Membrane potentials of respiratory neurones during dizocilpine-induced apneusis in adult cats

Journal of Physiology (1996), 495.3, pp.851-861

Membrane potentials of respiratory neurones duringdizocilpine-induced apheusis in adult cats

A. Haji *, 0. Pierrefiche, R. Takeda *, A. S. Foutz, J. Champagnatand M. Denavit-Saubie t

Institut Alfred Fessard, Biologie Fonctionnelle du Neurone, CNRS, 91198 Gif-sur-Yvette,France and *Department of Pharmacology, Faculty of Medicine, Toyama Medical

and Pharmaceutical University, Toyama 930-01, Japan

1. In the vagotomized cat, blockade of NMDA receptors by dizocilpine (MK-801) produces anapneustic pattern of respiration characterized by a large increase in the duration ofinspiration.

2. To identify dizocilpine-induced disfacilitations and disinhibitions in respiratory neuronesgenerating the respiratory rhythm, membrane potential and input resistance of augmentinginspiratory (I; n = 11) and post-inspiratory (PI; n = 9) neurones were examined in theventral respiratory group area, before and after administration of dizocilpine(0 1-0 3 mg kg-' i.v.) in decerebrate, vagotomized, paralysed and artificially ventilated cats.

3. In I neurones, dizocilpine decreased the ramp depolarization and an 82% increase in inputresistance was observed during inspiration. The inspiratory phase was prolonged, leading toa sustained level of depolarization during apneusis. The amplitude of stage 1 expiratoryhyperpolarization decreased and its decay, which is normally slow, was faster. Throughoutthe remainder of expiration (stage 2) the membrane potential levelled off and the inputresistance increased slightly (by 15 %).

4. In PI neurones, dizocilpine depressed depolarization and suppressed firing in eight out ofnine cells during the stage 1 expiratory phase. This was associated with a large (91 %)increase of input resistance. The membrane potential switched quickly to stage 2 expiratoryrepolarization, during which a slight (19 %) increase in input resistance occurred.

5. The hyperpolarization of PI neurones during early inspiration was reduced in amplitude bydizocilpine and input resistance was increased by 75% during inspiration, indicating thatdizocilpine reduced the activity of the presynaptic inhibitory early-inspiratory (eI) neurones.

6. We conclude that NMDA receptor blockade in the respiratory network disfacilitates eI, I andPI neurones during their active phase. Decreased inhibitory processes during the inspiratoryphase probably play a major role in the prolongation of inspiration.

Systemic administration of dizocilpine, a non-competitive,selective blocker of the N-methyl-D-aspartic acid (NMDA)subtype of glutamate receptors (Wong, Kemp, Priestly,Knight, Woodruff & Iversen, 1986; Huettner & Bean, 1988),produces an apneustic breathing pattern in cats deprived ofafferent input from the lung stretch receptors (Foutz,Champagnat & Denavit-Saubie, 1988a, b; Foutz, Champagnat& Denavit-Saubie, 1989). This breathing pattern,characterized by a prolonged inspiratory phase and areduction of post-inspiratory activity in the phrenic neuro-gram, can be produced reversibly by preventing inflation of

the lungs, or permanently by vagotomy (Pierrefiche, Foutz,Champagnat & Denavit-Saubie, 1992). This latter studyrevealed that the discharge patterns of the different classesof respiratory neurones (RNs) recorded extracellularlyshowed characteristic changes during dizocilpine-inducedapneusis. We observed a prolongation of the discharge ofearly-inspiratory (eI) neurones throughout the apneusticinspiratory phase, and a large reduction of activity of thepost-inspiratory (PI) neuronal pool which discharges duringstage 1 expiration defined by post-inspiratory activity ofthe phrenic nerve (Pierrefiche et al. 1992).

t To whom correspondence should be addressed.

