- Email: [email protected]
Ketamine anesthesia helps preserve neuronal viability
Journal of Neuroscience Methods 189 (2010) 230–232
Contents lists available at ScienceDirect
Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth
Short communication
Ketamine anesthesia helps preserve neuronal viability Ramatis B. de Oliveira a , Brett Graham a , Marcus C.H. Howlett a,b , Fernanda S. Gravina a , Max W.S. Oliveira a , Mohammad S. Imtiaz a,c , Robert J. Callister a , Rebecca Lim a , Alan M. Brichta a , Dirk F. van Helden a,∗ a b c
School of Biomedical Sciences and Pharmacy, University of Newcastle, University Drive, Newcastle, NSW 2308, Australia Retinal Signal Processing, Netherlands Institute of Neuroscience, Amsterdam 1105BA, The Netherlands Department of Physiology & Pharmacology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada
a r t i c l e
i n f o
Article history: Received 12 February 2010 Received in revised form 30 March 2010 Accepted 30 March 2010 Keywords: Ketamine Locus coeruleus Hypoglossal motor neurons Electrophysiological properties Input resistance Viability
a b s t r a c t The dissociative anesthetic ketamine that acts as an N-methyl-D-aspartate (NMDA) antagonist has been reported to improve neurological damage after experimental ischemic challenges. Here we show that deep anesthesia with ketamine before euthanasia by decapitation improves the quality of neonatal mouse neuronal brain slice preparations. Specifically we found that neurons of the locus coeruleus (LC) and hypoglossal motor neurons had significantly higher input resistances, and LC neurons that generally are difficult to voltage control, could be more reliably voltage clamped compared to control neurons. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ketamine is a well known dissociative anesthetic that acts as an N-methyl-D-aspartate (NMDA) antagonist (Collins et al., 1960). It is commonly used for elective surgeries (Bergman, 1999), as treatment for acute and chronic pain (Mathisen et al., 1995), and also to establish an animal model for psychosis (Newcomer et al., 1999). Ketamine has also been reported to improve neurological function and reduce damage after experimental ischemic challenges (Reeker et al., 2000). The effects of acute and chronic ketamine can vary among studies due to different methodological approaches. Acute treatments seem to present a higher incidence of “positive” outcomes, such as improvement in depressive symptoms (Berman et al., 2000), whereas chronic treatments have been associated with psychotic-like symptoms, dependence and other effects (Himmelseher and Durieux, 2005; Morgan and Curran, 2006; Stefanovic et al., 2009). Electrophysiological studies have shown that direct ketamine application in neurons results in a decreased firing frequency, due to an inhibition of Na+ and K+ channels (Schnoebel et al., 2005). An increase in input resistance and inhibition of excitatory postsynaptic potentials (EPSPs) has also been associated with direct
∗ Corresponding author. Tel.: +61 2 49215626. E-mail address: [email protected] (D.F. van Helden). 0165-0270/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2010.03.029
application of ketamine (Leong et al., 2004). Such action occurs during application of ketamine with these properties returning to control levels within minutes after removal of the ketamine. The present study examined electrophysiological properties of neurons of the locus coeruleus (LC), a key mood state-associated nucleus containing noradrenergic neurons that project widely across the brain (Aston-Jones et al., 1999; Ward and Gunn, 1976), with the studies repeated in hypoglossal motor neurons. Our specific aim was to evaluate if ketamine anesthesia before animal sacrifice would enhance neuronal viability, as without this we found that, while we could reasonably voltage clamp hypoglossal neurons, this was not the case for LC neurons. We found ketamine anesthesia before animal sacrifice reduced the conductance (i.e. leakiness) of both neuronal preparations and hence improved whole cell voltage control of LC neurons. 2. Methods Brain slices containing LC or hypoglossal neurons were prepared from Swiss mice (P7-12). The University of Newcastle Animal Care and Ethics Committee approved all procedures. Mice were either deeply anesthetized with ketamine (100 mg/kg i.p.) and then decapitated (n = 54 and 5 animals for LC and hypoglossal, respectively), or decapitated without anesthesia (n = 52 and 5 animals for LC and hypoglossal, respectively). The brain was rapidly removed and immersed in ice-cold “modified sucrose Ringer” con-
R.B. de Oliveira et al. / Journal of Neuroscience Methods 189 (2010) 230–232
231
Fig. 1. Example of successful (A) and unsuccessful (B) voltage clamp experiments. In (A) voltage control was good and there were no “breakthrough” inward spikes during the depolarizing ramp-evoked current, whereas in (B) voltage control was poor and many unclamped fast-activated voltage-dependent spikes occurred during the same ramp protocol. Holding potential for A and B was −58 mV.
