IK(Ca) was decreased in duration from to 1P94+0'12 s during anoxia.

Transcription

1 Journal of Physiology (1992) pp With 11 figures Printed in Great Britain IONIC BASIS OF MEMBRANE POTENTIAL CHANGES INDUCED BY ANOXIA IN RAT DORSAL VAGAL MOTONEURONES BY A. I. COWAN AND R. L. MARTIN From the Division of Neuroscience, The John Curtin School of Medical Research and the Division of Botany and Zoology, The Faculties, Australian National University, Canberra, ACT 2601, Australia (Received 17 September 1991) SUMMARY 1. The effects of anoxia on membrane properties of 119 dorsal vagal motoneurones (DVMs) were investigated in an in vitro slice preparation of the rat medulla. 2. Membrane potential was unaffected by anoxia in 11 % of DVMs. An hyperpolarization accompanied by a decrease in input resistance occurred in 44 % of DVMs; the remaining 45 % depolarized with either an increase (60 %) or decrease in input resistance (40 %). TTX at a concentration of /M did not significantly affect these responses. 3. Anoxic artificial cerebrospinal fluid (ACSF) containing 20 mm-tea reversed the response of DVMs that hyperpolarized in standard ACSF to reveal a depolarization of mv, and increased the anoxic depolarization from 5' to 8' mv. 4. Anoxic depolarization was converted to an hyperpolarization of 7' mv in ACSF containing 5 mm-4-aminopyridine (4-AP) and 1 JIM-TTX. A residual depolarization of 4'5 + 3'5 mv was then observed in ACSF containing 5 mm-4-ap, 1 /IM- TTX and 20 mm-tea. Anoxic hyperpolarization was increased from P8 to '9 mv in 5 mm-4-ap and 1 JIM-TTX and converted to a depolarization of mv in 5 mm-4-ap, 1 JIM-TTX and 20 mm-tea. 5. In anoxic ACSF containing TEA, the action potential width was increased from '04 to ms in hyperpolarizing DVMs, and from 0' to ms in depolarizing DVMs. The increase in width was prevented by 2-3 mm- Mn2+ 6. The long after-hyperpolarization (AHP) of DVMs, which is contributed to by both an apamin-sensitive IK(ca) and an apamin, charybdotoxin and TEA insensitive IK(Ca) was decreased in duration from to 1P94+0'12 s during anoxia. 7. It is concluded that anoxia enhances the delayed rectifier current (VK(Dn)) and an inward current, probably 'ca' but suppresses the A currents ('A) In DVMs that hyperpolarize during anoxia, the increase in IK(DR) outweighs the increase in lca and the decrease in IA, In depolarizing DVMs the decrease in IA and increase in 'Ca outweigh the increase in 'K(DR)* The change in input resistance is determined by the relative sizes of current enhancement or suppression. NIS

2 90 A. I. COWAN AND R. L. MARTIN INTRODUCTION Responses of the neuronal membrane potential to a reduction in oxygen supply are variable. For example, hippocampal CAI neurones primarily hyperpolarize (Hansen, Hounsgaard & Jahnsen, 1982; Fujiwara, Higashi, Shimoji & Yoshimura, 1987; Krnjevic & Leblond, 1989; Leblond & Krnjevic, 1989) whereas CA3 neurones (Lehmenkiihler, Caspers, Speckmann, Bingman, Lipinski & Kersting, 1988), dentate granule neurones (Somjen, Aitken, Balestrino & Schiff, 1987), dorsal root ganglion cells (Urban & Somjen, 1990) and hypoglossal motoneurones (Haddad & Donnelly, 1990) primarily depolarize. The ionic basis of these changes in membrane potential remains to be elucidated. There is general agreement from studies on hippocampal CAl neurones that an increased K+ conductance underlies membrane hyperpolarization, but the identity of the K+ conductance(s) involved is unclear. Hansen et al. (1982) noted complete block of anoxic hyperpolarization with 04 mm-4-aminopyridine (4-AP) and no effect of 3 mm-tetraethylammonium (TEA) whereas Fujiwara et al. (1987) reported that neither 1P5 mm-4-ap nor 10 mm-tea suppressed anoxic hyperpolarization. In the presence of mm-4-ap Leblond & Krnjevic (1989) observed a 'depolarizing trend' during anoxia. Apamin, which blocks one type of Ca2+-dependent K+ channel (Rudy, 1988) did not suppress anoxic hyperpolarization and neither did tolbutamide, an ATP-sensitive K+ channel antagonist (Leblond & Krnjevic, 1989). Similarly, neither intracellularly nor extracellularly applied Cs+ nor intracellular deposition of the Ca2+ chelator EGTA reliably blocked anoxic hyperpolarization (Fujiwara et al. 1987; Leblond & Krnjevic, 1989). However, in a voltage-clamp study Krnjevic & Leblond (1989) reported that in all neurones tested the outward anoxic current was blocked by intracellular Cs+ and often blocked by extracellular Cs+. The Q current (IQ), one of the Cs+-sensitive K+ conductances of hippocampal neurones (Halliwell & Adams, 1982), was not sensitive to anoxia whereas another Cs+-sensitive current, the A current (IA), was reduced by about 25%. IM' a muscarine-sensitive K+ current, was reduced by about 56%. In addition, the TEA- and charybdotoxin-sensitive Ca2+-dependent K+ current, Ic, (Lancaster & Nicoll, 1987) was reduced by 52 % and ICa was almost completely suppressed. The authors finally argued, without direct evidence, that anoxic membrane hyperpolarization is probably attributable to enhancement of a Ca2+_ dependent K+ current (IK(C.))' and that release of Ca2+ from intracellular stores by anoxia, rather than entry of Ca2+ through voltage-dependent Ca2+ channels, is the catalyst for activation of this current. The most recent literature concerning the ionic basis of anoxic hyperpolarization of CAl neurones (Krnjevic & Xu, 1990) suggests that enhancement of the M current (IM) is primarily responsible. In the present study we have re-examined the ionic basis of the membrane potential changes induced by anoxia because the existing literature, summarized above, is both contradictory and inconclusive. We have recorded from dorsal vagal motoneurones (DVMs) of the rat brainstem using the in vitro brain slice preparation. This region of the CNS has received scant attention in relation to the effects of oxygen deprivation. DVMs can be readily identified in the slice preparation by antidromic stimulation of their axons as they course through the reticular formation

