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Sodium pentobarbital abolishes bursting spontaneous activity of dorsal cochlear nucleus in rat brain slices
Hearing Research 149 (2000) 216^222 www.elsevier.com/locate/heares
Sodium pentobarbital abolishes bursting spontaneous activity of dorsal cochlear nucleus in rat brain slices Kejian Chen *, Donald A. Godfrey Department of Otolaryngology, Medical College of Ohio, 3065 Arlington Ave., Toledo, OH 43614, USA Received 28 February 2000; accepted 10 August 2000
Abstract There is evidence that pentobarbital, a commonly used anesthetic, can affect neuronal activity, but its effects on particular neurons of the dorsal cochlear nucleus (DCN) are not well known. Bursting (complex spiking) spontaneous activity has been observed in the DCN in brain slice preparations and in recordings from unanesthetized decerebrate animals, but seldom in experiments with anesthetized animals. This study investigated the effects of pentobarbital on spontaneous activity in the DCN in brain slices. Most extracellularly recorded bursting neurons decreased firing rates and reversibly changed their firing to simple spiking with irregular intervals during pentobarbital. Some reversibly stopped firing after the change to an irregular pattern. Most neurons with regular spontaneous activity (simple spiking) showed decreased firing rates and more irregular intervals during pentobarbital. The results also suggest some involvement of Q-aminobutyric acid type A receptors in the pentobarbital effects. ß 2000 Elsevier Science B.V. All rights reserved. Key words: Single unit recording; Anesthetic e¡ect; Q-Aminobutyric acid type A receptor; Q-Aminobutyric acid type B receptor; Regular ¢ring; Irregular ¢ring; Auditory
1. Introduction Sodium pentobarbital has long been used as an anesthetic for in vivo animal experiments. There is evidence that it can a¡ect neuronal activity, and that such e¡ects may involve Q-aminobutyric acid (GABA) receptors (Mathers and Barker, 1980; Study and Barker, 1981 ; West, 1998). Other studies, however, have suggested that pentobarbital e¡ects may involve mechanisms besides GABAA receptors (Antkowiak, 1999), such as depression of voltage-gated sodium channels (Rehberg et al., 1999). Several lines of evidence suggest that pentobarbital reduces inhibitory responses of dorsal cochlear nucleus (DCN) neurons to sound, although its e¡ects on ventral cochlear nucleus neurons were not obvious (Evans and Nelson, 1973; Young and Brownell, 1976). To date, little is known about its e¡ects on particular neuron types of the DCN.
* Corresponding author. Tel.: +1 (419) 383-6526; Fax: +1 (419) 383-3096; E-mail: [email protected]
Using extracellular recording to minimize electrode e¡ects on neuronal ¢ring, bursting spontaneous activity has been observed in the DCN in brain slice preparations of rats (Waller and Godfrey, 1994 ; Chen et al., 1994), mice (Chen et al., 1999b), and hamsters (unpublished observations), as well as in recordings from unanesthetized decerebrate cats (Parham and Kim, 1995), but seldom in experiments with anesthetized animals (Pfei¡er and Kiang, 1965 ; Godfrey et al., 1975). Also, only a few neurons with highly regular spontaneous activity were recorded in an in vivo study (Godfrey et al., 1975). This might be related to e¡ects of anesthetics on the spontaneous activity of DCN neurons. Using intracellular recording, bursting (complex spiking) patterns have been associated with cartwheel cells of the DCN, and regular (simple spiking) patterns have been associated with fusiform cells (Zhang and Oertel, 1993; Manis, 1990 ; Manis et al., 1994). Irregular simple spiking patterns have been recorded extracellularly, but have not been associated with a particular type of neuron (Waller and Godfrey, 1994). The aim of the present study was to determine the
0378-5955 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 0 0 ) 0 0 1 8 8 - X
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e¡ects of sodium pentobarbital on spontaneous ¢ring patterns of DCN neurons, especially bursting neurons. We also investigated possible involvement of GABA receptors in these e¡ects. Preliminary results of this study were previously presented in abstract form (Chen and Godfrey, 1999). 2. Methods The methods were similar to those of our previous studies (Chen et al., 1994, 1999a). Each Sprague^Dawley rat (from Harlan Sprague Dawley, Inc.), usually of 200^400 g body weight and of either sex, was anesthetized with sodium pentobarbital (52 mg/kg, i.p.), decapitated, and the head immersed in ice-cold oxygenated arti¢cial cerebrospinal £uid (ACSF). Each brain was quickly removed and bisected along the midsagittal plane. The halves were trimmed and sliced transversely at 450 Wm thickness with a McIlwain tissue chopper.
217
Slices containing each cochlear nucleus were immediately transferred onto a nylon mesh in an interface chamber (Haas et al., 1979) perfused at 1.2 ml/min with ACSF consisting of (in mM) KH2 PO4 1.25, KCl 3.0, MgSO4 1.5, CaCl2 2.5, NaCl 126, NaHCO3 26, glucose 10, pH 7.4, maintained at 32^34³C. After a recovery period (approximately 1.5 h), slices were explored extracellularly with glass micropipettes (1.5^2.5 Wm tip diameter, 5^10 M6) ¢lled with 1 M NaCl. Extracellular discharges of spontaneously active neurons in the DCN were recorded on magnetic tape and subsequently replayed for computer analysis of ¢ring patterns. Drugs were applied through the ACSF. The volume of the £ow path from the stopcock to the slice chamber was 1.15 ml, resulting in an uncorrected time delay for drug applications of approximately 1 min between the change of a solution and 50% rise of concentration in the chamber (Godfrey and Waller, 1992). Drugs used were sodium pentobarbital (20^2000 WM) from Abbott Laboratories, bicuculline methochloride
Table 1 E¡ects of sodium pentobarbital on DCN neurons Unit
Spontaneous activity pattern
8010701
bursting
8010901 8011502
bursting bursting
8012701 8012702 8062401 8062901 8070201 9011501 9012501 9020101 9030402 9030501 9031701 9031901 9032301 9032302 9040901 9041201 9041301 9041401 8010902 8011501 8062501 8062601 9010801 9030401 8010702
bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting regular regular regular regular regular regular irregular
a b
Pentobarbital concentration (WM)a 20 200 2000 1000 500 50 100 100 100 200 200 200 200 200 200 300 200 200 200 200 200 200 200 200 200 1000 500 200 200 200 300 2000
Multiple doses are listed in the order of presentation. `irregular/stopped' means changed to irregular pattern, then stopped ¢ring.