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A. Haji and others

These observations led to the hypothesis that dizocilpineaffected the el and PI neuroneswvhich are thought to play an

important role in determining inspiratory termination, byreciprocal inhibitions (Richter, 1982; Richter, Ballantyne &Remmers, 1986a; Duffin & Aweida, 1990). Inspiratorytermination would proceed as follows: the first, still reversiblestage of off-switch is produced by a class of late inspiratoryneurones termed 'off-switch neurones' (Pierrefiche et al. 1992)or 'burstIE neurones' (Oku, Tanaka & Ezure, 1992). Theonset of discharge of these neurones depends on disinhibitionfrom el neurones, whose activity thus sets the timing ofinspiration. Inspiration is then brought irreversibly to an endby the discharge of PI neurones.

Dizocilpine might produce apneusis by reducing inhibitionoriginating from a depressed PI neuronal pool, whileinhibition originating from the eI neuronal pool is decreasedin amplitude but maintained throughout the prolongedinspiratory phase. It was shown previously that during theapneusis induced by dizocilpine administered systemically,the activity of all classes of RNs except the stage 2expiratory (E2) neurones, is decreased (Pierrefiche et al.1992). This decrease probably arises mainly from a directblockade of NMDA receptors on RNs, since iontophoreticstudies have shown that endogenously activated NMDAreceptors contribute to the discharge of various types ofRNs (Pierrefiche, Schmid, Foutz & Denavit-Saubie, 1991).However, the large decrease in activity of PI neurones may

be partly due to indirect (network produced) effects. Thefiring of PI neurones is believed to be due to thecombination of excitatory synaptic drives and a post-

inhibitory rebound excitation (Richter, 1982) involving a

high-voltage-activated cation conductance (Takeda & Haji,1993), followed by an accommodation process leading to thearrest of their activity (Richter, Ballantyne & Mifflin, 1985;Richter et al. 1986a; Richter, Champagnat & Mifflin, 1986b;Pierrefiche, Champagnat & Richter, 1995). Thus, the effectsof intravenous dizoeilpine on I and PI neuronal pools wouldbe expected to reflect direct effects on the recorded neurones

as well as indirect effects coming from the reorganization ofthe bulbar respiratory network.

The aim of the present study was to test the effects of

dizoeilpine on PI and el neurones by intracellularmeasurements of early-inspiratory inhibition in PI neurones

and stage 1 expiratory inhibition in I neurones. To achievethis goal, we measured the membrane potential and input

resistance in bulbar RNs continuously before and after theapneustic pattern was induced by intravenous administrationof dizocilpine.

METHODSSurgical proceduresExperiments were carried out on adult cats weighing 2-5-3-8 kg in

accordance with the regulations fiomn the French agricultural

ministr-y. The animals were anaesthetized with halothane (2-0-2A5%halothane in 50% oxygen during induction and 1 5-1 8% during

surgery). The trachea was intubated and catheters were placed in

the femoral vein, the femoral artery and the urethra. The externalcarotid arteries were tied bilaterally distal to the branching of thelingual artery. The head of the animal was mounted on astereotaxic frame and the mid-collicular decerebration wasperformed according to the procedures described by Kirsten &St John (1978). After aspirating the brain rostral to the mid-collicular transection and securing haemostasis, a C2-C3laminectomy and an occipital craniotomy were performed. Phrenicnerves, cervical vagus nerves and superior laryngeal nerves wereexposed by a dorsal approach and cut bilaterally. A bilateral widepneumothorax was performed to reduce thoracic movements. Thenhalothane anaesthesia was discontinued.

The animals were paralysed with pancuronium bromide (Pavulon,03 mg kg-' initially and 01 mg kg-' h- i.v.) and the lungs weremechanically ventilated with oxygen-enriched air. Trachealpressure was kept below 8 cmH2O at maximum lung inflation. Anexpiratory flow resistance of 1-2 cmH2O was applied to preventcollapse of the lungs. The end-tidal concentrations of 02 and CO2were continuously monitored (Capnomac, Datex, Helsinki,Finland) and kept at 30-32% and 4-5%, respectively, by changingthe rate of ventilation (18-30 inflations min-') with a fixed tidalvolume of 10 ml kg-'. A 5% glucose-lactate Ringer solution wasinfused I.v. at the rate of 4 ml kg-' h-' and increased as required to20-30 ml kg-' h-' to keep arterial blood pressure higher than80 mmHg. Rectal temperature was maintained at 37-38°C byexternal heating.

