]
[7]
Acknowledgements We thank the John Curtin School of Medical Research, Australian National University, Australia. The Swedish Medical Research Council (Y0924), Vetenskapsra˚det ˚ ke Wibergs Stiftelse, Seger(K2002-33X-13408-03A), A falks Stiftelsen, Crafoordska Stiftelsen, Swa¨rds Stiftelse and the Medical Faculty Lund University for financial support.
[8]
[9] [10]
[11] [12]
References [1] N. Akaike, K. Hattori, N. Inomata, Y. Oomura, g-Aminobutyric-acidand pentobarbitone-gated chloride currents in internally perfused frog sensory neurones, J. Physiol. 360 (1985) 367 –386. [2] E.A. Barnard, P. Skolnick, R.W. Olsen, H. Mohler, W. Sieghart, G. Biggio, C. Braestrup, A.N. Bateson, S.Z. Langer, International Union of Pharmacology. XV. Subtypes of g-aminobutyric acid A receptors: classification on the basis of subunit structure and receptor function, Pharmacol. Rev. 50 (1998) 291–313. [3] B. Birnir, M.L. Tierney, J.E. Dalziel, G.B. Cox, P.W. Gage, A structural determinant of desensitization and allosteric regulation by
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pentobarbitone of the GABAA receptor, J. Membr. Biol. 155 (1997) 157 –166. A.R. Brooks-Kayal, M.D. Shumate, H. Jin, T.Y. Rikhter, M.E. Kelly, D.A. Coulter, g-Aminobutyric acid A receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development, J. Neurochem. 77 (2001) 1266– 1278. A.S. Cohen, D.D. Lin, G.L. Quirk, D.A. Coulter, Dentate granule cell GABAA receptors in epileptic hippocampus: enhanced synaptic efficacy and altered pharmacology, Eur. J. Neurosci. 17 (2003) 1607–1616. J.E. Dalziel, G.B. Cox, P.W. Gage, B. Birnir, Mutant human a1b1(T262Q) GABAA receptors are directly activated but not modulated by pentobarbital, Eur. J. Pharmacol. 385 (1999) 283 –286. N.P. Franks, W.R. Lieb, Molecular and cellular mechanisms of general anaesthesia, Nature 367 (1994) 607–614. J. Kapur, R.L. Macdonald, Rapid seizure-induced reduction of benzodiazepine and Zn2þ sensitivity of hippocampal dentate granule cell GABAA receptors, J. Neurosci. 17 (1997) 7532–7540. E.R. Korpi, G. Grunder, H. Luddens, Drug interactions at GABAA receptors, Prog. Neurobiol. 67 (2002) 113 –159. M.S.F. Lim, B. Birnir, Heterogeneity of functional GABAA receptors in rat dentate gyrus neurons revealed by a change in response to drugs during the whole-cell current time-course, Neuropharmacology 40 (2001) 1034–1043. R.A. Nicoll, J.M. Wojtowicz, The effects of pentobarbital and related compounds on frog motoneurons, Brain Res. 191 (1980) 225 –237. J.A. Peters, E.F. Kirkness, H. Callachan, J.J. Lambert, A.J. Turner, Modulation of the GABAA receptor by depressant barbiturates and pregnane steroids, Br. J. Pharmacol. 94 (1988) 1257–1269. J.M. Rho, S.D. Donevan, M.A. Rogawski, Direct activation of GABAA receptors by barbiturates in cultured rat hippocampal neurons, J. Physiol. 497 (1996) 509–522. B. Robertson, Actions of anaesthetics and avermectin on GABAA chloride channels in mammalian dorsal root ganglion neurones, Br. J. Pharmacol. 98 (1989) 167–176. K.A. Wafford, S.A. Thompson, D. Thomas, J. Sikela, A.S. Wilcox, P.J. Whiting, Functional characterization of human g-aminobutyric acid A receptors containing the a4 subunit, Mol. Pharmacol. 50 (1996) 670 –678.