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E¡ects Firing rate change (spikes/s)
Firing pattern
3.7C4.1 3.1C2.6 3.3C0 6.0C1.1 4.4C0 4.0C4.5 5.3C2.3 7.2C6.1 2.5C1.7 4.8C5.6 8.0C0 1.6C0.6 2.0C0.6 3.3C0 1.6C0 3.5C0 2.7C0.7 2.2C0 3.8C1.8 2.9C9.7 5.4C4.4 1.3C0 4.2C1.3 2.2C0.5 5.1C2.9 12.3C2.7 14.6C0 21.9C6.7 31.2C23.6 16.6C11.3 6.4C2.7 12.6C5.8
bursting irregular irregular/stoppedb irregular irregular/stopped bursting irregular irregular irregular irregular irregular/stopped irregular irregular irregular/stopped stopped irregular/stopped irregular irregular/stopped irregular irregular irregular irregular/stopped irregular irregular irregular irregular irregular/stopped irregular regular regular irregular irregular
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K. Chen, D.A. Godfrey / Hearing Research 149 (2000) 216^222
3. Results 3.1. E¡ects of sodium pentobarbital on DCN neurons Twenty-eight spontaneously active neurons (21 bursting, six regular and one irregular) from 22 adult rats were tested with sodium pentobarbital (20^2000 WM). Table 1 summarizes the e¡ects of sodium pentobarbital on these neurons. During 100^300 WM pentobarbital, intra-burst intervals of bursting neurons became longer over the initial 5^10 min. The number of spikes in each burst also decreased during pentobarbital, and the pattern gradually became simple spiking or no ¢ring. Of the 21 bursting neurons tested, the ¢ring patterns of 20 changed to irregular, i.e. simple spiking with irregular intervals (Fig. 1A). The irregular ¢ring lasted throughout the 20^30 min pentobarbital application, except for seven of these neurons, which stopped ¢ring after the change to an irregular pattern. One neuron stopped ¢ring without an obvious pattern change. All neurons resumed bursting ¢ring after pentobarbital was washed out. Most of the neurons (19/21) showed decreased ¢ring rates during 100 WM or higher pentobarbital concentration. Only two neurons showed increased ¢ring
Fig. 1. E¡ects of sodium pentobarbital on ¢ring patterns of DCN neurons. Each sweep represents 200 ms; time scale at the bottom of each section: 20 ms/division. Spike polarity is inverted so that negative is up. A: The ¢ring pattern of a bursting neuron (complex spiking) changed to irregular (simple spiking) during 200 WM sodium pentobarbital (SP). B: The ¢ring rate of a regular neuron (simple spiking) decreased during and after 200 WM SP. Spikes showed a gradual decrease in amplitude during the 60 min recording. The largest spikes (negative peak to positive peak) in both A and B represent approximately 150 WV.
(5^20 WM) from Sigma Chemical Co., and baclofen hydrochloride (1^3 WM), muscimol hydrobromide (3^ 20 WM), and saclofen (100 WM) from Research Biochemicals International. The care and use of animals reported in this study were approved by the National Institutes of Health (NIDCD Grant DC00172 ^ `Microchemistry of the Cochlear Nucleus') and by the Medical College of Ohio Institutional Animal Care and Use Committee.
Fig. 2. Sodium pentobarbital (SP) changed the ¢ring pattern of a bursting neuron in a dose-dependent manner. Each sweep represents 200 ms; time scale at the bottom: 20 ms/division. The largest spikes (negative [up] peak to positive peak) represent approximately 150 WV.
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Fig. 3. E¡ects of a GABAA receptor antagonist on responses of bursting neurons to sodium pentobarbital. A: 5 WM bicuculline (BIC) largely blocked responses of a bursting neuron to sodium pentobarbital (SP). B: 10 WM bicuculline (BIC) altered responses of a bursting neuron to sodium pentobarbital (SP). Each sweep represents 200 ms in A, 500 ms in B. Time scale at the bottom of A: 20 ms/division, B: 50 ms/division. The largest spikes (negative [up] peak to positive peak) represent approximately 150 WV in A and 500 WV in B.
rates during 200 WM pentobarbital. Simple spiking neurons with regular or irregular patterns were also affected by pentobarbital. All regular neurons showed decreased ¢ring rates; an example is shown in Fig. 1B. The ¢ring patterns of four regular neurons became less regular, but those of two others changed little. The ¢ring of the one tested irregular neuron remained irregular, but with a slightly higher ¢ring rate during and lower ¢ring rate after pentobarbital (data not shown). In the two cases tested, pentobarbital changed ¢ring patterns of bursting neurons in a dose-dependent manner. One changed its pattern during 200 WM and stopped ¢ring during 2 mM pentobarbital (Fig. 2). 3.2. E¡ects of GABA receptor antagonists on responses to pentobarbital In order to test for involvement of GABA receptors in the e¡ects of pentobarbital on DCN neurons, antag-
onists for either GABAA or GABAB receptors were used along with pentobarbital. Bicuculline, an antagonist for GABAA receptors, at 5^20 WM altered the responses to 200 WM pentobarbital of all six bursting neurons tested. Fig. 3 shows the e¡ects of 5 WM and 10 WM bicuculline on responses of two bursting neurons to 200 WM pentobarbital. Neurons usually responded to bicuculline alone with slightly increased ¢ring rates, but they responded to bicuculline plus pentobarbital with more signi¢cantly increased ¢ring rates and long-burst patterns (Fig. 3B), during which the inter-burst intervals were shorter than those during pentobarbital alone. Saclofen, an antagonist for GABAB receptors, showed little e¡ect on neuronal responses to pentobarbital (Fig. 4). Two bursting neurons remained bursting, but with decreased ¢ring rates (24^40% of control rates) during 100 WM saclofen alone. During saclofen plus pentobarbital, the ¢ring patterns of both bursting neu-
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4. Discussion In this study, we found that pentobarbital at 100^300 WM a¡ected the ¢ring pattern of every bursting neuron tested. For most bursting neurons during pentobarbital, ¢ring rate decreased, intra-burst interval increased, number of spikes per burst decreased, and the pattern gradually changed to irregular (simple spiking) or no ¢ring. Although only a few regular neurons were tested, the ¢ring rates of all decreased, and the patterns of most became less regular during pentobarbital. Evidence suggests that these ¢ring pattern changes occur at pentobarbital concentrations comparable to
Fig. 4. The GABAB receptor antagonist, saclofen (100 WM), did not change the response of a bursting neuron to sodium pentobarbital (SP). Each sweep represents 200 ms; time scale at the bottom: 20 ms/division. The largest spikes (negative [up] peak to positive peak) represent approximately 150 WV.