Recording and stimulating proceduresThe central end of phrenic nerve was desheathed and placed onbipolar recording electrodes. The amplified phrenic activity wasfiltered (0-1-3000 Hz), rectified and integrated with a leakyintegrator (time constant, 01 s). RNs were sought in the ventralrespiratory area, extending 2-0-4-3 mm lateral to the mid-line,0-30 mm rostral to the obex, and 2X6-4-0 mm below the dorsalsurface of the medulla oblongata. Membrane potentials wererecorded with sharp glass micropipettes filled with 2 M potassiumcitrate with a resistance of 20-40 MQ as measured in brain tissue.I and PI neurones were identified by their patterns of membranepotential fluctuation in relation to phrenic nerve activity (Richter,1982; Haji & Takeda, 1993). In order to identify the axonalprojection of the recorded neurone, the central ends of vagus andsuperior laryngeal nerves were stimulated using bipolar electrodeswith a 0-1 ms pulse of 0-1-0-3 mA intensity. The cervical spinalcord was stimulated with an array of five concentric bipolarstimulating electrodes inserted into the ventrolateral part of theC2-C3 spinal cord, using 0-2 ms pulses of 0-2-0-5 mA. For currentclamping and measurements of input resistance, currents wereapplied through the recording electrode using a high frequencycurrent injection and voltage sampling method (8100-1, Dagan,Minneapolis, AIN, USA). To measure the input resistance, square-wave pulses of a constant intensity (between -0 7 and -10 nA;for 100 ms at 2 Hz ) were applied intracellularly and the resultingmembrane potential shifts from baseline between pulses weremeasured at each phase of the respiratory cycle. Phrenic and bulbarneuronal signals were stored on magnetic tape with digitalrecorders (DTR-1800, Biologic, Claix, Isere, France and PC-204,Sony) and played back for computer analysis using signalprocessing software (CED, Cambridge and D8S98-SV, Canopus,Kobe, Japan). The timing of changes in membrane potentialrelative to phrenic nerve discharge were determined byperistimulus averaging, using trigger points at inspiratory onsetand termination (Pierrefiche et al. 1992). The powrer spectrumanalysis of synaptic noise was performed by Fast FourierTransform (FFT) using 256 points sampled at 250 Hz.

J Phystol. 495.3852



Respiratory neuron e activity during apneusis

Table 1. Effects of dizocilpine on the respiratory phase duration in decerebrate vagotomized cats

Respiratory period

T ~~T Tr TTIn Te-1 Te-2 Ttot(s) (s) (s) (s)

Before dizocilpine 1-38 + 0 07 0 99 + 0413 2-62 + 0-32 5 07 + 0-32After dizocilpine 4-54 + 0 50** 0 30 + 0-06** 2-86 + 0-28 7-71 + 0 59**Percentage change (%) 329 30 109 153

Tin, inspiratory; Te,-, stage 1 expiratory (post-inspiratory); Te-2, stage 2 expiratory; T7{,, total respiratoryperiod. Values are means + S.E.M. (n = 20), obtained before and 30-40 min after the injection of dizocilpine(041-0 3 mg kg-' I.V.). ** P < 0 01, versus before values (paired t tests).