3 DORSAL VAGAL MOTONEURONES AND ANOXIA (Yarom, Sugimori & Llinas, 1985). In the rat they primarily innervate gastrointestinal and thoracic smooth muscle but a small number innervate cardiac muscle (Nosaka, Yamamoto & Yasunaga, 1979; Dennison, O'Connor, Aprison, Merritt & Felten, 1981). Our data show that DVMs may either hyperpolarize or depolarize during anoxia and that these responses, in all likelihood, reflect simultaneous effects on two types of K+ currents and on Ica Anoxia also suppresses IK(Ca) but this probably does not contribute to the changes in membrane potential. 91 METHODS Slice preparation. Experiments were performed on 35- to 45-day-old male Wistar rats. Under halothane anaesthesia the skull was opened, the brain sectioned at the level of the inferior colliculus and the forebrain removed. After cutting the spinal cord at about C1 the brainstem was removed from the cranium. During these procedures the exposed surfaces of the brain were bathed with oxygenated ice-cold artificial cerebrospinal fluid (ACSF). In some experiments the animals were perfused through the heart with ice-cold ACSF after removal of the forebrain, to facilitate washout of blood and the demonstration of biocytin-stained neurones on a low level of background staining. The rostral end of the brainstem was fixed to a mounting block with cyanoacrylate glue, the mounting block placed in a chamber containing oxygenated ACSF at 2 'C, and 400,um thick transverse slices of the medulla cut using a vibroslice (Campden Instruments). One slice was immediately transferred to the recording chamber where it was placed on nylon net, immobilized by a weighted nylon net cover and illuminated from below by a fibre optic light source. It was viewed through a stereomicroscope (Wild Leitz, M5A). The slice was allowed to recover from surgical trauma for at least one hour before commencement of recording. The temperature in the recording chamber was maintained in the range 33X 'C. The other brain slices were placed in an incubation chamber at room temperature. The incubation and recording chambers were modifications of those described by Nicoll & Alger (1981). The flow rate of solution through the recording chamber was 2-3 ml/min. This flow rate and the small volume of the chamber (0 5 ml) allowed complete changes of the solution in the bath about 40 s after the new solution arrived at the bath inlet. Solutions. The standard ACSF used as a control solution and in the preparation of the slices contained (mm): NaCl, 123; NaHCO3, 25; KCl, 3; NaH2PO4, 1-25; Mg S04, 1-25; CaCl2, 2; glucose, 10. It was bubbled with 5 % CO2-95 % 02 to give a ph of 7-4. An anoxic solution was produced by bubbling the ACSF with 5 % CO2-95 % N2, resulting in a partial pressure of 02 (Po ) in solution of mmhg. Under these conditions the P0, was expected to be mmhg at a depth of 100 Itm into the tissue slice (Fujiwara et al. 1987). This solution and any containing drugs were applied by switching perfusion solution. Drugs used were TTX (0 3-1 ItM,), TEA (20 mm), 4-AP (20 fm-5 mm), manganese chloride (2-3 mm), apamin (100 nm), carbachol ( fm) and charybdotoxin (20-40 nm). All chemicals were from Sigma Chemical Company except the charybdotoxin which was from Alomone Laboratories. When drugs were added to the ACSF, the osmolarity was maintained by adjusting the concentration of NaCl. Recording and stimulation. Intracellular microelectrodes were pulled from either borosilicate or aluminosilicate glass. DC resistances, when filled with 3 M-KC1, were in the range MQ and MCI, respectively. For recording, electrodes were filled with 3 M-KCl, or 2-5 M-KCl containing % biocytin (Sigma). The membrane potential of neurones was measured from the intracellular microelectrode relative to an interstitial agar-3 M-KCl reference microelectrode. All experiments were performed in discontinuous current clamp mode using an Axoclamp II (Axon Instruments), DC amplifier. Switching rates were in the range 5-10 khz, and the signals were filtered at 1 khz. Input resistance was measured using hyperpolarizing current pulses of na and ms duration and action potentials were elicited with 3 ms depolarizing current pulses of na. The output of the amplifier was displayed on an oscilloscope, and a chart recorder, digitized and stored on hard disk of an IBM compatible PC for later analysis. The pen of the chart recorder did not follow the full amplitude of the action potential, and digitization of records on a long time base (500 ms) also truncated the action potential.

4 92 A. I. COWAN AND R. L. MARTIN Antidromic stimulation of DVMs was achieved using monopolar stimulating electrodes made from glass-insulated tungsten wire. Experimental protocol. The criteria used to determine that a satisfactory neuronal penetration had been achieved were a membrane potential more negative than -60 mv and action potential amplitude of at least 80 mv. Following neuronal penetration, input resistance and the action potential were recorded at resting membrane potential and the firing pattern in response to 400 ms depolarizing current pulses of various strengths was recorded at different membrane potentials. If the neurone was firing spontaneously at resting membrane potential minimal hyperpolarizing current was applied to prevent firing before any records were taken. Continuous recordings of membrane potential and input resistance were commenced and an anoxic insult of 6-10 min duration was applied. A recovery period of 15 min occurred before application of oxygenated ACSF containing drugs. After 10 min the effects of a given drug or combination of drugs on the action potential, membrane potential and input resistance were examined. Another anoxic insult was then initiated from the same membrane potential as that from which the control trial occurred, by injection of constant depolarizing or hyperpolarizing current if necessary. After 5 or 8 min of anoxia the action potential and firing pattern were recorded. Recovery in the oxygenated solution was permitted for at least 15 min before any further drug-containing solutions were used. The change in membrane potential during anoxia was measured as the difference between the membrane potential after 6 or 10 min of anoxia and the pre-anoxic membrane potential only in neurones whose membrane potential returned to pre-anoxic values during the recovery period. Histology. Each slice in which intracellular recordings using biocytin-containing electrodes were achieved was fixed in a 1% glutaraldehyde, 40% paraformaldehyde solution. After fixation, the slice was soaked in a 25 % sucrose-phosphate buffer solution, and embedded in a gelatin-albumen block. Sections 80,um thick were cut using a vibroslice (Campden Instruments). The sections were then washed in 01 M-phosphate buffer and incubated with avidin, and biotin conjugated horseradish peroxidase (ABC Kit, Vectastain) for 4 h in 01 M-phosphate buffer containing 1 % Triton. The sections were then reacted with diaminobenzidine (DAB) according to the method described by Horikawa & Armstrong (1988). Statistical analysis. Statistical analysis of the data was performed using a two-way analysis of variance for a randomized block design employing the Genstat 5 (Lawes Agricultural Trust) statistics package. Quantitative results are expressed as the mean+standard error of the mean. RESULTS Identification and characteristics of dorsal vagal motoneurones Antidromic stimulation of DVMs was achieved in 60 % of recorded neurones and reflected the instances when DVM axons ran in the plane of the tissue section. Based upon results when antidromic stimulation was achieved, the main criteria used for identification of DVMs were their anatomical location, action potential characteristics (spike amplitude, width at half-amplitude, maximum after-hyperpolarization (AHP) amplitude and duration of the AHP), and firing patterns in response to long (400 ms) depolarizing current pulses at various membrane potentials. In 119 DVMs at resting membrane potential ( mv) the amplitude of the action potential was mv, the width at half-amplitude ms, the maximum amplitude of the AHP mv, the duration of the AHP s and the input resistance MQ (range MQ). At resting membrane potential, a 400 ms pulse of na resulted in a firing pattern in which there was a delay before generation of an action potential (Fig. 1 A). An increase in the strength of the depolarizing current pulse to na (Fig. 1B), resulted in an increase in the frequency of firing and in some cases the neurone fired an action potential at the start of the current pulse, but the interspike interval remained irregular. Only when a current pulse of na was applied from a