taining (in mM): 25 NaHCO3 , 11 Glucose, 235 Sucrose, 2.5 KCl, 1 NaH2 PO4 , 1 MgCl2 and 2.5 CaCl2 , bubbled with 95% O2 /5% CO2 . While immersed, the cerebellum and brain stem were isolated and slices (270–300 m thick) were prepared using a vibrating tissue slicer (Leica VT1000S). Generally, the LC and hypoglossal structures were contained within one or two slices, respectively. Slices were transferred to a humidified and oxygenated incubation chamber at ∼23 ◦ C, allowed to equilibrate for 1.5–2 h, then moved to a recording bath positioned on an upright microscope (Olympus BX50). Slices were superfused with artificial cerebral spinal fluid at 34 ± 2 ◦ C containing (in mM): 120 NaCl, 25 NaHCO3 , 11 Glucose, 2.5 KCl, 1 NaH2 PO4 , 1 MgCl2 and 2.5 CaCl2 , constantly bubbled with 95% O2 /5% CO2 . Using infrared video microscopy, LC neurons were identified by their large size and location below the ventrolateral border of the 4th ventricle; hypoglossal neurons were identified by their large size and location relative to the central canal/4th ventricle (Graham et al., 2006). Whole-cell patch clamp recordings were made using an Axopatch-1C amplifier and Axograph X software. Patch electrode resistances ranged from 1.8–2.5 M when filled with internal solution composed of (in mM): 135 KCH3 SO4 , 8 NaCl, 10 HEPES, 2 Mg2 ATP, 0.3 Na3 GTP, 0.1 EGTA, pH 7.3. All recordings were made in voltage clamp, sampled at 100 kHz and filtered at 5 kHz (or 200 Hz for voltage ramps). Input resistance, cell capacitance and series resistance were measured online from exponential fits of the decay phase of the current response to −5 mV pulses under voltage clamp with measurements made ∼10 s after obtaining whole cell recording mode. Experiments involved applying depolarizing voltage ramps (40 mV/s) from a holding potential of −58 mV (after correction for liquid junction potential offset). Only one slice per animal containing either hypoglossal or LC neurons was used for each experiment. Usually 3–5 neurons were tested in slices containing LC neurons and 7–10 neurons in hypoglossal slices. Neurons were considered successfully voltage clamped when there were no breakthrough spikes during the voltage ramp (Fig. 1). In general, the success rate of voltage clamp
experiments for neurons in LC slices was “all or none”, more specifically: in a “good” slice >80% of the neurons could be successfully clamped and in a “bad” slice >80% of neurons could not be clamped even though the neurons were functional. This criterion was used for statistical analysis of the success of voltage clamping with comparisons made between slices and hence animals, as one slice was used for each animal (Table 1). Statistical analyses were performed with SPSS 17.0. A chi-square test assessed the difference in numbers of animals where LC neuronal voltage clamping was or was not successful, and an independent groups t-test assessed the difference in RIN , between the ketamine-anesthetized and control groups. 3. Results Ketamine improved LC neuron viability, in slice preparations from ketamine-anesthetized mice versus those that did not receive ketamine before euthanasia (hereafter termed controls). Recordings from LC neurons in ketamine-anesthetized mice exhibited better voltage control and higher input resistances (RIN ) compared with those from controls (Table 1). In voltage clamp experiments, depolarizing ramps were applied and the experiment was considered successful if unclamped action potential artifacts were not observed in the current trace (Fig. 1). Ketamine anesthesia, led to a marked improvement (p < 0.05) in the success rate of voltage clamp experiments in LC slices: 73% for the ketamine-anesthetized group (54 animals) versus 42% from controls (52 animals) as presented in Table 1. RIN was significantly higher (∼30%, p < 0.001) in the neurons from the ketamine-anesthetized group compared to controls (Table 1), suggesting improved cell viability. To determine whether the protective effect of ketamine generalized to other neuronal populations, we repeated the experiments on hypoglossal motor neurons. Though hypoglossal neurons are relatively large, good voltage control was always achieved during ramps; however, RIN was higher (∼25%) in neurons from ketamine-anesthetized animals (p < 0.05, Table 1). These results on hypoglossal neurons again indicate that ketamine improves neuron viability.