Effects of pentobarbital on GABA-activated currents in acutely-isolated rat dentate gyrus granule neurons M.S.F. Lima, C.E.L. Lindquistb, B. Birnira,b,* a
Membrane Biology Program, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, ACT 2601, Australia b Molecular and Cellular Physiology, Department of Physiological Sciences, Tornavagen 10 BMC F11, Lund University, 22184 Lund, Sweden Received 28 August 2003; received in revised form 17 September 2003; accepted 17 September 2003
Abstract Granule neurons from the rat dentate gyrus were acutely isolated and whole-cell currents recorded. Maximal enhancement of 7 mM gaminobutyric acid (GABA; EC30) evoked currents was obtained with 100 mM pentobarbital where the peak-current was 2.1 ^ 0.2 of control. One hundred mM pentobarbital alone evoked no current response whereas 1 mM pentobarbital elicited a current response that was 0.4 ^ 0.2 of the 100 mM GABA-activated peak current. In 100 mM pentobarbital, the GABA EC50 value shifted from 14 to 3 mM but the peaksaturating-current value was not altered. An off-current was recorded on removal of 100 mM and higher pentobarbital concentrations. Ten mM pentobarbital abolished the peak-current response to 7 and 100 mM GABA. The results show that in the granule neurons the drug potency differs for the different effects of pentobarbital at GABAA receptors with the modulatory and inhibitory effects requiring lower concentrations than the direct activation of the receptors. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: g-aminobutyric acidA; Hippocampus; Barbiturates; Inhibition; Receptor; Ion channel
g-aminobutyric acidA (GABAA) receptors are the most important neuronal inhibitory receptors in the mammalian brain. When GABA binds to the receptors it opens a chloride channel. These receptors are the target of therapeutic drugs such as the barbiturates, benzodiazepines and anaesthetics but the specific pharmacological profiles are dictated by the receptor subtype [2,9]. To date, 19 different mammalian subunits have been cloned and are grouped into different subfamilies (a1 – 6, b1 – 3, g1 – 3, r1 – 3, p, e, d and u). They assemble into hetero-pentameric receptors [2] that are functionally and pharmacologically distinct [2,9]. In the hippocampus the dentate gyrus normally functions as a filter and prevents propagation of synchronised activity into the hippocampus [4]. The hippocampal dentate gyrus granule neurons have been shown to express a variety of GABAA receptors [5,10]. In the epileptic hippocampus, the pharmacology of drugs that interact specifically with the GABAA receptors is altered. The GABA-activated current is less sensitive to modulation by benzodiazepines, more *
Corresponding author. Tel.: þ 46-46-222-0675; fax: þ46-46-222-7763. E-mail address: [email protected] (B. Birnir).
sensitive to inhibition by Zn2þ but the modulation by pentobarbital appears similar to what it was in healthy granule neurons [4,8]. The barbiturates are a group of drugs that have anaesthetic, anti-convulsant and anxiolytic properties. A commonly used barbiturate is pentobarbital. It has been demonstrated to have three major effects on whole-cell currents activated by GABA. GABA-activated currents may be potentiated or blocked by pentobarbital and at some GABAA receptors pentobarbital directly activates a current response [9]. The whole-cell current response in native neurons is set by the cumulative response of all the GABAA receptor subtypes that are expressed in the cell. In this study we examined in more detail the effects pentobarbital has on healthy, acutely-isolated dentate gyrus granule neurons. The results show that in a concentration-dependent manner pentobarbital modulated, activated and inhibited GABAA receptor currents. Experiments were performed on acutely-isolated granule neurons from the hippocampal dentate gyrus region from Wistar rats aged 16– 21 days. The method of isolating the neurons has been previously described [10]. Briefly, the rats were killed by decapitation, the brain removed and
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.09.025
140
M.S.F. Lim et al. / Neuroscience Letters 353 (2003) 139–142
immersed in ice-cold artificial cerebrospinal fluid (ACSF) containing in mM: 126 NaCl, 26 NaHCO3, 20 glucose, 3 KCl, 2.6 CaCl2, 2.5 NaH2PO4 and 1.3 MgSO4. The solution was equilibrated with carbogen gas resulting in a pH of 7.4. 500 mm thick hippocampal slices were cut and incubated in ACSF (35 8C) for 30 min. An enzyme solution was then added having a final concentration of 20 Units/ml Papain (Worthington), 1.1 mM cysteine, 0.22 mM EDTA and 13.4 mM mercaptoethanol and the incubation continued for an additional 30 min. The enzyme solution was then replaced with fresh carbogenated ACSF and the slices incubated for a further 30 min at room temperature (22 8C). The dentate gyri were dissected out of the hippocampal slices and triturated using a polished Pasteur pipette. Isolated neurons were placed in the recording chamber in the experimental bath solution and used for experiments. Whole-cell patch-clamp recordings were performed as described previously [10]. After establishing a whole-cell configuration, the cell was lifted up and positioned in front of a drug delivery tube. Drugs were applied using a fast perfusion system [10]. The currents were recorded with Axopatch amplifiers (1B or 200B) filtered, digitised