- Email: [email protected]
Effect of ketamine-anesthesia on N-methyl-D-aspartate-induced activation of type I nucleus accumbens neurons
Life Sciences, Vol. 54, pp. PL 35-38 Printed in the U S A
Pergamon Press
PHARMACOLOGY" LETTERS Accelerated Communication
EFFECT OF KETAMINE-ANESTHESIA ON N-METHYL-D-ASPARTATE-INDUCED ACTIVATION OF TYPE I NUCLEUS ACCUMBENS NEURONS
Mark D. Kelland and Louis A. Chiodo Cellular and Clinical Neurobiology Program, Department of Psychiatry, Wayne State University School of Medicine, Detroit, MI, USA (Submitted September29, 1993;acceptedOctober21, 1993; received in final form October 29, 1993)
Abstract. Extracellular single-unit recording and microiontophoretic techniques were used to determine the effects of ketamine-anesthesia on N-methyI-D-aspartate (NMDA)-induced excitation of Type I nucleus accumbens neurons. NMDA increased the firing rate of most neurons in this preparation. Thus, it may be concluded that ketamine-anesthesia does not result in blockade of central NMDA receptors. The excitation caused by NMDA was readily reversed in all cases by co-iontophoresis of MK 801, but was generally unaffected by coiontophoresis of ketamine. However, ketamine-anesthesia did significantly increase the current levels necessary for, and limited the magnitude of, NMDA-induced activation of these cells (as compared to urethane-anesthetized rats), suggesting that ketamine is not without effect on NMDA-receptors in vivo. Introduction It is often considered that the drug classifications dissociative-anesthetic (e.g., ketamine) and non-competitive, NMDA antagonist (e.g., dizocilpine [MK 801]) are synonymous. Although ketamine and MK 801 share many properties (e.g., see 1,2,3,4,7), there is growing evidence of differences between these compounds. For example, ketamine-anesthesia (requiring high doses of ketamine) does not block the ability of NMDA to induce swallowing responses in rats (6). Ketamine-anesthesia also failed to alter either apomorphine-induced stereotypy or apomorphine-induced excitation of Type II globus pallidus neurons, phenomena which were both disrupted by administration of MK 801 (5). Thus, when used as an anesthetic, ketamine does not appear to block NMDA receptors in vivo. In the present study, we utilize microiontophoretic and single-unit electrophysiological techniques to provide direct evidence that ketamine-anesthesia does not block the ability of NMDA to excite Type I nucleus accumbens neurons. Methods Standard, extracellular single-unit recording and iontophoretic techniques were used to sample the activity of Type I nucleus accumbens neurons in male Sprague-Dawley rats Corresponding Author: M.D. Kelland, Wayne St. Univ. Sch. Med., Dept. of Psychiat., 2309 Scott Hall, 540 E. Canfield, Detroit, MI 48201, USA. (313) 993-4270 FAX (313) 993-4269 0024-3205/94 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
PL-36
Ketamine-anesthesia Effects on NMDA
Vol. 54, No. 3, 1994
(250-400 gms, Hilltop Lab Animals). Neuronal signals were sampled with a five-barrel, glass micropipette. The center barrel was filled with 2 M NaCI containing 2% pontamine sky blue dye (~ vitro impedances at 135 Hz, 1.0-2.0 megohms). Three side-barrels were filled with either NMDA, ketamine or MK 801 (each compound: 10 mM, pH 4.0). The remaining barrel was used for balancing tip currents. Routine histological verification confirmed the location of recording sites within the nucleus accumbens. Animals were anesthetized with either ketamine (150 mg/kg, i.p.) or urethane (1.25 g/kg, i.p.). Since many accumbens neurons are relatively silent in anesthetized rats, most cells were located by pulsing NMDA from the iontophoretic pipette as it was advanced