5 DORSAL VAGAL MOTONEURONES AND ANOXIA membrane potential between -40 and -50 mv, did the interspike interval become more regular (Fig. 1 C). The typical DVM labelled with biocytin had a soma size approximately 20 x 20 Jim and a sparse dendritic tree (Fig. 2). In adjacent sections of the brainstem slice the 93 A -65 mv B -65 mv -49 mv 20 mv 50 ms Fig. 1. The firing pattern of a dorsal vagal motoneurone in response to depolarizing current for 400 ms (indicated by the bar). A shows the response to a 0-3 na and B to a 0-6 na current pulse initiated from the resting membrane potential, demonstrating a delay before action potential generation. C, the response to a 0-2 na current pulse initiated from a depolarized potential elicited a more regular firing pattern. Note that the action potentials are truncated due to digitization of the records. neurone was clearly bipolar. These morphological characteristics are similar to those described by Yarom et al. (1985) for guinea-pig DVMs. Effects of anoxia on membrane potential, input resistance and action potential In standard ACSF, DVMs exhibited various responses to anoxia. Of the 111 neurones tested 11% showed no change in membrane potential, 44 % primarily hyperpolarized and 45 % primarily depolarized. When hyperpolarization occurred, it was preceded in 59 % of the neurones by a transient depolarization occurring min after the onset of anoxia. The

6 94 A. I. COWAN AND R. L. MARTIN L I 20pum Fig. 2. A dorsal vagal motoneurone intracellularly labelled with biocytin. D, dorsal: L, lateral. Control TEA A 02 B N250 mns N2 N50 mns N~~~~~~~~~~~~~~~2-71 mv N2-71 mv N mv 02 2 ms Fig. 3. The effect of anoxia on the action potential of a dorsal vagal motoneurone. A, action potential elicited by a 3 ms depolarizing current of 1-0 na in oxygenated ACSF (02) and after 8 min of anoxia (N2). B, action potential of the same cell in ACSF containing 20 mm-tea. Note the large increase in duration of the action potential during anoxia. hyperpolarization was variable in latency, with a mean of min (range min), and had a mean amplitude of mv (range 1-12 mv). It was accompanied by a significant increase in input resistance of % (P < 0 005). In neurones which depolarized during anoxia the mean amplitude of the depolarization was mv (range 2-14 mv) with an onset at min. Of these neurones 36 % demonstrated a partial repolarization which occurred min after the onset of anoxia. In 60 % of depolarizing neurones there was an increase in input resistance of %, whereas the remaining 40% of neurones showed a decrease in input resistance of %. In the eight neurones tested, the hyperpolarizing or depolarizing response was again observed when the exposure to anoxia in standard ACSF was repeated. During anoxia changes in the shape and duration of the action potential occurred. There was a small but significant decrease in the amplitude and an increase in the width at half-amplitude. The maximum amplitude of the AHP was decreased and although the average duration of the AHP was reduced (Fig. 3A, Table 1), in 11/55 neurones the AHP increased in length. The decrease in AHP duration was similar in all DVMs regardless of the effects of anoxia on membrane potential and input resistance (Table 2).

7 DORSAL VAGAL MOTONEURONES AND ANOXIA TABLE 1. Effects of anoxia on the action potential and AHP After-hyperpolarization Action potential Maximum Input Number Amplitude Width amplitude Duration resistance of Perfusate (mv) (Ms) (mv) (s) (MCI) neurones Control ACSF ACSF, anoxia * t t * 55 TEA TEA, anoxia * t t t 11 4-AP AP, anoxia Apamin Apamin, anoxia P * Mn2+, TEA Mn2+, TEA, anoxia Statistical significance refers to the comparison of the action potential during anoxia and control in the same ACSF (t: P < 0-005; *: P < 0-01). Note the longer AHP duration in apamin reflects measurements made at more positive membrane potentials. TABLE 2. Effects of anoxia on the AHP duration AHP duration (s) Membrane response during Oxygenated Resting membrane Number of anoxia in standard ACSF ACSF Anoxia potential (mv) neurones Hyperpolarization t Depolarization, decreased RIN t Depolarization, increased RIN * Statistical significance refers to the comparison of the AHP duration during anoxia and control (t: P<0005; *: P<0-01; t: P<0-025). In addition, anoxia changed the response of DVMs to 400 ms depolarizing current pulses, so that there was a reduction in the delay before the generation of the first action potential (Fig. 4). A delay before the first action potential is attributed to an A current (Yarom et al. 1985). The decrease in the delay during anoxia suggests therefore that there is a reduction in the A current. On reoxygenation all neurones repolarized to resting membrane potential within 15 min, but at this time the input resistance was significantly increased from the preanoxic level ( %, n = 111, P < 0-005). The initial change in membrane potential commenced min after reoxygenation. Of the neurones that demonstrated an hyperpolarizing anoxic response 35 % exhibited a complex post-anoxic response which was characterized by an initial hyperpolarization of 2-18 mv (mean: mv), before repolarization to the pre-anoxic membrane potential. Effects of TTX In the presence of TTX ( JtM) the fast action potential was abolished and a slowly rising, small amplitude action potential could be observed. That this was a Ca2+-based action potential was confirmed by its abolition in 3 mm-mn2+. Such 95

8 96 A. I. COWAN AND R. L. MARTIN A B mv C D -60 mv 20 mvl 50 ms Fig. 4. Effect of anoxia on the firing pattern in response to a 400 ms current pulse. A shows a current pulse of 0-2 na and B 0-4 na in oxygenated ACSF resulted in a delay before generation of the first action potential. During anoxia there was no delay before the first action potential when the current pulse of 0-2 na (C) and 0 4 na (D) was applied. A TTX -60 mv 20 mv 30 ms B TTX + TEA 20 mv[ -60 mv ms Fig. 5. Tetrodotoxin resistant action potential. A, spontaneous slowly rising, small amplitude action potential in ACSF containing 1 jzm-ttx. B, spontaneous action potential in ACSF containing 1 /SM-TTX and 20 mm-tea. narrow spikes could not be evoked in the presence of 20 mm-tea, but instead longlasting Ca2+ spikes occurred (Fig. 5). TTX did not affect the resting membrane potential or input resistance of DVMs.