Table 1 The effect of ketamine on the success rate of ramp voltage clamp experiments and input resistance (RIN ). LC neurons in brain slices of mice deeply anesthetized before euthanasia by decapitation were successfully clamped in 73% of the slices, compared to 43% for non-anesthetized mice. Hypoglossal neurons did not present ramp voltage clamp control problems in the either group. RIN was higher in LC and hypoglossal neurons from ketamine-anesthetized mice, however, membrane capacitance (Cm ) and series resistance (Rs ) did not change. (+) Different from the control (i.e. no ketamine) group (Chi-square test; p < 0.05). (*, **) Different from the control (i.e. no ketamine) group (independent groups t-test; *p < 0.05, **p < 0.001). RIN , Cm and Rs values are presented as mean ± SEM. Locus coeruleus
No. animals with neurons that could be voltage clamped No. animals with neurons that could not be voltage clamped Input resistance (M) Membrane capacitance (pF) Series resistance (M) No. of cells
Hypoglossal
Ketamine
Control (no ketamine)
Ketamine
Control (no ketamine)
39 animals (73%)+ 15 animals (27%) 256.8 ± 9.8** 70 ± 0.9 4.9 ± 0.09 129
22 animals (425%) 30 animals (57.5%) 194 ± 7.3 68.3 ± 0.9 4.9 ± 0.1 159
5 animals (100%) 0 animals 64.9 ± 4.3* 83.7 ± 4.3 5 1 ± 0.2 38
5 animals (100%) 0 animals 51.6 ± 2.1 89.9 ± 4.0 4.8 ± 0.2 35
232
R.B. de Oliveira et al. / Journal of Neuroscience Methods 189 (2010) 230–232
4. Discussion Neuron viability is a key issue for experiments on slices of CNS tissue. The results presented here indicate ketamine-anesthesia, before euthanasia by decapitation, provides an important strategy for preserving neuron viability. Indeed, it significantly improved the success rate of our voltage clamp experiments in LC neurons. It is unlikely that the effects observed here arose through a residual ketamine effect, as it has been shown that direct application of ketamine in tissues washes out in minutes (Schnoebel et al., 2005). We have not evaluated if anesthesia by itself would exert similar outcomes. We chose to pre-treat with ketamine, because of its known protective action in limiting cell damage during ischemia–reperfusion (Reeker et al., 2000). This protective action most likely arises through its well-established antagonist action of ketamine on NMDA receptors (Collins et al., 1960). This, in combination with blockage of Na+ and K+ channels, probably decreases metabolic demands, caused by markedly increased neuronal firing and resultant Ca2+ loading, which occur during slice preparation. Such damage is likely to be reflected electrophysiologically as a decreased RIN . Importantly, membrane capacitance was unchanged by ketamine (Table 1). This argues against a role for factors such as altered gap junction-mediated coupling between neurons. Our findings therefore provide quantitative support for using ketamineanesthesia to improve the viability of neurons in brain slices. References Aston-Jones G, Rajkowski J, Cohen J. Role of locus coeruleus in attention and behavioral flexibility. Biol Psychiatry 1999;46:1309–20.