through the brain. Following the identification of a Type I accumbens neuron with a stable response to the excitatory effects of NMDA iontophoresis (at least 4 ejection periods), one of two procedures was followed. In the first experiment, NMDA was repeatedly administered for 40 sec pulses with a 60 sec interval between pulses. At various times, the NMDA antagonists MK 801 or ketamine were co-administered with NMDA. In this experiment, the antagonists were ejected throughout a period of two consecutive NMDA ejections, including the period between the NMDA ejection (to test the effects of the antagonists alone). In the second experiment, NMDA was administered iontophoretically onto each cell for 2 ejection periods at currents of 20, 40, 60, 80 and 100 nA (some cells did not receive the highest currents due to an apparent depolarization inactivation). Statistical differences between groups were analyzed either by a t-test or by ANOVA followed by a Student Newman-Keuls Multiple Range Test (with 95% confidence intervals; BMDP Statistical Software, Inc.). Results . Experiment 1. Iontophoretic administration of NMDA significantly increased the firing rate of all cells sampled in ketamine-anesthetized rats (one cell sampled in each of 10 rats; Fig. 1). The basal firing rate of these cells averaged 3.1 + 1.2 spikes/sec, whereas iontophoretic administration of NMDA (70-120 nA) increased the average firing rate to 10.4 + 2.5 spikes/sec (t18=2.6, p<0.05). Co-iontophoresis of ketamine did not significantly alter the excitatory effect of NMDA on these cells at ejections currents of either 20 nA (mean reduction in NMDA-induced firing rate of 0.2 + 0.8 spikes/sec) or 40 nA (mean reduction in NMDA-induced firing rate of 0.2 + 0.7 spikes/sec). In contrast, co-iontophoresis of MK 801 (20 nA) significantly blocked the effects of NMDA (mean reduction in NMDA-induced firing rate of 6.2 + 2.0 spikes/sec). Iontophoretic administration of MK 801 or ketamine alone had no significant effects on either the firing rates of Type I accumbens neurons or the amplitude/shape of the observed waveforms. The firing rates of cells during antagonist administration were as follows (basal rate given above): MK 801,3.6 + 1.2 s/sec; ketamine 20 nA, 3.8 + 1.2 s/sec; ketamine 40 nA, 4.0 + 1.2 s/sec. Experiment 2. Iontophoretic administration of NMDA increased the firing rate of all cells sampled in both ketamine-anesthetized rats (7 cells in 3 rats) and urethane-anesthetized rats (8 cells in 2 rats) (Fig. 2). However, ketamine-anesthesia significantly reduced the potency of NMDA at lower current levels, and significantly reduced the potency and/or efficacy of NMDA at higher current levels (Fig. 2).
Vol. 54, No. 3, 1994
Ketamine-anesthcsia Effects on NMDA
Fig. 1. A cumulative rate histogram .... demonstrates the response of a 2o representative cell to NMDA alone or in the presence of the antagonists ketamine (KET) and MK 801. This cell ~, 15 showed a robust response to NMDA ~ . (current of -70 nA), despite the ~ lO presence of ketamine as an anesthetic. Additional co-iontophoresis of ketamine ~- 5 (at 20 or 40 nA) had no effect. MK 801 (20 nA) significantly reduced the excitatory effect of NMDA. o ~-
40-
Fig. 2. Cumulative dose-response curves (_t. S.E.) demonstrate the reduced ability of NMDA 8 3o. to increase the firing rate of Type I accumbens -~. neurons in ketamine-anesthetized rats as ~ 2o. compared to rats anesthetized with urethane. The difference between the curves was lo. significant (Fl,ss=7.5, p<0.01). *p<0.05 vs. O urethane-anesthetized rats at the same current 0
PL-37
KET
KET
20..
~0.-
MKB01
20
..
/
• Time (5 min intervols) e--e
Urethane
A--A
Ketomine
2o
4'o eo Current (nA)
!