9 DORSAL VAGAL MOTONEURONES AND ANOXIA In 8/10 neurones tested, block of synaptic transmission with TTX did not change the maximum amplitude of the hyperpolarizing (n = 4) or depolarizing (n = 4) response to anoxia (Fig. 6). In the other two neurones, the amplitude of the hyperpolarization was increased by 6 and 9 mv respectively. Changes in input 97 A Control -70 mv B TTX -70 mv 10 mvl 60 s Fig. 6. Effect of TTX on the anoxic response. A, the control depolarizing response to a period of anoxia (indicated by the bar). B, the maximum amplitude of the depolarization was not affected by the presence of 1M,m-TTX, but the latency of the membrane potential change was increased. Input resistance was measured by regular 300 ms hyperpolarizing current pulses of 0-02 na. Note that the chart recorder was unable to follow the action potentials in A, but did record the AHP. resistance were similar to those observed in the absence of TTX. In about half of the neurones tested the latency of the hyperpolarizing response was decreased (mean decrease: minm n = 6) and the latency of the depolarizing response was increased (mean increase: min n = 4). This result is consistent with the early part of the anoxic responses having a component which is dependent upon activity in presynaptic neurones although the direct involvement of postsynaptic voltage-dependent sodium channels cannot be ruled out. Effects of TEA After 10 min in oxygenated ACSF containing 20 mm-tea, there was a membrane depolarization of mv (n = 25, P < 0-05), and input resistance was increased by % (P < 0 05). When measurement of the characteristics of the action potential was made at the same membrane potential before and after application of TEA, then TEA broadened the action potential and increased the maximum amplitude of the AHP (Table 1). The duration of the AHP was increased from to s (n = 11). Superfusion with ACSF containing 20 mm-tea consistently reversed the response of hyperpolarizing neurones to anoxia, to produce an average depolarization of mv (n = 10, P < 0 005, Fig. 7). Input resistance was decreased by % compared with a decrease of % in standard ACSF for the same neurones (P < 0 005). In DVMs that depolarized during anoxia in standard ACSF, the amplitude of the

10 98 A. I. COWAN AND R. L. MARTIN depolarization was increased in 20 mm-tea from to mv (n = 9, P < 0-01). Those DVMs that demonstrated a depolarization and an increase in input resistance during anoxia in standard ACSF ( %, n = 5), now demonstrated an average decrease in input resistance of %. Input resistance of the DVMs A Control -66 mv B TEA -66 mvv*. 10 mvl 60 s Fig. 7. Effect of TEA on the anoxic response. A, control hyperpolarizing response. B, in ACSF containing 20 mm-tea the membrane potential change during anoxia was reversed, and a depolarizing response resulted. Input resistance was measured with 300 ms hyperpolarizing pulses of 0 03 na in A and 0-01 na in B. that depolarized with a decrease in input resistance during anoxia in standard ACSF (1 1'6 +29%, n = 4) decreased slightly less in ACSF containing 20 mm-tea ( %). In anoxic ACSF containing TEA, hyperpolarizing neurones showed a dramatic increase in the width of the action potential from 0' to I 1 ms (n = 6, Fig. 3B). This was significantly greater (P <005) than the increase in width observed in depolarizing neurones (from to ms, n = 6). The maximum amplitude of the AHP, and the AHP duration were also decreased (Table 1) but the size of these changes was not related to the direction of the membrane potential shift during anoxia in standard ACSF. TEA did not necessarily prevent an increase in AHP duration during anoxia (2/11 neurones). ACSF containing TTX (0 3 J/M) as well as 20 mm-tea did not alter the anoxic response from that seen in TEA alone in one hyperpolarizing and one depolarizing neurone. Effect of 4-AP and TEA Almost all experiments involving 4-AP were carried out in the presence of 0'3-10 jtm-ttx to counteract some of the 4-AP-induced increase in membrane potential noise, which presumably resulted from increased neurotransmitter release as the width of the presynaptic action potential increased. Superfusion with oxygenated ACSF containing 20-50,tM-4-AP, which was expected to block the K+ current ID (Storm, 1990), and TTX did not affect the membrane potential or input resistance of DVMs (n = 3). The control anoxic response was also unaltered (n = 5).

11 DORSAL VAGAL MOTONEURONES AND ANOXIA 99 Oxygenated ACSF containing TTX and 5 mm-4-ap resulted in a membrane depolarization of mv (n = 10), accompanied by an increase in input resistance of In four neurones, 2 mm-4-ap alone resulted in a significant increase (P < 0 02) in the width of the action potential and decrease in the amplitude A Control -74 mv _ B TTX + 4-AP -74 mv C TTX + 4-AP + TEA -74 mv 10 mvl 60 s Fig. 8. Effect of 4-AP and TEA on the anoxic response. A, control hyperpolarizing response to anoxia. B, this response was not altered in ACSF containing 1,uM-TTX and 5 mm-4-ap. C, a depolarization during anoxia was demonstrated in ACSF containing 20 mm-tea, 1 /zm-ttx and 5 mm-4-ap. Input resistance was measured with 300 ms hyperpolarizing pulses of 0-02 na in all records. of the AHP (P < 005, Table 1) but there was no significant effect on the action potential amplitude. The duration of the AHP was not altered by 5-10 mm-4-ap. DVMs that hyperpolarized during anoxia in standard ACSF, demonstrated a nonsignificant increase in the amplitude of the hyperpolarization during anoxia in ACSF containing 5 mm-4-ap and TTX ( mv compared with mv, n = 5, Fig. 8A and B). However, in anoxic ACSF containing 20 mm-tea as well as 5 mm- 4-AP and TTX, these neurones depolarized (5-3 ± 4-5 mv, n = 4, Fig. 8C). Input resistance was decreased by % during anoxia in standard ACSF, by % during anoxia in ACSF containing 5 mm-4-ap and TTX and by % during anoxia in ACSF containing 5 mm-4-ap, TTX and 20 mm-tea. The response of all DVMs that depolarized during anoxia in standard ACSF was significantly altered in anoxic ACSF containing 5 mm-4-ap and TTX, such that the neurones hyperpolarized by mv (n = 7, P < 0 005, Fig. 9A and B). In four neurones this response was accompanied by a decrease in input resistance of % which was greater than the decrease in standard ACSF during anoxia ( %). In the three neurones that demonstrated an increase in input resistance during anoxia in standard ACSF ( %), the input resistance now

12 100 A. I. COWAN AND R. L. MARTIN decreased on average in anoxic ACSF containing 5 mm-4-ap and TTX ( %). In anoxic ACSF containing 5 mm-4-ap, TTX and 20 mm-tea, four neurones demonstrated a depolarization (mean: mv, Fig. 9 C) which was significantly different (P < 0 05) from the hyperpolarization during anoxia in ACSF containing A Control -63 mv T B TTX + 4-AP -63 mv C TTX + 4-AP + TEA 10 mvl 60 s -63 mv Fig. 9. Effect of 4-AP and TEA on the anoxic response. A, control depolarizing response to anoxia, which was not altered by the addition of 1 am-ttx (not shown). B, in ACSF containing 5 mm-4-ap and 1 /.M-TTX the same cell hyperpolarized during anoxia. C, a depolarization during anoxia was demonstrated in ACSF containing 20 mm-tea, 1 /LM- TTX and 5 mm-4-ap. Input resistance was measured with 300 ms hyperpolarizing pulses of 0-02 na in all records. 4-AP and TTX. The input resistance of these DVMs decreased by % in anoxic ACSF containing 5 mm-4-ap, TTX and 20 mm-tea compared with a decrease of % during anoxia in ACSF containing 5 mm-4-ap and TTX. During anoxia in ACSF containing 2 mm-4-ap the effects on the action potential and AHP amplitude were of a similar magnitude to those observed in standard anoxic ACSF, although in the small number of neurones studied the effects did not reach significance (Table 1). AHP duration was not measured in anoxic ACSF containing 4-AP because the potential trajectory was too noisy, and averaging procedures were not available at that time. Effects of apamin In oxygenated ACSF 100 nm-apamin had no effect on membrane potential or input resistance (n = 8). There was also no effect on the height or width of the action potential but the maximum amplitude of the AHP was significantly decreased from P2 to P5 mv (P < 0 01, Table 1). The duration of the AHP was