Bergman SA. Ketamine: review of its pharmacology and its use in pediatric anesthesia. Anesth Prog 1999;46:10–20. Berman RM, Cappiello A, Anand A, Oren DA, Heninger GR, Charney DS, et al. Antidepressant effects of ketamine in depressed patients. Biol Psychiatry 2000;47:351–4. Collins VJ, Gorospe CA, Rovenstine EA. Intravenous nonbarbiturate, nonnarcotic analgesics: preliminary studies. 1. Cyclohexylamines. Anesth Analg 1960;39:302–6. Graham BA, Schofield PR, Sah P, Margrie TW, Callister RJ. Distinct physiological mechanisms underlie altered glycinergic synaptic transmission in the murine mutants spastic, spasmodic, and oscillator. J Neurosci 2006;26:4880–90. Himmelseher S, Durieux ME. Revising a dogma: ketamine for patients with neurological injury? Anesth Analg 2005;101:524–34, table of contents. Leong D, Puil E, Schwarz D. Ketamine blocks non-N-methyl-D-aspartate receptor channels attenuating glutamatergic transmission in the auditory cortex. Acta Otolaryngol 2004;124:454–8. Mathisen LC, Skjelbred P, Skoglund LA, Oye I. Effect of ketamine, an NMDA receptor inhibitor, in acute and chronic orofacial pain. Pain 1995;61:215–20. Morgan CJ, Curran HV. Acute and chronic effects of ketamine upon human memory: a review. Psychopharmacology (Berl) 2006;188:408–24. Newcomer JW, Farber NB, Jevtovic-Todorovic V, Selke G, Melson AK, Hershey T, et al. Ketamine-induced NMDA receptor hypofunction as a model of memory impairment and psychosis. Neuropsychopharmacology 1999;20: 106–18. Reeker W, Werner C, Mollenberg O, Mielke L, Kochs E. High-dose S(+)-ketamine improves neurological outcome following incomplete cerebral ischemia in rats. Can J Anaesth 2000;47:572–8. Schnoebel R, Wolff M, Peters SC, Brau ME, Scholz A, Hempelmann G, et al. Ketamine impairs excitability in superficial dorsal horn neurones by blocking sodium and voltage-gated potassium currents. Br J Pharmacol 2005;146:826–33. Stefanovic A, Brandner B, Klaassen E, Cregg R, Nagaratnam M, Bromley LM, et al. Acute and chronic effects of ketamine on semantic priming: modeling schizophrenia? J Clin Psychopharmacol 2009;29:124–33. Ward DG, Gunn CG. Locus coeruleus complex: elicitation of a pressor response and a brain stem region necessary for its occurrence. Brain Res 1976;107: 401–6.
Contents lists available at ScienceDirect
Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth
Short communication
Ketamine anesthesia helps preserve neuronal viability Ramatis B. de Oliveira a , Brett Graham a , Marcus C.H. Howlett a,b , Fernanda S. Gravina a , Max W.S. Oliveira a , Mohammad S. Imtiaz a,c , Robert J. Callister a , Rebecca Lim a , Alan M. Brichta a , Dirk F. van Helden a,∗ a b c
School of Biomedical Sciences and Pharmacy, University of Newcastle, University Drive, Newcastle, NSW 2308, Australia Retinal Signal Processing, Netherlands Institute of Neuroscience, Amsterdam 1105BA, The Netherlands Department of Physiology & Pharmacology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada
a r t i c l e
i n f o
Article history: Received 12 February 2010 Received in revised form 30 March 2010 Accepted 30 March 2010 Keywords: Ketamine Locus coeruleus Hypoglossal motor neurons Electrophysiological properties Input resistance Viability
a b s t r a c t The dissociative anesthetic ketamine that acts as an N-methyl-D-aspartate (NMDA) antagonist has been reported to improve neurological damage after experimental ischemic challenges. Here we show that deep anesthesia with ketamine before euthanasia by decapitation improves the quality of neonatal mouse neuronal brain slice preparations. Specifically we found that neurons of the locus coeruleus (LC) and hypoglossal motor neurons had significantly higher input resistances, and LC neurons that generally are difficult to voltage control, could be more reliably voltage clamped compared to control neurons. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ketamine is a well known dissociative anesthetic that acts as an N-methyl-D-aspartate (NMDA) antagonist (Collins et al., 1960). It is commonly used for elective surgeries (Bergman, 1999), as treatment for acute and chronic pain (Mathisen et al., 1995), and also to establish an animal model for psychosis (Newcomer et al., 1999). Ketamine has also been reported to improve neurological function and reduce damage after experimental ischemic challenges (Reeker et al., 2000). The effects of acute and chronic ketamine can vary among studies due to different methodological approaches. Acute treatments seem to present a higher incidence of “positive” outcomes, such as improvement in depressive symptoms (Berman et al., 2000), whereas chronic treatments have been associated with psychotic-like symptoms, dependence and other effects (Himmelseher and Durieux, 2005; Morgan and Curran, 2006; Stefanovic et al., 2009). Electrophysiological studies have shown that direct ketamine application in neurons results in a decreased firing frequency, due to an inhibition of Na+ and K+ channels (Schnoebel et al., 2005). An increase in input resistance and inhibition of excitatory postsynaptic potentials (EPSPs) has also been associated with direct
∗ Corresponding author. Tel.: +61 2 49215626. E-mail address: [email protected] (D.F. van Helden). 0165-0270/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2010.03.029
application of ketamine (Leong et al., 2004). Such action occurs during application of ketamine with these properties returning to control levels within minutes after removal of the ketamine. The present study examined electrophysiological properties of neurons of the locus coeruleus (LC), a key mood state-associated nucleus containing noradrenergic neurons that project widely across the brain (Aston-Jones et al., 1999; Ward and Gunn, 1976), with the studies repeated in hypoglossal motor neurons. Our specific aim was to evaluate if ketamine anesthesia before animal sacrifice would enhance neuronal viability, as without this we found that, while we could reasonably voltage clamp hypoglossal neurons, this was not the case for LC neurons. We found ketamine anesthesia before animal sacrifice reduced the conductance (i.e. leakiness) of both neuronal preparations and hence improved whole cell voltage control of LC neurons. 2. Methods Brain slices containing LC or hypoglossal neurons were prepared from Swiss mice (P7-12). The University of Newcastle Animal Care and Ethics Committee approved all procedures. Mice were either deeply anesthetized with ketamine (100 mg/kg i.p.) and then decapitated (n = 54 and 5 animals for LC and hypoglossal, respectively), or decapitated without anesthesia (n = 52 and 5 animals for LC and hypoglossal, respectively). The brain was rapidly removed and immersed in ice-cold “modified sucrose Ringer” con-
R.B. de Oliveira et al. / Journal of Neuroscience Methods 189 (2010) 230–232
231
Fig. 1. Example of successful (A) and unsuccessful (B) voltage clamp experiments. In (A) voltage control was good and there were no “breakthrough” inward spikes during the depolarizing ramp-evoked current, whereas in (B) voltage control was poor and many unclamped fast-activated voltage-dependent spikes occurred during the same ramp protocol. Holding potential for A and B was −58 mV.
taining (in mM): 25 NaHCO3 , 11 Glucose, 235 Sucrose, 2.5 KCl, 1 NaH2 PO4 , 1 MgCl2 and 2.5 CaCl2 , bubbled with 95% O2 /5% CO2 . While immersed, the cerebellum and brain stem were isolated and slices (270–300 m thick) were prepared using a vibrating tissue slicer (Leica VT1000S). Generally, the LC and hypoglossal structures were contained within one or two slices, respectively. Slices were transferred to a humidified and oxygenated incubation chamber at ∼23 ◦ C, allowed to equilibrate for 1.5–2 h, then moved to a recording bath positioned on an upright microscope (Olympus BX50). Slices were superfused with artificial cerebral spinal fluid at 34 ± 2 ◦ C containing (in mM): 120 NaCl, 25 NaHCO3 , 11 Glucose, 2.5 KCl, 1 NaH2 PO4 , 1 MgCl2 and 2.5 CaCl2 , constantly bubbled with 95% O2 /5% CO2 . Using infrared video microscopy, LC neurons were identified by their large size and location below the ventrolateral border of the 4th ventricle; hypoglossal neurons were identified by their large size and location relative to the central canal/4th ventricle (Graham et al., 2006). Whole-cell patch clamp recordings were made using an Axopatch-1C amplifier and Axograph X software. Patch electrode resistances ranged from 1.8–2.5 M when filled with internal solution