and stored on a computer. The data were analysed by Channel 2 (M. Smith, ANU, Australia) or pClamp (Axon Instruments, USA) software analysis packages. Dose-response plots were constructed from the modulated GABA-activated peak currents and the peak offcurrents. Values are presented as mean ^ SEM for three to nine neurons and differences between groups are considered significant for P , 0:05 using Student’s t-test. We first examined what concentration range of pentobarbital was effective in modulating the current response to low concentration of GABA. Fig. 1A shows typical current responses to 7 mM GABA (GABA EC30 concentration [10]) and several (3, 10, 50 100 and 1000 mM) test concentrations of pentobarbital. The data are from different cells but normalised to the 7 mM GABA peakcurrent response in the same cell. Pentobarbital concentrations from 0.1 to 3 mM (n ¼ 15) did not significantly affect the amplitude of the peak-current response to the 7 mM GABA but in 10 mM pentobarbital (n ¼ 3) the peakcurrent amplitude was reduced to 0.6 ^ 0.1 of the 7 mM GABA response (Fig. 1Aa,c,B, P , 0:05). In the higher pentobarbital concentrations (50, 100 mM and 1 mM) the 7 mM GABA-activated peak current was enhanced. The greatest current enhancement was measured in 100 mM pentobarbital (n ¼ 8) where it was 2.1 ^ 0.2 of control (Figs. 1Aa,e,B). In 1 mM pentobarbital (n ¼ 3, Figs. 1Af, B) the peak-current enhancement was not significantly different from that of the 100 mM pentobarbital but there was an apparent increase in the initial rate of the current decay and an off-current was recorded when the drugs were removed (Fig. 1Af). Both the increased rate of the current decay and the presence of the off-current indicate a significant inhibitory effect of 1 mM pentobarbital in addition to the enhancing effect of the GABA-activated current response. Due to the clear evidence of inhibition at
Fig. 1. Pentobarbital modulation of the GABA-activated current. (A) Representative current responses to 7 mM GABA (a.) plus test concentrations of pentobarbital (b. 3 mM, c. 10 mM, d. 50 mM, e. 100 mM, f. 1 mM). The currents were normalised to the 7 mM GABA in the same cell. (B) Effects of pentobarbital concentrations on the 7 mM GABAactivated peak-current response. Currents were normalised to the 7 mM peak current in the same cell. Data points represent the averages from 3 to 9 neurons and SEM is shown if larger than the symbol. (C) Effects of 100 mM pentobarbital on GABA-activated currents. Data points represents the averages from three to six neurons and SEM is shown if larger than the symbol. The currents were normalised to the 1 mM GABA plus pentobarbital peak amplitude. The solid line shows the fit of a Hill-type equation to the data. The broken line is from Lim et al. [10] and shows the GABA-activation curve.
10 mM and again at 1 mM pentobarbital we did not attempt to fit the data with a concentration-response curve and the lines in Fig. 1B simply connect the data points. The effective concentration of pentobarbital for modulation of the GABA-activated current response in this study is similar to what has been reported for frog sensory neurons [1], chromaffin cells [12] and cultured rat hippocampal neurons [13] where it ranges between 10 and 300 mM pentobarbital. The concentration for the greatest enhancement of the peak-current amplitude ranged from 100 and 300 mM in these studies. The clinical half-saturation concentration (EC50) of pentobarbital for generating anaesthesia in mammals is 50 mM [7]. In this study the fractional enhancement of the GABA-activated current by 50 mM pentobarbital was 1.7 ^ 0.1 (n ¼ 3) of control. We then varied the GABA concentration in the presence of 100 mM pentobarbital. The results are shown in Fig. 1C and were fitted with a Hill-type equation: I00 ¼ Imax £ ½GABA^h =ð½EC50 ^h þ ½GABA^h Þ where Imax is the saturating peak-current amplitude, EC50 is the half-maximal concentration for the activation of the receptors and h is the Hill coefficient. All currents were normalised to the saturating 1 mM GABA plus pentobarbital currents. The data points are the averages of three or more experiments. The curve in the presence of GABA alone is taken from Lim et al. [10]. The apparent affinity for GABA increased about four times in the presence of 100 mM pentobarbital. The half-saturating concentration of
M.S.F. Lim et al. / Neuroscience Letters 353 (2003) 139–142
GABA (EC50) was shifted from 14 to 3 mM in the presence of 100 mM pentobarbital but the Hill coefficients were similar, 1.3 and 1.2, respectively. Pentobarbital directly activates some GABAA receptor subtypes [1,2,11,13 –15]. We examined if pentobarbital directly activated GABAA receptors expressed in the dentate gyrus granule neurons. In 12 cells 100 mM pentobarbital failed to evoke a current response whereas all the cells responded to 100 mM GABA. We applied 1 mM pentobarbital to another six cells and in all cases an inward current was evoked. The 1 mM pentobarbital evoked current was on the average 0.4 ^ 0.2 (n ¼ 4) of the 100 mM GABA evoked current in the same cell. The concentration required for direct activation appears to vary widely in native cells. In chromaffin cells and mammalian dorsal root ganglion neurons pentobarbital concentrations as low as 30 and 60 mM, respectively, were effective in evoking a current response [12,14] whereas in cultured