Drug administration and data analysisRecordings of neuronal activity started at least 4 h afterdiscontinuation of halothane anaesthesia. Dizocilpine (purchasedfrom Research Biochemicals International) was given intravenouslyat doses of 0 1-0 3 mg kg-', injected slowly over a period of5-10 min. These doses produced consistent effects both on bulbarneuronal and phrenic activity without any significant change inblood pressure. Thus intracellular recordings of neurones could bemaintained continuously before and after dizocilpine injection.Since the effects of dizocilpine lasted more than 12 h, only oneneurone per animal could be tested with dizocilpine. Membranepotentials were evaluated at three points in the respiratory cycle,i.e. during inspiration (In), stage 1 expiration (e-1) and stage 2expiration (e-2). The peak membrane potential measured duringthe active or depolarizing phase was designated as Dmax, and thatmeasured during the inactive or hyperpolarizing phase as Hmax.Relatively constant membrane potentials obtained during stage 2 ofexpiration were designated as Vei2 and used as a reference potentialto evaluate relative amplitudes of the active phase depolarizationand of the inactive phase hyperpolarization. AMembrane potentialand input resistance were measured before and 30-45 min afterdizocilpine injection, when a steady-state apneustic pattern wasestablished in the phrenic neurogram. Quantitative data werepresented as means + S.E.M. Differences in the mean values beforeand after the drug administration were analysed by two-tailedStudent's paired t tests. Values of membrane potential at differentmoments of the respiratory cycle were compared before and afterdizocilpine, by analysis of variance for repeated measures, followedby multiple comparisons.

RESULTSStable intracellular recordings were achieved in eleven I andnine PI neurones. All the neurones had membranepotentials more negative than -50 mV and action potentialswith overshoots and after-hyperpolarizations during thecontrol period and a stable record was maintainedcontinuously for at least 30 min after dizocilpineadministration. Of these twenty neurones, ten cells wereidentified as non-antidromically activated (NAA) neurones(6 I and 4 PI neurones) and ten cells as laryngeal motor(LM) neurones (5 I and 5 PI neurones). No bulbospinalneurones were encountered. Membrane potential responsesto dizocilpine did not differ between LM and NAA neurones.

As previously shown (Foutz et al. 1988a, b, 1989), intra-venous doses of dizocilpine (0 1-0 3 mg kg-') increased theduration of phrenic nerve inspiratory discharges byprolonging the initial ramp-like discharge with a constant'plateau-like' discharge (Figs 1-5). The peak amplitude ofintegrated phrenic discharge decreased to 54 6 + 4 7%(al = 20) of the pre-dizocilpine value. In addition, thephrenic post-inspiratory discharge decreased in bothamplitude and duration, while the duration of stage 2expiratory silence remained unchanged (Table 1). Theseeffects of dizocilpine appeared 5-10 min after the injectionand progressively increased in the following 5-10 min.Thus, a typical apneustic pattern was attained in10-20 min and continued for more than 12 h after the drugadministration.

Membrane potentials of inspiratory neuronesAt the onset of inspiration in eupnoea, I neurones started todepolarize 30-50 ms before the corresponding dischargeburst in the phrenic nerve. This pattern was unchanged bydizocilpine. Thereafter, dizocilpine prolonged the ramp-likeinspiratory depolarization (Fig. IA and B). The slope of thisramp changed from rectilinear to curvilinear as inspirationprogressed. This led to a constant plateau-like depolarization,a most striking effect consistently observed in I neuronesduring apneusis (Figs IA and 2A-C). In both eupnoea andapneusis, membrane potential started to repolarize30-50 ms before cessation of phrenic inspiratory discharge.The delays between the discharge of I neurones and phrenicnerve were therefore maintained after dizocilpine at bothinspiratory onset and termination (Fig. 1B). In eupnoea,the onset of expiration was marked by a large and rapidhyperpolarization lasting 100 ms, followed by a gradualrepolarization during the stage 1 expiratory phase (Fig. 1B,right panel). Following this the membrane potential slowlydepolarized during stage 2 expiration, but did not reach thefiring threshold (Figs 1 and 2). In apneusis, the membranepotential became relatively constant throughout theexpiratory phase (Fig. 2A-C).