composed of (in mM): 135 KCH3 SO4 , 8 NaCl, 10 HEPES, 2 Mg2 ATP, 0.3 Na3 GTP, 0.1 EGTA, pH 7.3. All recordings were made in voltage clamp, sampled at 100 kHz and filtered at 5 kHz (or 200 Hz for voltage ramps). Input resistance, cell capacitance and series resistance were measured online from exponential fits of the decay phase of the current response to −5 mV pulses under voltage clamp with measurements made ∼10 s after obtaining whole cell recording mode. Experiments involved applying depolarizing voltage ramps (40 mV/s) from a holding potential of −58 mV (after correction for liquid junction potential offset). Only one slice per animal containing either hypoglossal or LC neurons was used for each experiment. Usually 3–5 neurons were tested in slices containing LC neurons and 7–10 neurons in hypoglossal slices. Neurons were considered successfully voltage clamped when there were no breakthrough spikes during the voltage ramp (Fig. 1). In general, the success rate of voltage clamp
experiments for neurons in LC slices was “all or none”, more specifically: in a “good” slice >80% of the neurons could be successfully clamped and in a “bad” slice >80% of neurons could not be clamped even though the neurons were functional. This criterion was used for statistical analysis of the success of voltage clamping with comparisons made between slices and hence animals, as one slice was used for each animal (Table 1). Statistical analyses were performed with SPSS 17.0. A chi-square test assessed the difference in numbers of animals where LC neuronal voltage clamping was or was not successful, and an independent groups t-test assessed the difference in RIN , between the ketamine-anesthetized and control groups. 3. Results Ketamine improved LC neuron viability, in slice preparations from ketamine-anesthetized mice versus those that did not receive ketamine before euthanasia (hereafter termed controls). Recordings from LC neurons in ketamine-anesthetized mice exhibited better voltage control and higher input resistances (RIN ) compared with those from controls (Table 1). In voltage clamp experiments, depolarizing ramps were applied and the experiment was considered successful if unclamped action potential artifacts were not observed in the current trace (Fig. 1). Ketamine anesthesia, led to a marked improvement (p < 0.05) in the success rate of voltage clamp experiments in LC slices: 73% for the ketamine-anesthetized group (54 animals) versus 42% from controls (52 animals) as presented in Table 1. RIN was significantly higher (∼30%, p < 0.001) in the neurons from the ketamine-anesthetized group compared to controls (Table 1), suggesting improved cell viability. To determine whether the protective effect of ketamine generalized to other neuronal populations, we repeated the experiments on hypoglossal motor neurons. Though hypoglossal neurons are relatively large, good voltage control was always achieved during ramps; however, RIN was higher (∼25%) in neurons from ketamine-anesthetized animals (p < 0.05, Table 1). These results on hypoglossal neurons again indicate that ketamine improves neuron viability.
Table 1 The effect of ketamine on the success rate of ramp voltage clamp experiments and input resistance (RIN ). LC neurons in brain slices of mice deeply anesthetized before euthanasia by decapitation were successfully clamped in 73% of the slices, compared to 43% for non-anesthetized mice. Hypoglossal neurons did not present ramp voltage clamp control problems in the either group. RIN was higher in LC and hypoglossal neurons from ketamine-anesthetized mice, however, membrane capacitance (Cm ) and series resistance (Rs ) did not change. (+) Different from the control (i.e. no ketamine) group (Chi-square test; p < 0.05). (*, **) Different from the control (i.e. no ketamine) group (independent groups t-test; *p < 0.05, **p < 0.001). RIN , Cm and Rs values are presented as mean ± SEM. Locus coeruleus
No. animals with neurons that could be voltage clamped No. animals with neurons that could not be voltage clamped Input resistance (M) Membrane capacitance (pF) Series resistance (M) No. of cells
Hypoglossal
Ketamine
Control (no ketamine)
Ketamine
Control (no ketamine)