13 DORSAL VAGAL MOTONEURONES AND ANOXIA unaffected, although its shape was consistently altered over the first s (Fig. 10). It therefore appears that an apamin-sensitive IK(Ca) is present in DVMs and contributes to the AHP within the first 1 s. The membrane potential response to anoxia was unaffected by apamin and the increase in width of the action potential, the reduction of the duration of the AHP lot -65 macmv 20 mvl 500 ms X ~~~~~~20 mvl -. ~~~~~50 ms Apamin -a-65mv Fig. 10. Effect of apamin' on the AHP of a dorsal vagal motoneurone. Apamin (100 nm) decreased the amplitude of the AHP over the first 1 s, but did not affect the AHP duration. and the reduction in the amplitude of the AHP were of similar magnitude as those observed in standard anoxic ACSF (Table 1). In 1/8 neurones treated with apamin the AHP duration increased during anoxia. Effects of tolbutamide In oxygenated ACSF ItM-tolbutamide, which is known to block ATPsensitive K+ channels (Ashcroft, 1988), had no effect on resting membrane potential, input resistance or on the action potential (n = 5). Further, the anoxic response was not changed in the presence of tolbutamide. Effects of Mn2' and TEA The addition of 2-3 mm-mn21 to oxygenated ACSF containing TEA resulted in an hyperpolarization of P8 mv, and an increase in input resistance of % (n = 5). In oxygenated ACSF containing 2-3 mm-mn2+ and 20 mm-tea, no significant effects on the amplitude of the action potential or on the maximum amplitude of the AHP were observed. The duration of the AHP following an action potential was reduced relative to that in 20 mm-tea, and the action potential width at half-amplitude was decreased (Table 1). This result suggests that IK(ca) underlies the later phases of the AHP (beyond 0 5 s). To confirm this finding the duration of the AHP was measured in neurones penetrated with electrodes containing 300 mm- EGTA. In six such neurones the AHP lasted 1X02 +0X14 s. This was longer than that observed in Mn2+ but approximated the time at which the AHP was no longer

14 102 A. I. COWAN AND R. L. MARTIN affected by 100 nm-apamin. It appears that chelation of intracellular Ca2+ with EGTA was insufficient to prevent expression of the apamin-sensitive IK(Ca)' but sufficient to prevent expression of a longer duration (beyond 1 s) IK(ca). Neither charybdotoxin (20-40 nm, n = 3) nor carbachol ( JM, n =3) affected the duration of the AHP. A TTX -65 mv B TTX+Mn mv C TTX+Mn2+ +TEA -5 mv 10 mvl 60 s Fig. 11. Effect of manganese and TEA on the anoxic response. A, depolarizing response to anoxia in ACSF containing 1,FM-TTX. B, this response was not significantly altered in ACSF containing 1 /tm-ttx and 3 mm-manganese. C, an enhanced depolarization was consistently observed in ACSF containing 20 mm-tea, 1,#M-TTX and 3 mm-manganese. Input resistance was measured with 300 ms hyperpolarizing pulses of 0 03 na in A and 0-02 na in B and C. During anoxia in ACSF containing Mn2+ (2-3 mm) and 20 mm-tea, the depolarizing response of all neurones to anoxia was greatly enhanced (n = 10, Fig. 11). In five of these experiments TTX was added before TEA and Mn2±. The mean maximum amplitude, at mv, was significantly greater (P < 005) than the anoxic response in ACSF containing TEA (mean amplitude: mv, n = 5) or Mn2+ and TTX (mean amplitude: mv, n = 5). The depolarization was accompanied by a significant decrease in input resistance of % (P < 0-001) compared with a decrease of % during anoxia in ACSF containing TEA and a decrease of % in anoxic ACSF containing Mn2+ and TTX. An initial slow depolarization (rate: mv/min) occurred with a latency of 11±+ 0 4 min, a later rapid depolarization (rate: P7 mv/min) occurred at min. Repolarization following reoxygenation commenced at min, which was

15 DORSAL VAGAL MOTONEURONES AND ANOXIA consistently faster than seen in the absence of Mn2+ and TEA (IP min). In some neurones the rapid repolarization continued so that the pre-anoxic membrane potential was achieved within 5 min of reoxygenation whereas in other neurones repolarization to resting membrane potential was delayed for as much as 25 min. In three neurones, addition of TTX to the ACSF containing 2-3 mm-mn2' and 20 mm-tea had inconsistent effects on the depolarization during anoxia. In one neurone, the depolarization was prevented, and there was no change in input resistance during anoxia. In another neurone, the anoxic depolarization and decrease in input resistance were not affected by TTX. In a third neurone, there was a 69 % decrease in the size of the depolarization, and a 50 % attenuation of the decrease in input resistance. The AHP amplitude and duration were decreased in anoxic ACSF containing TEA and Mn2" (Table 1), most probably reflecting the effects of anoxia on IA. 103 DISCUSSION This study has demonstrated that DVMs either hyperpolarize or depolarize in response to anoxia. By using a variety of ion channel blockers, singly or in combination, we have sought to identify the major voltage-dependent ion channels of DVMs in the rat, and thus to understand the probable origin of the membrane potential changes induced by anoxia. The fast action potential was blocked by TTX to reveal a slower, smaller amplitude action potential which was abolished by Mn2. In the presence of 20 mm- TEA or 2 mm-4-ap the TTX-sensitive action potential was broadened. The long duration AHP was not affected by 20 mm-tea or 5-10 mm-4-ap but in 2-3 mm- Mn2+ the AHP was reduced to about 25 % of its usual length. These results are consistent with presence of a TTX-sensitive current (INa) responsible for the upstroke of the fast action potential and a low-threshold ICa responsible for a slow TTXresistant action potential. A high-threshold ICa and a slow INa may exist but we have not specifically looked for their presence. Repolarization of the action potential is apparently contributed to by two K+ currents, one sensitive to TEA and therefore presumably a delayed rectifier (IK(DR)) and the other sensitive to 2 mm-4-ap and therefore presumably IA. The presence of IA is further suggested by the change in firing pattern in response to prolonged depolarizing current pulses of different magnitudes in 4-AP. The reduction in the duration of the AHP in Mn2+ and by EGTA would suggest that the long duration AHP is due to IK(ca)* The first 1 s of the AHP is significantly reduced in amplitude by apamin but not TEA, indicating that over this time period the AHP is contributed to by an apamin-sensitive IK(ca)* The later part of the AHP is apparently due to an apamin, TEA and charybdotoxininsensitive IK(Ca)* Our conclusions regarding the array of voltage-dependent ion channels in rat DVMs closely parallel those reported by Yarom et al. (1985) for guinea-pig DVMs. However, they did not use apamin or charybdotoxin to identify components, if any, of the long duration AHP. Membrane potential changes after the first 2 min of anoxia appear to be independent of activity in presynaptic neurones as block of presynaptic action potentials with TTX had no effect beyond this time period. Involvement of a slow