rons changed to irregular and then stopped ¢ring as with pentobarbital alone. 3.3. E¡ects of GABA receptor agonists on ¢ring patterns of bursting neurons During application of 1 WM baclofen, an agonist for GABAB receptors, the ¢ring of a bursting neuron remained largely bursting, with a lower ¢ring rate. During 3 WM baclofen, three bursting neurons stopped ¢ring without signi¢cant pattern change (Fig. 5). During 3 WM muscimol, an agonist for GABAA receptors, a bursting neuron did not change either its rate or pattern, but during 10 WM muscimol, its ¢ring rate decreased and its intra-burst intervals became much longer than control, resembling those in some irregular simple spiking neurons (Fig. 5). Two bursting neurons stopped ¢ring during 20 WM muscimol.
Fig. 5. E¡ects of GABA receptor agonists on the ¢ring pattern of a bursting neuron. The spontaneous ¢ring stopped during 3 WM baclofen, was una¡ected during 3 WM muscimol, and changed to an irregular (simple spiking) pattern during 10 WM muscimol. Each sweep represents 200 ms; time scale at the bottom: 20 ms/division. The largest spikes (negative [up] peak to positive peak) represent approximately 450 WV.
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those in the brain during anesthesia. Studies that have measured concentrations of pentobarbital in the brain and cerebrospinal £uid of anesthetized animals report values from 17.8 to 73.1 mg/kg, corresponding to approximately 72^294 WM (Hatanaka et al., 1988 ; Bolander et al., 1984). These studies have varied in route of drug administration, species of animal, and stages of anesthesia at which samples were collected. A study by Bolander et al. (1984), using an EEG threshold method, evaluated the potency and pharmacokinetic properties of pentobarbital as well as several other barbiturates. The concentration of pentobarbital in the brain was reported as about 294 WM during the `silent second', a well speci¢ed EEG criterion which was considered a reliable measure of anesthesia. This pentobarbital concentration is comparable to those which elicited ¢ring pattern changes of bursting neurons in our study. Thus, the absence of bursting activity in the previous in vivo studies using pentobarbital or other barbiturate anesthesia may at least partly have resulted from e¡ects of the anesthetics. Although both GABAA and GABAB receptors have been reported in the DCN (Juiz et al., 1994), the e¡ects of pentobarbital on bursting neurons were a¡ected by application of bicuculline, an antagonist for GABAA receptors, but not by saclofen, an antagonist for GABAB receptors. This ¢nding may suggest that the e¡ects of pentobarbital were at least partly through GABAA receptors on cartwheel cells. Although muscimol has been reported to increase ¢ring of cartwheel cells in mouse brain slices (Golding and Oertel, 1996), only decreased ¢ring of bursting neurons was observed during 10^20 WM muscimol in our study. Most bursting neurons showed decreased ¢ring during pentobarbital and increased ¢ring during bicuculline. Instead of showing less e¡ect of pentobarbital in the presence of bicuculline, the ¢ring rates of all bursting neurons increased greatly, more than with bicuculline alone. This suggests, in agreement with previous studies (Antkowiak, 1999 ; Rehberg et al., 1999), that the e¡ects of pentobarbital cannot be explained simply as agonist e¡ects on GABAA receptors. Rather, the results suggest that pentobarbital and bicuculline might interact in a more complicated way, such as through an allosteric e¡ect on GABAA receptors (Liljequist and Tabako¡, 1986). A question arises as to whether the loss of bursting activity results from a direct e¡ect of pentobarbital on cartwheel cells or whether the action is indirect through an e¡ect on granule cells. We have previously found that bursting activity can often be abolished by blocking the granule cell (parallel ¢ber) input to cartwheel cells with drugs such as 6,7-dinitroquinoxaline-2,3-dione (DNQX), an antagonist for non-N-methyl-D-aspartate glutamate receptors (Waller et al., 1996). However, during 5 WM DNQX, as the ¢ring rates of bursting
221
neurons declined, changes of ¢ring patterns were not observed ; the patterns remained bursting (Chen et al., 1999a, and unpublished results). Further, stimulation of parallel ¢ber populations with single electric shocks often leads to burst ¢ring in bursting neurons, suggesting that bursting activity results from intrinsic membrane properties of cartwheel cells rather than properties of granule cells (Waller et al., 1996). Thus, the available evidence suggests that pentobarbital a¡ects bursting activity mainly by a direct action on cartwheel cells. Our ¢nding of often decreased and less regular ¢ring of regular neurons during pentobarbital suggests that the rare occurrence of highly regular spontaneous activity in vivo might also have some basis in anesthetic e¡ects. Assuming that bursting activity represents cartwheel cells in the DCN, which are inhibitory interneurons (Berrebi and Mugnaini, 1991; Mugnaini et al., 1987), the decrease and sometimes total loss of their spontaneous activity during pentobarbital may also contribute to less inhibitory responses to sound with in vivo recordings using barbiturate anesthesia (Evans and Nelson, 1973; Young and Brownell, 1976). In conclusion, anesthetic drug e¡ects on DCN neuronal spontaneous activity may have been underestimated in past studies. Among other di¡erences, anesthetic effects have to be considered when comparing in vivo results with in vitro results. The e¡ects of other commonly used anesthetics, such as ketamine and xylazine, also need to be studied. Acknowledgements The authors are grateful to Dr. Hardress J. Waller for computer programming. Supported by NIH Grant DC00172.