39 animals (73%)+ 15 animals (27%) 256.8 ± 9.8** 70 ± 0.9 4.9 ± 0.09 129
22 animals (425%) 30 animals (57.5%) 194 ± 7.3 68.3 ± 0.9 4.9 ± 0.1 159
5 animals (100%) 0 animals 64.9 ± 4.3* 83.7 ± 4.3 5 1 ± 0.2 38
5 animals (100%) 0 animals 51.6 ± 2.1 89.9 ± 4.0 4.8 ± 0.2 35
232
R.B. de Oliveira et al. / Journal of Neuroscience Methods 189 (2010) 230–232
4. Discussion Neuron viability is a key issue for experiments on slices of CNS tissue. The results presented here indicate ketamine-anesthesia, before euthanasia by decapitation, provides an important strategy for preserving neuron viability. Indeed, it significantly improved the success rate of our voltage clamp experiments in LC neurons. It is unlikely that the effects observed here arose through a residual ketamine effect, as it has been shown that direct application of ketamine in tissues washes out in minutes (Schnoebel et al., 2005). We have not evaluated if anesthesia by itself would exert similar outcomes. We chose to pre-treat with ketamine, because of its known protective action in limiting cell damage during ischemia–reperfusion (Reeker et al., 2000). This protective action most likely arises through its well-established antagonist action of ketamine on NMDA receptors (Collins et al., 1960). This, in combination with blockage of Na+ and K+ channels, probably decreases metabolic demands, caused by markedly increased neuronal firing and resultant Ca2+ loading, which occur during slice preparation. Such damage is likely to be reflected electrophysiologically as a decreased RIN . Importantly, membrane capacitance was unchanged by ketamine (Table 1). This argues against a role for factors such as altered gap junction-mediated coupling between neurons. Our findings therefore provide quantitative support for using ketamineanesthesia to improve the viability of neurons in brain slices. References Aston-Jones G, Rajkowski J, Cohen J. Role of locus coeruleus in attention and behavioral flexibility. Biol Psychiatry 1999;46:1309–20.
Bergman SA. Ketamine: review of its pharmacology and its use in pediatric anesthesia. Anesth Prog 1999;46:10–20. Berman RM, Cappiello A, Anand A, Oren DA, Heninger GR, Charney DS, et al. Antidepressant effects of ketamine in depressed patients. Biol Psychiatry 2000;47:351–4. Collins VJ, Gorospe CA, Rovenstine EA. Intravenous nonbarbiturate, nonnarcotic analgesics: preliminary studies. 1. Cyclohexylamines. Anesth Analg 1960;39:302–6. Graham BA, Schofield PR, Sah P, Margrie TW, Callister RJ. Distinct physiological mechanisms underlie altered glycinergic synaptic transmission in the murine mutants spastic, spasmodic, and oscillator. J Neurosci 2006;26:4880–90. Himmelseher S, Durieux ME. Revising a dogma: ketamine for patients with neurological injury? Anesth Analg 2005;101:524–34, table of contents. Leong D, Puil E, Schwarz D. Ketamine blocks non-N-methyl-D-aspartate receptor channels attenuating glutamatergic transmission in the auditory cortex. Acta Otolaryngol 2004;124:454–8. Mathisen LC, Skjelbred P, Skoglund LA, Oye I. Effect of ketamine, an NMDA receptor inhibitor, in acute and chronic orofacial pain. Pain 1995;61:215–20. Morgan CJ, Curran HV. Acute and chronic effects of ketamine upon human memory: a review. Psychopharmacology (Berl) 2006;188:408–24. Newcomer JW, Farber NB, Jevtovic-Todorovic V, Selke G, Melson AK, Hershey T, et al. Ketamine-induced NMDA receptor hypofunction as a model of memory impairment and psychosis. Neuropsychopharmacology 1999;20: 106–18. Reeker W, Werner C, Mollenberg O, Mielke L, Kochs E. High-dose S(+)-ketamine improves neurological outcome following incomplete cerebral ischemia in rats. Can J Anaesth 2000;47:572–8. Schnoebel R, Wolff M, Peters SC, Brau ME, Scholz A, Hempelmann G, et al. Ketamine impairs excitability in superficial dorsal horn neurones by blocking sodium and voltage-gated potassium currents. Br J Pharmacol 2005;146:826–33. Stefanovic A, Brandner B, Klaassen E, Cregg R, Nagaratnam M, Bromley LM, et al. Acute and chronic effects of ketamine on semantic priming: modeling schizophrenia? J Clin Psychopharmacol 2009;29:124–33. Ward DG, Gunn CG. Locus coeruleus complex: elicitation of a pressor response and a brain stem region necessary for its occurrence. Brain Res 1976;107: 401–6.