]e
8'o
1~0
Discussion These data provide direct electrophysiological evidence that ketamine-anesthesia (highdose ketamine) does not completely block the excitatory effect of NMDA on Type I nucleus accumbens neurons. This is in agreement with behavioral observations by Kessler et al. (6). Ketamine-anesthesia did, however, reduce the potency and/or efficacy of NMDA, suggesting that ketamine is not without effect in the present paradigm. As expected, MK 801 significantly blocked the effect of NMDA, in agreement with earlier demonstrations that MK 801 is an effective NMDA antagonist in ketamine-anesthetized rats (5). The difficulty with interpreting the present data lies in the fact that ketamine typically exhibits NMDA antagonist properties, and does so at doses significantly lower than those required for anesthesia (see 1). However, when Kelland et al. (5) directly compared ketamine with MK 801, significant differences were observed. Low-dose i.v. ketamine mimicked the effects of MK 801 on apomorphine-induced stereotypy and apomorphineinduced excitation of Type II globus pallidus neurons, but ketamine-anesthesia did not (5). Thus, the method of administration of ketamine appears to be a significant factor in the subsequent pharmacological effects of this compound in vivo. The failure of ketamine coiontophoresis to block NMDA in the present study was predictable, since the ketamineanesthesia likely resulted in tissue concentrations of ketamine so high that the additional ketamine was insignificant. Based on these findings, it is also important to address the pharmacological specificity of MK 801. In the present study, MK 801 had no effect on the firing rate of Type I
PL-38
Ketamine-anesthesia Effects on NMDA
Vol. 54, No. 3, 1994
accumbens neurons during the interval between NMDA ejection periods. MK 801 also had no effect on the shape or amplitude of the waveforms, indicating that MK 801 did not reduce the effects of NMDA due to a non-specific or local anesthetic effect. Indeed, iontophoretically applied MK 801 has previously been shown to selectively block the excitatory effects of NMDA, as opposed to MK 801 's lack of effect on excitation caused by kainate (10). In the absence of literature suggesting that MK 801 acts at receptors other than the NMDA receptor complex, we must conclude that the reduction of NMDA's excitatory effect on Type I accumbens neurons by MK 801 was a blockade of the NMDA receptor complex. The question which remains is: What are the different properties of ketamine and MK 801 ? While we cannot yet answer this question, there is at least one intriguing possibility. As with most other neurotransmitter systems, subtypes of the NMDA receptor complex have recently been proposed (8,9). It is possible, perhaps even likely, that ketamine and MK 801 have significantly different actions at these receptor subtypes. Thus, further examination of the properties of the NMDA receptor complex itself may lead to an understanding of the distinctions between various classes of NMDA receptor antagonists. Acknowledqments The authors thank Ms. J. Rubin for technical assistance. This research was supported by the Tourette Syndrome Association (MDK) and PHS Grant MH41557 (LAC). References .
2. 3. 4. 5. 6. 7.
.
9.
10.
J. CHURCH and D. LODGE, Status of Ketamine in Anesthesiology, E.F. Domino (Ed), 501-519, NPP Books, Ann Arbor, MI (1990). G.E. EVONIUK, R.P. HERTZMAN and P. SKOLNICK, Psychopharmacology 105 125-128 (1991 ). R.F. HALLIWELL, J.A. PETERS and J.J. LAMBERT, Br. J. Pharmacol. 9.6_6480-494 (1989). C.R. HONEY, Z. MILJKOVIC and J.F. MACDONALD, Neurosci. Lett. 6._!.1135-139 (1985). M.D. KELLAND, R.P. SOLTIS, R.C. BOLDRY and J.R. WALTERS, Physiol. Behav. 54 547-554 (1993). J.P. KESSLER, N. CHERKAOUI, D. CATALIN and A. JEAN, Exp. Brain Res. 83 151-158 (1990). J.F. MACDONALD, M.C. BARTLETT, I. MODY, P. PAHAPILL, J.N. REYNOLDS, M.W. SALTER, J.H. SCHNEIDERMAN and P.S. PENNEFATHER, J. Physiol. 432 483-508 (1991 ). D.T. MONAGHAN, Neurosci. Lett. 122. 21-24 (1991). H. MONYER, R. SPRENGEL, R. SCHOEPFER, A. HERB, M. HIGUCHI, H. LOMELI, N. BURNASHEV, B. SAKMANN and P.H. SEEBURG, Science 256 12171221 (1992). J. ZHANG, L.A. CHIODO and A.S. FREEMAN, Brain Res. 590 153-163 (1992).