References Antkowiak, B., 1999. Di¡erent actions of general anesthetics on the ¢ring patterns of neocortical neurons mediated by the GABAA receptor. Anesthesiology 91, 500^511. Berrebi, A.S., Mugnaini, E., 1991. Distribution and targets of the cartwheel cell axon in the dorsal cochlear nucleus of the guinea pig. Anat. Embryol. 183, 427^454. Bolander, H.G., Wahlstro«m, G., Norberg, L., 1984. Reevaluation of potency and pharmacokinetic properties of some lipid-soluble barbiturates with an EEG-threshold method. Acta Pharmacol. Toxicol. 54, 33^40. Chen, K., Godfrey, D.A., 1999. Sodium pentobarbital abolishes bursting spontaneous activity of dorsal cochlear nucleus in rat brain slices. Abstr. Soc. Neurosci. 25, 394. Chen, K., Waller, H.J., Godfrey, D.A., 1994. Cholinergic modulation of spontaneous activity in rat dorsal cochlear nucleus. Hear. Res. 77, 168^176.
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Chen, K., Waller, H.J., Godfrey, T.G., Godfrey, D.A., 1999a. Glutamatergic transmission of neuronal responses to carbachol in rat dorsal cochlear nucleus slices. Neuroscience 90, 1043^1049. Chen, K., Sprunger, L.K., Meisler, M.H., Waller, H.J., Godfrey, D.A., 1999b. Reduced spontaneous activity in the dorsal cochlear nucleus of Scn8a mutant mice. Brain Res. 847, 85^89. Evans, E.F., Nelson, P.G., 1973. The responses of single neurons in the cochlear nucleus of the cat as a function of their location and the anesthetic state. Exp. Brain Res. 17, 402^427. Godfrey, D.A., Waller, H.J., 1992. Sampling £uid from slice chambers by microsiphoning. J. Neurosci. Methods 41, 167^173. Godfrey, D.A., Kiang, N.Y., Norris, B.E., 1975. Single unit activity in the dorsal cochlear nucleus of the cat. J. Comp. Neurol. 162, 269^ 284. Golding, N.L., Oertel, D., 1996. Context-dependent synaptic action of glycinergic and GABAergic inputs in the dorsal cochlear nucleus. J. Neurosci. 16, 2208^2219. Haas, H.L., Schaerer, B., Vosmansky, M., 1979. A simple perfusion chamber for the study of nervous tissue slices in vitro. J. Neurosci. Methods 1, 323^325. Hatanaka, T., Sato, S., Endoh, M., Katayama, K., Kakemi, M., Koizumi, T., 1988. E¡ect of chlorpromazine on the pharmacokinetics and pharmacodynamics of pentobarbital in rats. J. Pharmacobiodyn. 11, 18^30. Juiz, J.M., Albin, R.L., Helfert, R.H., Altschuler, R.A., 1994. Distribution of GABAA and GABAB binding sites in the cochlear nucleus of the guinea pig. Brain Res. 639, 193^201. Liljequist, S., Tabako¡, B., 1986. Bicuculline-pentobarbital interactions on [35 S]TBPS binding in various brain areas. Life Sci. 39, 851^855. Manis, P.B., 1990. Membrane properties and discharge characteristics of guinea pig dorsal cochlear nucleus neurons studied in vitro. J. Neurosci. 10, 2338^2351. Manis, P.B., Spirou, G.A., Wright, D.D., Paydar, S., Ryugo, D.K., 1994. Physiology and morphology of complex spiking neurons in the guinea pig dorsal cochlear nucleus. J. Comp. Neurol. 348, 261^ 276.
Mathers, D.A., Barker, J.L., 1980. (3)Pentobarbital opens ion channels of long duration in cultured mouse spinal neurons. Science 209, 507^509. Mugnaini, E., Berrebi, A.S., Dahl, A.L., Morgan, J.I., 1987. The polypeptide PEP-19 is a marker for Purkinje neurons in cerebellar cortex and cartwheel neurons in the dorsal cochlear nucleus. Arch. Ital. Biol. 126, 41^67. Parham, K., Kim, D.O., 1995. Spontaneous and sound-evoked discharge characteristics of complex-spiking neurons in the dorsal cochlear nucleus of the unanesthetized decerebrate cat. J. Neurophysiol. 73, 550^561. Pfei¡er, R.R., Kiang, N.Y.-S., 1965. Spike discharge patterns of spontaneous and continuously stimulated activity in the cochlear nucleus of anesthetized cats. Biophys. J. 5, 301^316. Rehberg, B., Wittmann, M., Urban, B.W., 1999. Sodium channels (from rat, mouse, and man) in neuroblastoma cells and di¡erent expression systems have similar sensitivities to pentobarbital. Neurosci. Lett. 264, 81^84. Study, R.E., Barker, J.L., 1981. Diazepam and (3)-pentobarbital: Fluctuation analysis reveals di¡erent mechanisms for potentiation of Q-aminobutyric acid responses in cultured central neurons. Proc. Natl. Acad. Sci. USA 78, 7180^7184. Waller, H.J., Godfrey, D.A., 1994. Functional characteristics of spontaneously active neurons in rat dorsal cochlear nucleus in vitro. J. Neurophysiol. 71, 467^478. Waller, H.J., Godfrey, D.A., Chen, K., 1996. E¡ects of parallel ¢ber stimulation on neurons of rat dorsal cochlear nucleus. Hear. Res. 98, 169^179. West, M.O., 1998. Anesthetics eliminate somatosensory-evoked discharges of neurons in the somatotopically organized sensorimotor striatum of the rat. J. Neurosci. 18, 9055^9068. Young, E.D., Brownell, W.E., 1976. Responses to tones and noise of single cells in dorsal cochlear nucleus of unanesthetized cats. J. Neurophysiol. 39, 282^300. Zhang, S., Oertel, D., 1993. Cartwheel and super¢cial stellate cells of the dorsal cochlear nucleus of mice: intracellular recordings in slices. J. Neurophysiol. 69, 1384^1397.