hippocampal neurons concentrations greater than 100 mM were required with an EC50 for activation of 330 mM [13]. The sensitivity of cells to pentobarbital depends on the pentobarbital EC50 of the receptors expressed in the cell but also on the relative fraction of receptors expressed that are not activated by pentobarbital. In dentate gyrus granule neurons the a4 subunit is prominent [4,9]. a4 containing GABAA receptors are not directly activated by pentobarbital and the relative insensitivity to pentobarbital in this study suggests a significant population of a4 containing GABAA receptors in these cells. The results are in accordance with our previous study on dentate gyrus granule neurons where we showed that the GABA-activated whole-cell currents were only slightly sensitive to the benzodiazepine, diazepam and a significant part of the whole-cell plateau current was inhibited by 3 mM zinc, both indicative of receptors containing the a4 subunit [10]. In epileptic dentate gyrus neurons the altered pharmacology correlates with an even higher level of a4 containing receptors in the cells [4]. Our results with the 10 mM pentobarbital plus GABA indicated that an inhibition by pentobarbital was present in low mM pentobarbital plus GABA but was masked as the pentobarbital concentration was increased to 50 or 100 mM by the greater current-enhancement effects of higher pentobarbital concentrations. However, a transient offcurrent response was produced when 1 mM pentobarbital plus GABA was washed off the cells (see Fig. 1A) and is indicative of channel block [14]. We examined if we could use the off-current response as a tool to detect channel block that was not revealed due to the greater potentiation of the whole-cell current by pentobarbital. An off-current may not be recorded and therefore overlooked if the drugs are not rapidly removed off the cell. The isolated neurons are particularly well suited to test for the off-current response of the granule neurons as the drugs can be applied and removed rapidly with a fast application system [10]. The results are shown in Fig. 2. The lowest concentration that evoked an off-current when washed rapidly off the cells was 100 mM
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Fig. 2. Pentobarbital inhibition of the GABA-activated current. (A) Offcurrents were recorded when 7 mM GABA plus 100 mM (a.), 1 mM (b.) or 10 mM (c.) pentobarbital or 100 mM GABA plus 10 mM pentobarbital (d.) were washed rapidly off the cells. (B) Peak amplitude of the off-current as a fraction of the 7 mM GABA-activated peak-current. The data are averages from three to eight neurons and SEM is shown if larger than the symbol.
pentobarbital (Fig. 2Aa). The average off-current amplitude as a fraction of the peak 7 mM GABA-activated current is shown in Fig. 2B. The off-current amplitude increased as the pentobarbital concentration was raised from 100 mM to 10 mM pentobarbital where it was on average 1.0 ^ 0.1 (n ¼ 8) of the 7 mM GABA-activated peak-current amplitude. In addition to producing a large off-current response, 10 mM pentobarbital also abolished the GABAactivated current response (Fig. 2Ac). We examined if the inhibition of the GABA-activated current was reduced if a higher concentration of GABA (100 mM) plus 10 mM pentobarbital was applied to the cells (Fig. 2Ad, n ¼ 3) but the level of the peak GABA-activated current inhibition was similar to that recorded in 7 mM GABA plus 10 mM pentobarbital (Fig. 2Ac and d). Transient rebound-currents (off-currents) have been recorded in frog sensory neurons (3 mM pentobarbital [1]), chromaffin cells (5 mM pentobarbital [12]), mammalian dorsal root ganglion neurons (600 mM pentobarbital [14]) and in cultured hippocampal neurons (1 mM pentobarbital ^ 1 mM GABA [13]) when the drugs were washed off the cells. The degree of block and current enhancement by pentobarbital has been shown to be influenced by the nature of the amino acid located at the 120 position in the second transmembrane region in a1b1 GABAA receptors [3,6]. Whether the inhibition represented by the reduced peak-current at 10 mM pentobarbital and the inhibition represented by the off-current are due to the same or different mechanism cannot be determined from our studies. If the different subtypes of GABAA receptors
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expressed in a neuron differ in their susceptibility to inhibition by pentobarbital this will influence the effective pentobarbital concentration range for potentiation and the level of current enhancement of the respective GABAactivated current. We have shown that GABAA receptors expressed in the dentate gyrus granule neuron are potentiated, directly activated and blocked by pentobarbital. The drug sensitivity of the three effects of pentobarbital differs with the modulatory and inhibitory effects requiring lower concentration (10 – 100 mM) than direct activation of the receptors.
[4]
[5]
[6]
[7]
Acknowledgements We thank the John Curtin School of Medical Research, Australian National University, Australia. The Swedish Medical Research Council (Y0924), Vetenskapsra˚det ˚ ke Wibergs Stiftelse, Seger(K2002-33X-13408-03A), A falks Stiftelsen, Crafoordska Stiftelsen, Swa¨rds Stiftelse and the Medical Faculty Lund University for financial support.
[8]
[9] [10]
[11] [12]
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