Pergamon Press
PHARMACOLOGY" LETTERS Accelerated Communication
EFFECT OF KETAMINE-ANESTHESIA ON N-METHYL-D-ASPARTATE-INDUCED ACTIVATION OF TYPE I NUCLEUS ACCUMBENS NEURONS
Mark D. Kelland and Louis A. Chiodo Cellular and Clinical Neurobiology Program, Department of Psychiatry, Wayne State University School of Medicine, Detroit, MI, USA (Submitted September29, 1993;acceptedOctober21, 1993; received in final form October 29, 1993)
Abstract. Extracellular single-unit recording and microiontophoretic techniques were used to determine the effects of ketamine-anesthesia on N-methyI-D-aspartate (NMDA)-induced excitation of Type I nucleus accumbens neurons. NMDA increased the firing rate of most neurons in this preparation. Thus, it may be concluded that ketamine-anesthesia does not result in blockade of central NMDA receptors. The excitation caused by NMDA was readily reversed in all cases by co-iontophoresis of MK 801, but was generally unaffected by coiontophoresis of ketamine. However, ketamine-anesthesia did significantly increase the current levels necessary for, and limited the magnitude of, NMDA-induced activation of these cells (as compared to urethane-anesthetized rats), suggesting that ketamine is not without effect on NMDA-receptors in vivo. Introduction It is often considered that the drug classifications dissociative-anesthetic (e.g., ketamine) and non-competitive, NMDA antagonist (e.g., dizocilpine [MK 801]) are synonymous. Although ketamine and MK 801 share many properties (e.g., see 1,2,3,4,7), there is growing evidence of differences between these compounds. For example, ketamine-anesthesia (requiring high doses of ketamine) does not block the ability of NMDA to induce swallowing responses in rats (6). Ketamine-anesthesia also failed to alter either apomorphine-induced stereotypy or apomorphine-induced excitation of Type II globus pallidus neurons, phenomena which were both disrupted by administration of MK 801 (5). Thus, when used as an anesthetic, ketamine does not appear to block NMDA receptors in vivo. In the present study, we utilize microiontophoretic and single-unit electrophysiological techniques to provide direct evidence that ketamine-anesthesia does not block the ability of NMDA to excite Type I nucleus accumbens neurons. Methods Standard, extracellular single-unit recording and iontophoretic techniques were used to sample the activity of Type I nucleus accumbens neurons in male Sprague-Dawley rats Corresponding Author: M.D. Kelland, Wayne St. Univ. Sch. Med., Dept. of Psychiat., 2309 Scott Hall, 540 E. Canfield, Detroit, MI 48201, USA. (313) 993-4270 FAX (313) 993-4269 0024-3205/94 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
PL-36
Ketamine-anesthesia Effects on NMDA
Vol. 54, No. 3, 1994
(250-400 gms, Hilltop Lab Animals). Neuronal signals were sampled with a five-barrel, glass micropipette. The center barrel was filled with 2 M NaCI containing 2% pontamine sky blue dye (~ vitro impedances at 135 Hz, 1.0-2.0 megohms). Three side-barrels were filled with either NMDA, ketamine or MK 801 (each compound: 10 mM, pH 4.0). The remaining barrel was used for balancing tip currents. Routine histological verification confirmed the location of recording sites within the nucleus accumbens. Animals were anesthetized with either ketamine (150 mg/kg, i.p.) or urethane (1.25 g/kg, i.p.). Since many accumbens neurons are relatively silent in anesthetized rats, most cells were located by pulsing NMDA from the iontophoretic pipette as it was advanced through the brain. Following the identification of a Type I accumbens neuron with a stable