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Sodium pentobarbital abolishes bursting spontaneous activity of dorsal cochlear nucleus in rat brain slices Kejian Chen *, Donald A. Godfrey Department of Otolaryngology, Medical College of Ohio, 3065 Arlington Ave., Toledo, OH 43614, USA Received 28 February 2000; accepted 10 August 2000
Abstract There is evidence that pentobarbital, a commonly used anesthetic, can affect neuronal activity, but its effects on particular neurons of the dorsal cochlear nucleus (DCN) are not well known. Bursting (complex spiking) spontaneous activity has been observed in the DCN in brain slice preparations and in recordings from unanesthetized decerebrate animals, but seldom in experiments with anesthetized animals. This study investigated the effects of pentobarbital on spontaneous activity in the DCN in brain slices. Most extracellularly recorded bursting neurons decreased firing rates and reversibly changed their firing to simple spiking with irregular intervals during pentobarbital. Some reversibly stopped firing after the change to an irregular pattern. Most neurons with regular spontaneous activity (simple spiking) showed decreased firing rates and more irregular intervals during pentobarbital. The results also suggest some involvement of Q-aminobutyric acid type A receptors in the pentobarbital effects. ß 2000 Elsevier Science B.V. All rights reserved. Key words: Single unit recording; Anesthetic e¡ect; Q-Aminobutyric acid type A receptor; Q-Aminobutyric acid type B receptor; Regular ¢ring; Irregular ¢ring; Auditory
1. Introduction Sodium pentobarbital has long been used as an anesthetic for in vivo animal experiments. There is evidence that it can a¡ect neuronal activity, and that such e¡ects may involve Q-aminobutyric acid (GABA) receptors (Mathers and Barker, 1980; Study and Barker, 1981 ; West, 1998). Other studies, however, have suggested that pentobarbital e¡ects may involve mechanisms besides GABAA receptors (Antkowiak, 1999), such as depression of voltage-gated sodium channels (Rehberg et al., 1999). Several lines of evidence suggest that pentobarbital reduces inhibitory responses of dorsal cochlear nucleus (DCN) neurons to sound, although its e¡ects on ventral cochlear nucleus neurons were not obvious (Evans and Nelson, 1973; Young and Brownell, 1976). To date, little is known about its e¡ects on particular neuron types of the DCN.
* Corresponding author. Tel.: +1 (419) 383-6526; Fax: +1 (419) 383-3096; E-mail: [email protected]
Using extracellular recording to minimize electrode e¡ects on neuronal ¢ring, bursting spontaneous activity has been observed in the DCN in brain slice preparations of rats (Waller and Godfrey, 1994 ; Chen et al., 1994), mice (Chen et al., 1999b), and hamsters (unpublished observations), as well as in recordings from unanesthetized decerebrate cats (Parham and Kim, 1995), but seldom in experiments with anesthetized animals (Pfei¡er and Kiang, 1965 ; Godfrey et al., 1975). Also, only a few neurons with highly regular spontaneous activity were recorded in an in vivo study (Godfrey et al., 1975). This might be related to e¡ects of anesthetics on the spontaneous activity of DCN neurons. Using intracellular recording, bursting (complex spiking) patterns have been associated with cartwheel cells of the DCN, and regular (simple spiking) patterns have been associated with fusiform cells (Zhang and Oertel, 1993; Manis, 1990 ; Manis et al., 1994). Irregular simple spiking patterns have been recorded extracellularly, but have not been associated with a particular type of neuron (Waller and Godfrey, 1994). The aim of the present study was to determine the
0378-5955 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 0 0 ) 0 0 1 8 8 - X
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K. Chen, D.A. Godfrey / Hearing Research 149 (2000) 216^222
e¡ects of sodium pentobarbital on spontaneous ¢ring patterns of DCN neurons, especially bursting neurons. We also investigated possible involvement of GABA receptors in these e¡ects. Preliminary results of this study were previously presented in abstract form (Chen and Godfrey, 1999). 2. Methods The methods were similar to those of our previous studies (Chen et al., 1994, 1999a). Each Sprague^Dawley rat (from Harlan Sprague Dawley, Inc.), usually of 200^400 g body weight and of either sex, was anesthetized with sodium pentobarbital (52 mg/kg, i.p.), decapitated, and the head immersed in ice-cold oxygenated arti¢cial cerebrospinal £uid (ACSF). Each brain was quickly removed and bisected along the midsagittal plane. The halves were trimmed and sliced transversely at 450 Wm thickness with a McIlwain tissue chopper.
217
Slices containing each cochlear nucleus were immediately transferred onto a nylon mesh in an interface chamber (Haas et al., 1979) perfused at 1.2 ml/min with ACSF consisting of (in mM) KH2 PO4 1.25, KCl 3.0, MgSO4 1.5, CaCl2 2.5, NaCl 126, NaHCO3 26, glucose 10, pH 7.4, maintained at 32^34³C. After a recovery period (approximately 1.5 h), slices were explored extracellularly with glass micropipettes (1.5^2.5 Wm tip diameter, 5^10 M6) ¢lled with 1 M NaCl. Extracellular discharges of spontaneously active neurons in the DCN were recorded on magnetic tape and subsequently replayed for computer analysis of ¢ring patterns. Drugs were applied through the ACSF. The volume of the £ow path from the stopcock to the slice chamber was 1.15 ml, resulting in an uncorrected time delay for drug applications of approximately 1 min between the change of a solution and 50% rise of concentration in the chamber (Godfrey and Waller, 1992). Drugs used were sodium pentobarbital (20^2000 WM) from Abbott Laboratories, bicuculline methochloride
Table 1 E¡ects of sodium pentobarbital on DCN neurons Unit
Spontaneous activity pattern
8010701
bursting
8010901 8011502
bursting bursting
8012701 8012702 8062401 8062901 8070201 9011501 9012501 9020101 9030402 9030501 9031701 9031901 9032301 9032302 9040901 9041201 9041301 9041401 8010902 8011501 8062501 8062601 9010801 9030401 8010702
bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting bursting regular regular regular regular regular regular irregular
a b
Pentobarbital concentration (WM)a 20 200 2000 1000 500 50 100 100 100 200 200 200 200 200 200 300 200 200 200 200 200 200 200 200 200 1000 500 200 200 200 300 2000
Multiple doses are listed in the order of presentation. `irregular/stopped' means changed to irregular pattern, then stopped ¢ring.