Dizocilpine exerted a significantly different effect on themembrane potential in the different phases of the

853J Physiol.495.3



A. Haji and others

Table 2. Effects of dizocilpine on membrane potential of inspiratory and post-inspiratory neurones

Membrane potential

Dmax Hmax Ve2 AMP1 AMP2(mV) (mV) (mV) (mV) (mV)

I neurone (n= 11)Before dizocilpine -61P6 + 2-1 -73-3 + 2-5 -69-9 + 2-0 11P7 + 1P4 3-4 + 0 7After dizocilpine -70-1 + 2-2 * -75-6 + 2-1 -74-2 + 2-1 * 55 + 0 9 ** 14 + 05 **

PI neurone (n = 9)Before dizocilpine -63-9 + 1P8 -75-2 + 2-3 -70-1 + 2-0 11P3 + 1P1 5-1 + 0 5After dizocilpine -68-8 + 24 ** -74-8 + 2-6 -72-1 + 2'4 6-0 + 09 ** 2-7 + 0 4 **

Dmax, membrane potentials measured at the most depolarized point (during inspiration for I neurones,during stage 1 expiration for Pl neurones); Hmax, at the most hyperpolarized point (during stage 1expiration for I neurones, during inspiration for PI neurones); Ke2, during stage 2 expiration for both I andPI neurones. AMP1, the difference between Dmax and Hmax; AMP2, the difference between Ve 2 and Hmax.Values are means + S.E.M., obtained before and 30-45 min after dizocilpine injection. n, number of cells.** P < 0-01, * P < 0 05, significantly different from the corresponding before values (paired t tests).

A

-60 [

MP (mV)

-80 L

PN mV[

Before

11 II!~~~-- .

mm

Dizocilpine, 0.3 mg kg-' I.V.

I

I s__

e-2

MP

-74V)

-77 -

In

Before

I Dizocilpine

.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~l

Dizocilpine a.1= _.W----iL.Uk iJ

_^ "- .--

I qm.. "Ir il-

aU4ih~IIIdAlilip- qmuprw- .

e-1

12 mV

60 Ms

1 IIIu IAAi L.LJ. aI

.L. .

Figure 1. Membrane potential of a laryngeal I motoneuronePanels A and B were recorded at different amplifications and recording speeds, obtained from the same

I neurone before and 30 min after dizocilpine administration. MP, membrane potential; PN, phrenicneurogram; e-2, stage 2 expiration; In, inspiration; e-1, stage 1 expiration.

PN

6AL

I

A..

vII

01

854 J: Phy8iol.495.3

RL.



Respiratory neurone activity during apneusis

respiratory cycle (F(2,20) = 29-4, P < 0001, analysis ofvariance). Values of the membrane potential fluctuationsmeasured at both maximal depolarization (Dmax) ininspiration and Ve2 became more negative in apneusis(Table 2; Fig. 2), but the effects of dizocilpine on themaximal hyperpolarization (Hmax, measured during stage 1expiration) were inconsistent. Hmax shifted slightly in thehyperpolarizing direction in six I neurones and in thedepolarizing direction in five other I neurones. Figure 2illustrates variations of these dizocilpine-induced effectsamong the I neuronal population. Dizocilpine decreasedinspiratory depolarization (the difference between Dmax andVe 2) by 51% (from 8-3 to 441 mV, P< 0 01), and stage 1expiratory hyperpolarization (AMP2; the difference betweenVe 2 and Hmax) by 59% (from 3-4 to 1P4 mV, P < 0 01;Table 2). The amplitude of membrane potential fluctuations

A Before

(AMP1) evaluated as the difference between maximal hyper-polarization (Hmax) and maximal depolarization (Dma1)therefore also decreased significantly after dizocilpine(Table 2). Spontaneous cell discharge ceased in eight out ofeleven I neurones. In the remaining three I neurones, spikedischarge continued to occur at a lower frequency and with amore constant firing rate (Fig. 2A-C).

Input resistance measurements in inspiratoryneuronesInput resistance (R) was measured before and afterdizocilpine administration in five I neurones. Figure 3Aillustrates a typical response of an I neurone which showeda large increase of input resistance during inspiration and asmall increase during stage 1 expiration. Table 3 summarizesthe changes in input resistance throughout the respiratory

Dizocilpine, 0-2 mg kg-1 i.v.

_70[_

PN 1 mV 441I iLp_

50s o [rJ _AP ~ [ ( f . R W u LJ L w .(spikes s ) o

Before Dizocilpine, 0-3 mg kg-' I.V.