16 104 A. I. COWAN AND R. L. MARTIN INa in the anoxic response is ruled out because this current is TTX sensitive (Adams & Galvan, 1986). We cannot in all certainty attribute the changes in membrane potential to intrinsic properties of DVMs because anoxia may act directly upon presynaptic terminals to cause spontaneous release of neurotransmitters or neuromodulators, or cause increased release by shifting the activation range of Ca21 channels located in the presynaptic terminal. Neurotransmitters or neuromodulators could result in activation of a postsynaptic potassium current which may be sensitive IM to TEA or 4-AP e.g. or Is (serotonin-activated K+ current). Discrimination of a presynaptic effect by use of Mn21 or other divalent cations is not possible because both pre- and postsynaptic Ca2+ channels will be blocked. Presynaptic effects can only be eliminated by studying isolated neurones. Interpretation of the shifts in membrane potential observed, either at rest or during anoxia, in the presence of TEA and 4-AP must be considered in relation to the specificity of these drugs. Potentially, mm-tea could block IK(DR)' 'A'IK(Ca) (BK channel), IK(ATP)'IIR (inwardly rectifying K+ current), andik(na) (sodiumactivated K+ current) (Rudy, 1988; Castle, Haylett & Jenkinson 1989). Data given in the results argue strongly mm-tea that 20 did not block IA to any extent: in anoxia the depolarization observed after application of TEA was 2-3 mv larger (in both DVMs which depolarized and those that hyperpolarized in standard anoxic ACSF) than that observed after application of 4-AP and TEA (with TTX). This is consistent with our belief (discussed below) that anoxia causes a decrease in 'A which, in the presence of TEA, is not counteracted by an outward current and will therefore cause a larger depolarization. There is no indicationofik(ca) (BK) in DVMs since the BK channel blocker, charybdotoxin, did not affect the action potential width (not stated in Results) or the AHP (Adams & Galvan, 1986). Similarly, it appears that DVMs do not express ATP-sensitiveK+ channels because tolbutamide, an apparently specific blocker of these channels (Ashcroft, 1988) did not affect resting membrane potential or the anoxic response. Indeed, studies concerning the distribution of ATPsensitiveK+ channels in the rat brain suggest a relatively low expression in the medulla (Mourre, Ben Ari, Bernardi, Fosset & Lazdunski, 1989). Application of hyperpolarizing pulses of varying sizes to DVMs (data not shown), which changed membrane potential through the range -60 to -90 mv, did not provide classical evidence of inward rectification, i.e. inward sag and rebound excitation (Brown, Gahwiler, Griffith & Halliwell, 1990). This result is in agreement with studies on guinea-pig DVMs (Nitzan, Segev & Yarom, 1990) and indicates that inwardly channels rectifyingk+ also not present. When INa and were 'Ca blocked, hyperpolarizing current pulses from membrane potentials between -25 and -55 mv revealed a sag and rebound which remained unchanged by application of /t#mcarbachol but was significantly suppressed by mm-tea (data shown). 20 not This result is interpreted as evidence that DVMs do not express ImM It is not easy to detect sodium-activated K+ channels but their presence generally in mammalian neurones may require re-evaluation in the light of the recent study by Dreyer (1991). 4-AP potentially also affects some K+ conductances which have been considered above and ('K(ATP) 'K(Na); Rudy, 1988). An effect 'K(DR) on is possible but again the given data argue strongly against this occurring to any significant extent. Most importantly, we consistently observed that mm-tea produced effects additional

17 DORSAL VA GAL MOTONE UR ONES AND ANOXIA to those seen after application of 2-5 mm-4-ap. In addition, we tested for the presence of ID, a slowly inactivating K+ current with great sensitivity to 4-AP. 4-AP at um, which blocks this current in hippocampal CAt neurones (Storm, 1990), had no effect on resting membrane potential or on the anoxic response. Anoxic hyperpolarization. which was observed in 44% of neurones studied, was accompanied by a decrease in input resistance. 4-AP (5 mm) alone had no significant effect on the membrane hyperpolarization but 20 mm-tea reversed it to reveal a small but distinct depolarization. We conclude that anoxic hyperpolarization primarily reflects an increase in IK(DR)' which is counteracted to a limited extent by a reduction in IA and by an inward current (discussed below). Forty-five per cent of DVMs depolarized during anoxia: this depolarization was accompanied by a decrease in input resistance in 40 % of such neurones, but by an increase in input resistance in the remaining 60%. A small but significant effect of 20 mm-tea alone was observed but in 5 mm-4-ap an anoxic hyperpolarization rather than a depolarization occurred. TEA (20 mm) in combination with 5 mm-4-ap then led to a small depolarization during anoxia. Thus DVMs which depolarize in response to anoxia apparently do so because IA is reduced. However, anoxia also increases IK(DR) in DVMs which depolarize in anoxia but this can best be observed when A channels are blocked. Further differences between DVMs which hyperpolarized and those that depolarized in anoxia were observed in anoxic ACSF containing 20 mm-tea: the width of the action potential was increased significantly more in hyperpolarizing neurones than in depolarizing neurones. An increase in action potential width may reflect an increase in 'Ca or a reduction in TEA-insensitive K+ conductances which contribute to repolarization of the action potential (possibly through an effect of neurotransmitters; Jacquin, Denavit-Saubie & Champagnat, 1989). The major TEAinsensitive K+ current involved in repolarization of the action potential is 'A The greater reduction in this current in depolarizing DVMs should produce a greater increase, not smaller increase, in action potential width than that observed in hyperpolarizing DVMs. Thus anoxie enhancement of 'ca probably occurs. The contribution of Ia to the membrane potential changes in anoxia, which may be larger in hyperpolarizing DVMs, has proved hard to identify because in the presence of Mn2" and the major K+ channel antagonists an extraordinarily large depolarization occurred. Nevertheless, when both IA and IK(DR) were blocked a residual depolarization was observed in all DVMs, which was accompanied by a decrease in input resistance. Anoxic depolarization associated with a decrease in input resistance is only possible if there is enhancement of an inward current in addition to a reduction in IA and an increase in IK(DR) We cannot rule out the possibility that the inward current is carried by Cl- since diffusion of KCl from the electrodes may have reversed the normal direction of Cl- movement. Anoxia also appears to cause a reduction in the apamin-insensitive IK(Ca)* This conclusion is based on the fact that in a large proportion of DV'Ms tested, a reduction in the AHP duration was observed during anoxia. which persisted in the presence of apamin. The effect was significant only when a large number of neurones had been studied, and was stronger when each neurone acted as its own control. The closing of only a small fraction of channels may appear to occur since there was not complete 105