response to the excitatory effects of NMDA iontophoresis (at least 4 ejection periods), one of two procedures was followed. In the first experiment, NMDA was repeatedly administered for 40 sec pulses with a 60 sec interval between pulses. At various times, the NMDA antagonists MK 801 or ketamine were co-administered with NMDA. In this experiment, the antagonists were ejected throughout a period of two consecutive NMDA ejections, including the period between the NMDA ejection (to test the effects of the antagonists alone). In the second experiment, NMDA was administered iontophoretically onto each cell for 2 ejection periods at currents of 20, 40, 60, 80 and 100 nA (some cells did not receive the highest currents due to an apparent depolarization inactivation). Statistical differences between groups were analyzed either by a t-test or by ANOVA followed by a Student Newman-Keuls Multiple Range Test (with 95% confidence intervals; BMDP Statistical Software, Inc.). Results . Experiment 1. Iontophoretic administration of NMDA significantly increased the firing rate of all cells sampled in ketamine-anesthetized rats (one cell sampled in each of 10 rats; Fig. 1). The basal firing rate of these cells averaged 3.1 + 1.2 spikes/sec, whereas iontophoretic administration of NMDA (70-120 nA) increased the average firing rate to 10.4 + 2.5 spikes/sec (t18=2.6, p<0.05). Co-iontophoresis of ketamine did not significantly alter the excitatory effect of NMDA on these cells at ejections currents of either 20 nA (mean reduction in NMDA-induced firing rate of 0.2 + 0.8 spikes/sec) or 40 nA (mean reduction in NMDA-induced firing rate of 0.2 + 0.7 spikes/sec). In contrast, co-iontophoresis of MK 801 (20 nA) significantly blocked the effects of NMDA (mean reduction in NMDA-induced firing rate of 6.2 + 2.0 spikes/sec). Iontophoretic administration of MK 801 or ketamine alone had no significant effects on either the firing rates of Type I accumbens neurons or the amplitude/shape of the observed waveforms. The firing rates of cells during antagonist administration were as follows (basal rate given above): MK 801,3.6 + 1.2 s/sec; ketamine 20 nA, 3.8 + 1.2 s/sec; ketamine 40 nA, 4.0 + 1.2 s/sec. Experiment 2. Iontophoretic administration of NMDA increased the firing rate of all cells sampled in both ketamine-anesthetized rats (7 cells in 3 rats) and urethane-anesthetized rats (8 cells in 2 rats) (Fig. 2). However, ketamine-anesthesia significantly reduced the potency of NMDA at lower current levels, and significantly reduced the potency and/or efficacy of NMDA at higher current levels (Fig. 2).
Vol. 54, No. 3, 1994
Ketamine-anesthcsia Effects on NMDA
Fig. 1. A cumulative rate histogram .... demonstrates the response of a 2o representative cell to NMDA alone or in the presence of the antagonists ketamine (KET) and MK 801. This cell ~, 15 showed a robust response to NMDA ~ . (current of -70 nA), despite the ~ lO presence of ketamine as an anesthetic. Additional co-iontophoresis of ketamine ~- 5 (at 20 or 40 nA) had no effect. MK 801 (20 nA) significantly reduced the excitatory effect of NMDA. o ~-
40-
Fig. 2. Cumulative dose-response curves (_t. S.E.) demonstrate the reduced ability of NMDA 8 3o. to increase the firing rate of Type I accumbens -~. neurons in ketamine-anesthetized rats as ~ 2o. compared to rats anesthetized with urethane. The difference between the curves was lo. significant (Fl,ss=7.5, p<0.01). *p<0.05 vs. O urethane-anesthetized rats at the same current 0
PL-37
KET
KET
20..
~0.-
MKB01
20
..
/
• Time (5 min intervols) e--e
Urethane
A--A
Ketomine
2o
4'o eo Current (nA)
!