_||__|_~.."L"..b-G~~~~~~_ 7-NMM'

-55

MP (mV)

-65

PN 1 mV[

AP 313[ T(spikes s-1) °

CBefore

-65 F PMP(mV)_L

PN 1 mV

(spikes s-1) O[ rLr' Lj

Dizocilpine, 0-3 mg kg- i.v.

jIIii_W" "

1~~~ -V

,r .r,,iW ,JA

2 s

Figure 2. Changes in membrane potential in I neurones after dizocilpine injebtionMP, membrane potential; PN, phrenic neurogram; AP, discharge frequency (spikes s-1). Records A, B andC were obtained from three different I neurones before (left panel) and 30-45 min after iv. injection ofdizocilpine (right panel). The neurone in C was identified as a laryngeal motoneurone. Action potentialswere truncated.

E

855J Physiol.495.3



A. Haji and others

cycle before and after dizocilpine injection. Input resistanceincreased preferentially (by 82%) during the depolarizedphase (apneustic inspiration) but also increased during themost hyperpolarized phase (stage 1 expiration; Re1).

In summary, in I neurones dizocilpine decreaseddepolarization and hyperpolarization in the respiratorycycle and increased input resistance.

Membrane potentials of post-inspiratory neuronesIn eupnoea, PI neurones showed an abrupt hyper-polarization during early inspiration, which started30-50 ms before the onset of phrenic inspiratory dischargeand was followed by a progressive repolarization during theremainder of inspiration. This was followed in stage 1expiration by an abrupt depolarization which started30-50 ms before the termination of phrenic inspiratorydischarge and reached a peak depolarization within 100 ms

A Before

(Fig. 4). This depolarization was associated with a burst ofaction potentials. Then, repolarization occurred duringstage 2 expiration (Fig. 4). An occasional spike appearedduring this stage of expiration in some PI neurones(Fig. 5A). The timing of the onset of the stage 1 expiratorydepolarization in relation to the termination of phrenicnerve discharge was not modified by dizocilpine (Fig. 4B).However, the stage 1 expiratory depolarization was severelydepressed in duration and quickly reached the stage 2expiratory level of polarization (Fig. 5). Thus Dmaxconsistently shifted in the hyperpolarizing direction whileHmax did not change consistently after dizocilpine. Thisresulted in a decline of the amplitude of membrane potentialfluctuations (Figs 4 and 5; Table 2).

As in I neurones, dizocilpine exerted a significantlydifferent effect on membrane potential in the three phasesof the respiratory cycle (F(2,16) = 19f3; P< 0 001, analysis

Dizocilpine, 0-1 mg kg-1 i.v.

-65

MP (mV)

-80

PN mV [

10 mV

e-1 e-2 In e-1

A

BBefore Dizocilpine, 0-3 mg kg-' I.V.

-60-

-75

PN 1 mV [ 4A. .' l~~~~~~~~~~~~~~~~~~~~~~ sr~= ~In e-1 e-2 In e-1 e-2

i0 m v[N

100 ms

Figure 3. Changes in input resistance in an I laryngeal motoneurone (A) and a PI neurone (B)Records were taken before and 20-30 min after the injection of dizocilpine. Lower panels represent themembrane potential shifts induced by intracellular currents of a constant intensity (-1t0 nA for 100 ms,2 Hz, for both neurones), taken at a greater amplification and faster recording speed. Arrows indicate theonset of the abrupt downward deflection of the potential at the onset of the pulse. In, inspiration; e-1,

stage 1 expiration; e-2, stage 2 expiration.

e-2

100 ms

J: Physiol.495.3




Table 3. Effects of dizocilpine on input resistance of inspiratory and post-inspiratory neurones

Input resistance

RBn Re-1 Re-2(MQ) (MQl) (MQ)

I neurone (n = 5)Before 3X4 + 0X6 5-8 + 0 9 6-4 + 1P0After dizocilpine 6'2 + 0.9** 6-8 + 0.8* 7-4 + 0.6*Percentage change (%) 182 117 115

PI neurone (n = 5)Before 3-2+004 3-4+007 5-3+1 1After dizocilpine 5,6 + 07** 6-5 + 0 9** 6-3 + 0.8*Percentage change (%) 175 191 119

Rtn, input resistance during inspiration; Rei,- input resistance during stage 1 expiration; Re-2, inputresistance during stage 2 expiration. Values are means + S.E.M. n, the number of cells. ** P <0-01,* P < 0 05, significantly different from the corresponding before values (paired t test). Note the largeincrease in input resistance during the active phase in I and PI neurones, and during inspiration inPI neurones.