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E¡ects Firing rate change (spikes/s)
Firing pattern
3.7C4.1 3.1C2.6 3.3C0 6.0C1.1 4.4C0 4.0C4.5 5.3C2.3 7.2C6.1 2.5C1.7 4.8C5.6 8.0C0 1.6C0.6 2.0C0.6 3.3C0 1.6C0 3.5C0 2.7C0.7 2.2C0 3.8C1.8 2.9C9.7 5.4C4.4 1.3C0 4.2C1.3 2.2C0.5 5.1C2.9 12.3C2.7 14.6C0 21.9C6.7 31.2C23.6 16.6C11.3 6.4C2.7 12.6C5.8
bursting irregular irregular/stoppedb irregular irregular/stopped bursting irregular irregular irregular irregular irregular/stopped irregular irregular irregular/stopped stopped irregular/stopped irregular irregular/stopped irregular irregular irregular irregular/stopped irregular irregular irregular irregular irregular/stopped irregular regular regular irregular irregular
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3. Results 3.1. E¡ects of sodium pentobarbital on DCN neurons Twenty-eight spontaneously active neurons (21 bursting, six regular and one irregular) from 22 adult rats were tested with sodium pentobarbital (20^2000 WM). Table 1 summarizes the e¡ects of sodium pentobarbital on these neurons. During 100^300 WM pentobarbital, intra-burst intervals of bursting neurons became longer over the initial 5^10 min. The number of spikes in each burst also decreased during pentobarbital, and the pattern gradually became simple spiking or no ¢ring. Of the 21 bursting neurons tested, the ¢ring patterns of 20 changed to irregular, i.e. simple spiking with irregular intervals (Fig. 1A). The irregular ¢ring lasted throughout the 20^30 min pentobarbital application, except for seven of these neurons, which stopped ¢ring after the change to an irregular pattern. One neuron stopped ¢ring without an obvious pattern change. All neurons resumed bursting ¢ring after pentobarbital was washed out. Most of the neurons (19/21) showed decreased ¢ring rates during 100 WM or higher pentobarbital concentration. Only two neurons showed increased ¢ring
Fig. 1. E¡ects of sodium pentobarbital on ¢ring patterns of DCN neurons. Each sweep represents 200 ms; time scale at the bottom of each section: 20 ms/division. Spike polarity is inverted so that negative is up. A: The ¢ring pattern of a bursting neuron (complex spiking) changed to irregular (simple spiking) during 200 WM sodium pentobarbital (SP). B: The ¢ring rate of a regular neuron (simple spiking) decreased during and after 200 WM SP. Spikes showed a gradual decrease in amplitude during the 60 min recording. The largest spikes (negative peak to positive peak) in both A and B represent approximately 150 WV.
(5^20 WM) from Sigma Chemical Co., and baclofen hydrochloride (1^3 WM), muscimol hydrobromide (3^ 20 WM), and saclofen (100 WM) from Research Biochemicals International. The care and use of animals reported in this study were approved by the National Institutes of Health (NIDCD Grant DC00172 ^ `Microchemistry of the Cochlear Nucleus') and by the Medical College of Ohio Institutional Animal Care and Use Committee.
Fig. 2. Sodium pentobarbital (SP) changed the ¢ring pattern of a bursting neuron in a dose-dependent manner. Each sweep represents 200 ms; time scale at the bottom: 20 ms/division. The largest spikes (negative [up] peak to positive peak) represent approximately 150 WV.
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Fig. 3. E¡ects of a GABAA receptor antagonist on responses of bursting neurons to sodium pentobarbital. A: 5 WM bicuculline (BIC) largely blocked responses of a bursting neuron to sodium pentobarbital (SP). B: 10 WM bicuculline (BIC) altered responses of a bursting neuron to sodium pentobarbital (SP). Each sweep represents 200 ms in A, 500 ms in B. Time scale at the bottom of A: 20 ms/division, B: 50 ms/division. The largest spikes (negative [up] peak to positive peak) represent approximately 150 WV in A and 500 WV in B.
rates during 200 WM pentobarbital. Simple spiking neurons with regular or irregular patterns were also affected by pentobarbital. All regular neurons showed decreased ¢ring rates; an example is shown in Fig. 1B. The ¢ring patterns of four regular neurons became less regular, but those of two others changed little. The ¢ring of the one tested irregular neuron remained irregular, but with a slightly higher ¢ring rate during and lower ¢ring rate after pentobarbital (data not shown). In the two cases tested, pentobarbital changed ¢ring patterns of bursting neurons in a dose-dependent manner. One changed its pattern during 200 WM and stopped ¢ring during 2 mM pentobarbital (Fig. 2). 3.2. E¡ects of GABA receptor antagonists on responses to pentobarbital In order to test for involvement of GABA receptors in the e¡ects of pentobarbital on DCN neurons, antag-
onists for either GABAA or GABAB receptors were used along with pentobarbital. Bicuculline, an antagonist for GABAA receptors, at 5^20 WM altered the responses to 200 WM pentobarbital of all six bursting neurons tested. Fig. 3 shows the e¡ects of 5 WM and 10 WM bicuculline on responses of two bursting neurons to 200 WM pentobarbital. Neurons usually responded to bicuculline alone with slightly increased ¢ring rates, but they responded to bicuculline plus pentobarbital with more signi¢cantly increased ¢ring rates and long-burst patterns (Fig. 3B), during which the inter-burst intervals were shorter than those during pentobarbital alone. Saclofen, an antagonist for GABAB receptors, showed little e¡ect on neuronal responses to pentobarbital (Fig. 4). Two bursting neurons remained bursting, but with decreased ¢ring rates (24^40% of control rates) during 100 WM saclofen alone. During saclofen plus pentobarbital, the ¢ring patterns of both bursting neu-
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4. Discussion In this study, we found that pentobarbital at 100^300 WM a¡ected the ¢ring pattern of every bursting neuron tested. For most bursting neurons during pentobarbital, ¢ring rate decreased, intra-burst interval increased, number of spikes per burst decreased, and the pattern gradually changed to irregular (simple spiking) or no ¢ring. Although only a few regular neurons were tested, the ¢ring rates of all decreased, and the patterns of most became less regular during pentobarbital. Evidence suggests that these ¢ring pattern changes occur at pentobarbital concentrations comparable to
Fig. 4. The GABAB receptor antagonist, saclofen (100 WM), did not change the response of a bursting neuron to sodium pentobarbital (SP). Each sweep represents 200 ms; time scale at the bottom: 20 ms/division. The largest spikes (negative [up] peak to positive peak) represent approximately 150 WV.