]e
8'o
1~0
Discussion These data provide direct electrophysiological evidence that ketamine-anesthesia (highdose ketamine) does not completely block the excitatory effect of NMDA on Type I nucleus accumbens neurons. This is in agreement with behavioral observations by Kessler et al. (6). Ketamine-anesthesia did, however, reduce the potency and/or efficacy of NMDA, suggesting that ketamine is not without effect in the present paradigm. As expected, MK 801 significantly blocked the effect of NMDA, in agreement with earlier demonstrations that MK 801 is an effective NMDA antagonist in ketamine-anesthetized rats (5). The difficulty with interpreting the present data lies in the fact that ketamine typically exhibits NMDA antagonist properties, and does so at doses significantly lower than those required for anesthesia (see 1). However, when Kelland et al. (5) directly compared ketamine with MK 801, significant differences were observed. Low-dose i.v. ketamine mimicked the effects of MK 801 on apomorphine-induced stereotypy and apomorphineinduced excitation of Type II globus pallidus neurons, but ketamine-anesthesia did not (5). Thus, the method of administration of ketamine appears to be a significant factor in the subsequent pharmacological effects of this compound in vivo. The failure of ketamine coiontophoresis to block NMDA in the present study was predictable, since the ketamineanesthesia likely resulted in tissue concentrations of ketamine so high that the additional ketamine was insignificant. Based on these findings, it is also important to address the pharmacological specificity of MK 801. In the present study, MK 801 had no effect on the firing rate of Type I
PL-38
Ketamine-anesthesia Effects on NMDA
Vol. 54, No. 3, 1994
accumbens neurons during the interval between NMDA ejection periods. MK 801 also had no effect on the shape or amplitude of the waveforms, indicating that MK 801 did not reduce the effects of NMDA due to a non-specific or local anesthetic effect. Indeed, iontophoretically applied MK 801 has previously been shown to selectively block the excitatory effects of NMDA, as opposed to MK 801 's lack of effect on excitation caused by kainate (10). In the absence of literature suggesting that MK 801 acts at receptors other than the NMDA receptor complex, we must conclude that the reduction of NMDA's excitatory effect on Type I accumbens neurons by MK 801 was a blockade of the NMDA receptor complex. The question which remains is: What are the different properties of ketamine and MK 801 ? While we cannot yet answer this question, there is at least one intriguing possibility. As with most other neurotransmitter systems, subtypes of the NMDA receptor complex have recently been proposed (8,9). It is possible, perhaps even likely, that ketamine and MK 801 have significantly different actions at these receptor subtypes. Thus, further examination of the properties of the NMDA receptor complex itself may lead to an understanding of the distinctions between various classes of NMDA receptor antagonists. Acknowledqments The authors thank Ms. J. Rubin for technical assistance. This research was supported by the Tourette Syndrome Association (MDK) and PHS Grant MH41557 (LAC). References .
2. 3. 4. 5. 6. 7.
.
9.
10.
J. CHURCH and D. LODGE, Status of Ketamine in Anesthesiology, E.F. Domino (Ed), 501-519, NPP Books, Ann Arbor, MI (1990). G.E. EVONIUK, R.P. HERTZMAN and P. SKOLNICK, Psychopharmacology 105 125-128 (1991 ). R.F. HALLIWELL, J.A. PETERS and J.J. LAMBERT, Br. J. Pharmacol. 9.6_6480-494 (1989). C.R. HONEY, Z. MILJKOVIC and J.F. MACDONALD, Neurosci. Lett. 6._!.1135-139 (1985). M.D. KELLAND, R.P. SOLTIS, R.C. BOLDRY and J.R. WALTERS, Physiol. Behav. 54 547-554 (1993). J.P. KESSLER, N. CHERKAOUI, D. CATALIN and A. JEAN, Exp. Brain Res. 83 151-158 (1990). J.F. MACDONALD, M.C. BARTLETT, I. MODY, P. PAHAPILL, J.N. REYNOLDS, M.W. SALTER, J.H. SCHNEIDERMAN and P.S. PENNEFATHER, J. Physiol. 432 483-508 (1991 ). D.T. MONAGHAN, Neurosci. Lett. 122. 21-24 (1991). H. MONYER, R. SPRENGEL, R. SCHOEPFER, A. HERB, M. HIGUCHI, H. LOMELI, N. BURNASHEV, B. SAKMANN and P.H. SEEBURG, Science 256 12171221 (1992). J. ZHANG, L.A. CHIODO and A.S. FREEMAN, Brain Res. 590 153-163 (1992).