18 106 A. 1. CO WAN AND R. L. MARTIN abolition of the long duration AHP. However, if anoxia induces opening of Ca2" channels, then under these conditions the intracellular concentration of Ca2" is likely to be higher than usual. As a consequence activation of additional Kca channels may occur, or those KCa channels normally activated after the action potential may stay open longer. These processes will oppose the reduction Of IK(Ca), to the extent that the AH1P in some neurones may actually be longer in anoxia. The anoxic depolarization of all DVMs in 20 mm-tea was significantly greater in the presence of Mnn2+. This depolarization was associated with a decrease in input resistance and was only occasionally prevented by the presence of TTX. We presume that when extracellular Ca21 cannot enter the neurones a separate series of pathological processes is set in motion. Further experiments are needed to understand the mechanism of this phenomenon. In the study of t)onnelly & Haddad (1989) DVMs demonstrated only a depolarization during anoxia. It is possible that they obtained this result because they only recorded from a few DVMs and in doing so encountered just the one type of anoxic response. However, the heterogeneous responses we observed are similar to those described for hippocampal CAl neurones by Fujiwara et al. (1987), and Leblond & Krnjevic (1989). In these studies, the percentage of hyperpolarizing neurones was about 65 % and in the study of Leblond & Krnjevi6 (1989), about 34 % of CA1 neurones demonstrated a depolarization. In contrast to CAt neurones and DVMs, hypoglossal motoneurones (Haddad & Donnelly, 1990), dorsal root ganglion cells (Urban & Somjen, 1990) and hippocampal CA3 neurones (Ben Ari, 1989) demonstrated primarily a depolarizing response to anoxia. The depolarization was associated with a decrease in input resistance in CA3 neurones (Ben Ari, 1989), with an increase in input resistance in hypoglossal motoneurones (Haddad & Donnelly, 1990) but with no consistent change in input resistance in dorsal root ganglion cells (Urban & Somjen, 1990). Potassium channel involvement in the anoxic response An increase of IK(DR) during anoxia may also occur in hypoglossal motoneurones since Haddad & Donnelly (1990) noted that mm-tea enhanced the anoxic depolarization. Although hippocampal CAI neurones have a TEA-sensitive IK(DR) (Rudy, 1988) an effect of 10 mm-tea on the anoxic hyperpolarization has not been demonstrated (Hansen et al. 1982; Fujiwara et al. 1987). In CAl neurones mm- TEA blocks IK(DR) (Storm, 1990), suggesting that concentrations higher than 10 mm may be required to demonstrate a significant contribution of IK(DR) in these neurones. In all DVMs IA is also affected by anoxia, but in contrast to the TEA-sensitive current mediating the hyperpolarization, the results suggest that IA is decreased. A decrease in IA during anoxia has also been observed in voltage clamp studies of CAI neurones (Krnjevi6 & Leblond, 1989) but this was not thought to contribute to the membrane potential changes. Prevention of anoxic hyperpolarization in CAl neurones with 10 mm-tea and 2-3 mm-cs' led Krnjevi6 & Leblond (1987) to suggest that enhancement Of IK(ca) is primarily responsible for the hyperpolarization. However, in DVMs, concentrations of TEA which did not affect the IK(ca)-dependent AHP, completely abolished the anoxic hyperpolarization and although closure of Kca channels open at rest could

19 DORSAL VAGAL MOTONEURONES AND ANOXIA underlie anoxic depolarization, 4-AP at concentrations which also did not affect the AHP duration completely prevented this response. On the other hand, a reduction in AHP duration was clearly evident during anoxia, indicating a reduction in IK(ca) We could not demonstrate an involvement of IK(ATP) in the anoxic response of DVMs and neither could Leblond & Krnjevic (1989) in CAI neurones. However, Jiang & Haddad (1991) noted that two specific K(ATP) channel blockers prevented considerable K+ loss during anoxia and enhanced anoxic depolarization in hypoglossal motoneurones. Finally, in CAI neurones the outward current induced by anoxia when the membrane potential was voltage clamped at -30 mv was blocked by carbachol (Krnjevic & Xu, 1990). Thus IM may contribute to anoxic hyperpolarization in those neurones which possess such a current. Inward currents during anoxia Although not proven, our results strongly indicate that anoxia enhances an inward current which is most likely to be ICa- In contrast, an almost complete suppression of ICa has been observed in voltage clamp studies of CAI neurones (Krnjevic & Leblond, 1989). Differences between neurone types could reflect differences in the effects of anoxia on intracellular levels of Ca2" or ATP, since Krnjevic & Leblond (1987, 1989) suggested that the inactivation of Caa2+ channels during anoxia is due either to an increase in intracellular Ca2+ or to a decrease in phosphorylation of Ca2+ channels as a result of a reduction in intracellular ATP. It may also be significant that the CAl neurones were subjected to only 2-4 min of anoxia. After this time, and thus at times comparable with our study, there is a decrease in extracellular Ca2+ concentration (Hansen, 1985; Silver & Erecin'ska, 1990) and neuronal depolarization (Silver & Erecinska, 1990) which is suggestive of an increase in ICa Both NMDA receptor activation (Choi, 1988; Silver & Ereciniska, 1990) and voltage-dependent Ca2+ channels may be involved (Choi, 1988; Amagasa, Ogawa & Yoshimoto, 1990). Differences between DVMs that hyperpolarize and depolarize during anoxia Given our arguments regarding the identity of various membrane currents which contribute to the membrane potential changes induced by anoxia, we can explain the responses in the following way. Those DVMs which hyperpolarize do so because enhancement of IK(DR) outweighs the combined depolarizing effects of a reduction in IA and an increase in Ica Input resistance always decreases and this implies that the increase in input resistance caused by the reduction in IA is less than the combined decrease in input resistance caused by increases in ICa and IK(DR). DVMs which depolarize in anoxia do so because the reduction in IA and increase in Ica outweighs any enhancement Of IK(DR)* Input resistance will increase when suppression of IA dominates but will decrease when the combined effects of ICa and IK(DR) dominate. The results of our study raise the possibility that individual DVMs have substantially different relative proportions of the various ion channels. Measurements of membrane potential and of the action potential characteristics reveal an apparently homogeneous population but these measures may be too coarse for the detection of different relative numbers of particular channels. Functionally, however, DVMs are not a homogeneous population and therefore the appropriate physiological response could readily be determined by varying the proportions of different 107