rons changed to irregular and then stopped ¢ring as with pentobarbital alone. 3.3. E¡ects of GABA receptor agonists on ¢ring patterns of bursting neurons During application of 1 WM baclofen, an agonist for GABAB receptors, the ¢ring of a bursting neuron remained largely bursting, with a lower ¢ring rate. During 3 WM baclofen, three bursting neurons stopped ¢ring without signi¢cant pattern change (Fig. 5). During 3 WM muscimol, an agonist for GABAA receptors, a bursting neuron did not change either its rate or pattern, but during 10 WM muscimol, its ¢ring rate decreased and its intra-burst intervals became much longer than control, resembling those in some irregular simple spiking neurons (Fig. 5). Two bursting neurons stopped ¢ring during 20 WM muscimol.
Fig. 5. E¡ects of GABA receptor agonists on the ¢ring pattern of a bursting neuron. The spontaneous ¢ring stopped during 3 WM baclofen, was una¡ected during 3 WM muscimol, and changed to an irregular (simple spiking) pattern during 10 WM muscimol. Each sweep represents 200 ms; time scale at the bottom: 20 ms/division. The largest spikes (negative [up] peak to positive peak) represent approximately 450 WV.
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those in the brain during anesthesia. Studies that have measured concentrations of pentobarbital in the brain and cerebrospinal £uid of anesthetized animals report values from 17.8 to 73.1 mg/kg, corresponding to approximately 72^294 WM (Hatanaka et al., 1988 ; Bolander et al., 1984). These studies have varied in route of drug administration, species of animal, and stages of anesthesia at which samples were collected. A study by Bolander et al. (1984), using an EEG threshold method, evaluated the potency and pharmacokinetic properties of pentobarbital as well as several other barbiturates. The concentration of pentobarbital in the brain was reported as about 294 WM during the `silent second', a well speci¢ed EEG criterion which was considered a reliable measure of anesthesia. This pentobarbital concentration is comparable to those which elicited ¢ring pattern changes of bursting neurons in our study. Thus, the absence of bursting activity in the previous in vivo studies using pentobarbital or other barbiturate anesthesia may at least partly have resulted from e¡ects of the anesthetics. Although both GABAA and GABAB receptors have been reported in the DCN (Juiz et al., 1994), the e¡ects of pentobarbital on bursting neurons were a¡ected by application of bicuculline, an antagonist for GABAA receptors, but not by saclofen, an antagonist for GABAB receptors. This ¢nding may suggest that the e¡ects of pentobarbital were at least partly through GABAA receptors on cartwheel cells. Although muscimol has been reported to increase ¢ring of cartwheel cells in mouse brain slices (Golding and Oertel, 1996), only decreased ¢ring of bursting neurons was observed during 10^20 WM muscimol in our study. Most bursting neurons showed decreased ¢ring during pentobarbital and increased ¢ring during bicuculline. Instead of showing less e¡ect of pentobarbital in the presence of bicuculline, the ¢ring rates of all bursting neurons increased greatly, more than with bicuculline alone. This suggests, in agreement with previous studies (Antkowiak, 1999 ; Rehberg et al., 1999), that the e¡ects of pentobarbital cannot be explained simply as agonist e¡ects on GABAA receptors. Rather, the results suggest that pentobarbital and bicuculline might interact in a more complicated way, such as through an allosteric e¡ect on GABAA receptors (Liljequist and Tabako¡, 1986). A question arises as to whether the loss of bursting activity results from a direct e¡ect of pentobarbital on cartwheel cells or whether the action is indirect through an e¡ect on granule cells. We have previously found that bursting activity can often be abolished by blocking the granule cell (parallel ¢ber) input to cartwheel cells with drugs such as 6,7-dinitroquinoxaline-2,3-dione (DNQX), an antagonist for non-N-methyl-D-aspartate glutamate receptors (Waller et al., 1996). However, during 5 WM DNQX, as the ¢ring rates of bursting
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neurons declined, changes of ¢ring patterns were not observed ; the patterns remained bursting (Chen et al., 1999a, and unpublished results). Further, stimulation of parallel ¢ber populations with single electric shocks often leads to burst ¢ring in bursting neurons, suggesting that bursting activity results from intrinsic membrane properties of cartwheel cells rather than properties of granule cells (Waller et al., 1996). Thus, the available evidence suggests that pentobarbital a¡ects bursting activity mainly by a direct action on cartwheel cells. Our ¢nding of often decreased and less regular ¢ring of regular neurons during pentobarbital suggests that the rare occurrence of highly regular spontaneous activity in vivo might also have some basis in anesthetic e¡ects. Assuming that bursting activity represents cartwheel cells in the DCN, which are inhibitory interneurons (Berrebi and Mugnaini, 1991; Mugnaini et al., 1987), the decrease and sometimes total loss of their spontaneous activity during pentobarbital may also contribute to less inhibitory responses to sound with in vivo recordings using barbiturate anesthesia (Evans and Nelson, 1973; Young and Brownell, 1976). In conclusion, anesthetic drug e¡ects on DCN neuronal spontaneous activity may have been underestimated in past studies. Among other di¡erences, anesthetic effects have to be considered when comparing in vivo results with in vitro results. The e¡ects of other commonly used anesthetics, such as ketamine and xylazine, also need to be studied. Acknowledgements The authors are grateful to Dr. Hardress J. Waller for computer programming. Supported by NIH Grant DC00172.
References Antkowiak, B., 1999. Di¡erent actions of general anesthetics on the ¢ring patterns of neocortical neurons mediated by the GABAA receptor. Anesthesiology 91, 500^511. Berrebi, A.S., Mugnaini, E., 1991. Distribution and targets of the cartwheel cell axon in the dorsal cochlear nucleus of the guinea pig. Anat. Embryol. 183, 427^454. Bolander, H.G., Wahlstro«m, G., Norberg, L., 1984. Reevaluation of potency and pharmacokinetic properties of some lipid-soluble barbiturates with an EEG-threshold method. Acta Pharmacol. Toxicol. 54, 33^40. Chen, K., Godfrey, D.A., 1999. Sodium pentobarbital abolishes bursting spontaneous activity of dorsal cochlear nucleus in rat brain slices. Abstr. Soc. Neurosci. 25, 394. Chen, K., Waller, H.J., Godfrey, D.A., 1994. Cholinergic modulation of spontaneous activity in rat dorsal cochlear nucleus. Hear. Res. 77, 168^176.
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K. Chen, D.A. Godfrey / Hearing Research 149 (2000) 216^222
Chen, K., Waller, H.J., Godfrey, T.G., Godfrey, D.A., 1999a. Glutamatergic transmission of neuronal responses to carbachol in rat dorsal cochlear nucleus slices. Neuroscience 90, 1043^1049. Chen, K., Sprunger, L.K., Meisler, M.H., Waller, H.J., Godfrey, D.A., 1999b. Reduced spontaneous activity in the dorsal cochlear nucleus of Scn8a mutant mice. Brain Res. 847, 85^89. Evans, E.F., Nelson, P.G., 1973. The responses of single neurons in the cochlear nucleus of the cat as a function of their location and the anesthetic state. Exp. Brain Res. 17, 402^427. Godfrey, D.A., Waller, H.J., 1992. Sampling £uid from slice chambers by microsiphoning. J. Neurosci. Methods 41, 167^173. Godfrey, D.A., Kiang, N.Y., Norris, B.E., 1975. Single unit activity in the dorsal cochlear nucleus of the cat. J. Comp. Neurol. 162, 269^ 284. Golding, N.L., Oertel, D., 1996. Context-dependent synaptic action of glycinergic and GABAergic inputs in the dorsal cochlear nucleus. J. Neurosci. 16, 2208^2219. Haas, H.L., Schaerer, B., Vosmansky, M., 1979. A simple perfusion chamber for the study of nervous tissue slices in vitro. J. Neurosci. Methods 1, 323^325. Hatanaka, T., Sato, S., Endoh, M., Katayama, K., Kakemi, M., Koizumi, T., 1988. E¡ect of chlorpromazine on the pharmacokinetics and pharmacodynamics of pentobarbital in rats. J. Pharmacobiodyn. 11, 18^30. Juiz, J.M., Albin, R.L., Helfert, R.H., Altschuler, R.A., 1994. Distribution of GABAA and GABAB binding sites in the cochlear nucleus of the guinea pig. Brain Res. 639, 193^201. Liljequist, S., Tabako¡, B., 1986. Bicuculline-pentobarbital interactions on [35 S]TBPS binding in various brain areas. Life Sci. 39, 851^855. Manis, P.B., 1990. Membrane properties and discharge characteristics of guinea pig dorsal cochlear nucleus neurons studied in vitro. J. Neurosci. 10, 2338^2351. Manis, P.B., Spirou, G.A., Wright, D.D., Paydar, S., Ryugo, D.K., 1994. Physiology and morphology of complex spiking neurons in the guinea pig dorsal cochlear nucleus. J. Comp. Neurol. 348, 261^ 276.
Mathers, D.A., Barker, J.L., 1980. (3)Pentobarbital opens ion channels of long duration in cultured mouse spinal neurons. Science 209, 507^509. Mugnaini, E., Berrebi, A.S., Dahl, A.L., Morgan, J.I., 1987. The polypeptide PEP-19 is a marker for Purkinje neurons in cerebellar cortex and cartwheel neurons in the dorsal cochlear nucleus. Arch. Ital. Biol. 126, 41^67. Parham, K., Kim, D.O., 1995. Spontaneous and sound-evoked discharge characteristics of complex-spiking neurons in the dorsal cochlear nucleus of the unanesthetized decerebrate cat. J. Neurophysiol. 73, 550^561. Pfei¡er, R.R., Kiang, N.Y.-S., 1965. Spike discharge patterns of spontaneous and continuously stimulated activity in the cochlear nucleus of anesthetized cats. Biophys. J. 5, 301^316. Rehberg, B., Wittmann, M., Urban, B.W., 1999. Sodium channels (from rat, mouse, and man) in neuroblastoma cells and di¡erent expression systems have similar sensitivities to pentobarbital. Neurosci. Lett. 264, 81^84. Study, R.E., Barker, J.L., 1981. Diazepam and (3)-pentobarbital: Fluctuation analysis reveals di¡erent mechanisms for potentiation of Q-aminobutyric acid responses in cultured central neurons. Proc. Natl. Acad. Sci. USA 78, 7180^7184. Waller, H.J., Godfrey, D.A., 1994. Functional characteristics of spontaneously active neurons in rat dorsal cochlear nucleus in vitro. J. Neurophysiol. 71, 467^478. Waller, H.J., Godfrey, D.A., Chen, K., 1996. E¡ects of parallel ¢ber stimulation on neurons of rat dorsal cochlear nucleus. Hear. Res. 98, 169^179. West, M.O., 1998. Anesthetics eliminate somatosensory-evoked discharges of neurons in the somatotopically organized sensorimotor striatum of the rat. J. Neurosci. 18, 9055^9068. Young, E.D., Brownell, W.E., 1976. Responses to tones and noise of single cells in dorsal cochlear nucleus of unanesthetized cats. J. Neurophysiol. 39, 282^300. Zhang, S., Oertel, D., 1993. Cartwheel and super¢cial stellate cells of the dorsal cochlear nucleus of mice: intracellular recordings in slices. J. Neurophysiol. 69, 1384^1397.
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