20 108 A. I. COWAN AND R. L. MARTIN channels. In this regard cardiac vagal efferent activity is known to be increased during systemic hypoxia (Potter & McCloskey, 1986) whereas presumably, nonessential gastrointestinal function is inhibited. Alternatively, effects of anoxia on the various ion channels may reflect the metabolic history of each DVM and its ability to buffer the increase in intracellular Ca2+ or decrease in intracellular ph which occurs (Silver & Ereciniska, 1990). We are grateful for technical assistance from Garry Rhodda and Laura O'Carroll. Our thanks go also to Steve Redman for reviewing the manuscript. This research was generously funded by the John Curtin School of Medical Research. REFERENCES ADAMS, P. R. & GALVAN, M. (1986). Voltage-dependent currents of vertebrate neurons and their role in membrane excitability. Advances in Neurology 44, AMAGASA. M.. OGAWA. A. & YOSHIMOTO, T. (1990). Effects of calcium and calcium antagonists against deprivation of glucose and oxygen in guinea pig hippocampal slices. Brain Research 526, 1-7. ASHCROFT, F. M. (1988). Adenosine 5'-triphosphate-sensitive potassium channels. Annual Review of Neuroscience BEN ARI, Y. (1989). Effect of glibenclamide, a selective blocker of an ATP-K' channel, on the anoxic response of hippocampal neurones. Pfiuigers Archiv 414, supply. 1, SI I-114. BROWN, D. A., GXHWILER, B. H., GRIFFITH, W. H. & HALLIWELL, J. V. (1990). Membrane currents in hippocampal neurons. Progress in Brain Research 83, CASTLE, N. A., HAYLETT, D.G. & JENKINSON, D. H. (1989). Toxins in the characterization of potassium channels. Trends in Neurosciences 12, CHoi. D. W. (1988). Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage. Trends in Neurosciences 11, DENNISON. S. J., O'CONNOR, B. L., APRISON, M. H., MERRITT, V. E. & FELTEN, D. L. (1981). Viscerotropic localization of preganglionic parasympathetic cell bodies of origin of the anterior and posterior subdiaphragmatic vagus nerves. Journal of Comparative Neurology 197, DONNELLY, D. F. & HADDAD,G.G. (1989). Hypoxia-induced changes in membrane potential and repetitive firing properties of medullary neurons: In-vitro studies in adult rats. Federation of the American Society for Experimental Biology Journal 3, A403. DREYER, S. E. (1991). Na'-activated K+ channels and voltage-evoked ionic currents in brain stem and parasympathetic neurones of the chick. Journal of Physiology 435, FUJIWARA, N., HIGASHI, H., SmMoJI, K. & YOSHIMURA, M. (1987). Effects of hypoxia on rat hippocampal neurons in vitro. Journal of Physiology 384, HADDAD,G. G. & DONNELLY, D. F. (1990). 02 deprivation induces a major depolarization in brain stem neurons in the adult but not in the neonatal rat. Journal of Physiology 429, HALLIWELL, J. V. & ADAMS, P. R. (1982). Voltage-clamp analysisof muscarinic excitation in hippocampal neurons. Brain Research 250, HANSEN, A. J. (1985). Effects of anoxia on ion distribution in the brain. Physiological Reviews 65, HANSEN, A. J., HOUNSGAARD, J. & JAHNSEN, H. (1982). Anoxia increases potassium conductance in hippocampal nerve cells. Acta Physiologica Scandinavica 115, HORIKAWA, K. & ARMSTRONG, W. E. (1988). A versatile means of intracellular labelling: injection of biocytin and its detection with avidin conjugates. Journal of Neuroscience Methods 25, JACQUIN, T., DENAVIT-SAUBI1E, M. & CHAMPAGNAT, J. (1989). Substance P and serotonin mutually reverse their excitatory effects in the rat nucleus tractus solitarius. Brain Research 502, JIANG, C. & HADDAD,G. G. (1991). Effect of anoxia on intracellular and extracellular potassium activity in hypoglossal neurons in vitro. Journal of Neurophysiology 66, KRNJEVId, K. & LEBLOND, J. (1987). Anoxia reversibly suppresses calcium currents in rat hippocampal slices. Canadian Journal of Physiology and Pharmacology 65,

21 DORSAL VA GAL MOTONEURONES AND A\NOXIAX10109 KRNJEVI6, K. & LEBLOND, J. (1989). Changes in membrane currents of hippocampal neurons evoked by brief anoxia. Journal of Neurophysiology 62, KRNJEVI6, K. & Xu, Y. (1990). Mechanisms underlying anoxic hyperpolarization of hippocampal neurons. Canadian Journal of Physiology and Pharmacology 68, LANCASTER, B. & NICOLL, R. A. (1987). Properties of two calcium-activated hyperpolarizations in rat hippocampal neurones. Journal of Physiology 389, LEBLOND, J. & KRNJEVIc, K. (1989). Hypoxic changes in hippocampal neurons. Journal of Neurophysiology 62, LEHMENKQ;HLER, A., CASPERS, H., SPECKMANN, E.-J., BINGMAN, D., LIPINSKI, H. G. & KERSTING, U. (1988). Neurons, glia and ions in hypoxia, hypercapnia and acidosis. In echanisms of Cerebral Hypoxia and Stroke, ed. SOMJEN, G., pp Plenum Press, New York. MOURRE, C., BEN ARI, Y., BERNARDI, H., FoSSET, M. & LAZDUNSKI, M. (1989). Antidiabetic sulfonylureas: localization of binding sites in the brain and effects on the hyperpolarization induced by anoxia in hippocampal slices. Brain Research 486, NICOLL, R. A. & ALGER, B. E. (1981). A simple chamber for recording from submerged brain slices. Journal of Neuroscience Methods 4, NITZAN, R., SEGEV, I. & YAROM, Y. (1990). Voltage behaviour along the irregular dendritic structure of morphologically and physiologically characterized vagal motoneurons in the guinea pig. Journal of Neurophysiology 63, NoSAKA, S., YAMAMOTO, T. & YASUNAGA, K. (1979). Localization of vagal cardioinhibitory preganglionic neurons within the rat brain stem. Journal of Comparative Neurology 186, POTTER, E. K. & MCCLOSKEY, D. I. (1986). Effects of hypoxia on cardiac vagal efferent activity and on the action of the vagus nerve at the heart in the dog. Journal of the Autonomic Nervous System 17, RUDY, B. (1988). Diversity and ubiquity of K channels. Neuroscience 25, SILVER, I. A. & ERECIJSKA, M. (1990). Intracellular and extracellular changes of [Ca2+] in hypoxia and ischaemia in rat brain in vivo. Journal of General Physiology 95, SOMJEN, G. G., AITKEN, P. G., BALESTRINO, M. & SCHIFF, S. J. (1987). Uses and abuses of in vitro systems in the study of pathophysiology of the central nervous system. In Brain Slices: Fundamentals, Applications and Implications, ed. SCHURR, A., TEYLER, T. J. & TSENG, M. T., pp Karger, Basel. STORM, J. F. (1990). Potassium currents in hippocampal pyramidal cells. Progress inbrainresearch 83, URBAIN, L. & SOMJEN, G. G. (1990). Reversible effects of hypoxia on neurons in mouse dorsal root ganglia in vitro. Brain Research 520, YAROM, Y., SUGIMORI, M. & LLINAIS, R. (1985). Ionic currents and firing patterns of mammalian vagal motoneurones in vitro. Neuroscience 6,