ABefore Dizocilpine, 0-3 mg kg-1 i.v.

-60

MP (mV)

-80

PN 1 mV[1 si s

B

MP

(mV)-69-71

PN

Figure 4. Membrane potential of a PI neurone before and 30 min after dizocilpine injectionRecords A and B were taken from the same PI laryngeal motoneurone at different amplifications andrecording speeds. In, inspiration; e-1, stage 1 expiration; e-2, stage 2 expiration. Note in A the progressivedecrease of hyperpolarization throughout the prolonged inspiratory phase.

--40-IFI

857J Physiol.495.3



A. Haji and others

of variance). Both stage 1 expiratory depolarization (thedifference between Dmax and Ve-2) and inspiratory hyper-polarization (AMP2; the difference between Ve2 and Hmax)decreased by 47%; from 6-2 to 3-3 mV and from 5*1 to2 7 mV, respectively (P < 0 01; Table 2). Thus theamplitude of inspiratory hyperpolarization, particularly theearly inspiratory hyperpolarization, was decreased in PIneurones by dizocilpine (Figs 4 and 5; Table 2). However theonset of hyperpolarization relative to inspiratory onset wasnot significantly affected. The membrane potential becamerelatively flat during the apneustic phase of the phrenicneurogram and the slope of repolarization developed moreslowly during apneustic inspiration than in eupnoea. Thefiring of action potentials ceased in eight out of nine PIcells, three of which first displayed a low frequencydischarge throughout the respiratory cycle during 1-5 minafter dizocilpine injection and then became silent (Fig. 5A).

The membrane potential of PI neurones frequentlydisplayed oscillating waves characteristic of high frequencyoscillation (HFO), (Mitchell & Herbert 1974; Remmers,

ABefore

-50 I

MP (mV)

-65 L

PN 1 mV [ _

50

AP (spikes s1

Takeda, Schultz & Haji, 1985; Takeda & Haji, 1992). Themost prominent oscillation rate corresponded to 80 Hzduring eupnoea and remained unaltered during dizocilpine-induced apneusis. However, the amplitude of each wave wasconsiderably decreased (Fig. 4B). Similar effects of dizocilpineon HFO waves in membrane potential were observed in fivePI neurones. We did not observe any other type of oscillatingsynaptic wave during dizocilpine-induced apneusis.

Input resistance measurements in post-inspiratoryneuronesIn five PI neurones, input resistance was measuredthroughout the respiratory cycle before and after dizocilpine.Input resistance increased by 75% during inspiratoryhyperpolarization (Rin) and by 91% during stage 1expiratory depolarization (Fig. 3B; Table 3, Rei). Thus, asin I neurones, the resistance in PI neurones increased afterdizocilpine in both the most depolarized and the mosthyperpolarized states, which were also decreased inamplitude.

Dizocilpine, 0-3 mg kg-' i.v. After 30 min

m ba.wII-

.YK.JLJ.fl.K5 s

BBefore Dizocilpine, 0.1 mg kg-' i.v. After 20 min

MP (mV) :[

-65mVX[

PN 1 mV__50

AP (spikes s-1)0

5 s

Figure 5. Changes in membrane potential in two different PI neurones before and afterdizoCilpine injectionLeft panels in A and B were taken before, and the middle and right panels were taken 2-5 min and20-30 min after dizocilpine injection, respectively. The neurone in B was identified as a laryngealmotoneurone.

J Phy-siol.495.3858

vO




DISCUSSIONThe present study describes large changes in membranepotentials and input resistance in I and PI neurones afteri.v. administration of dizocilpine, which has been shown toselectively block the NMDA receptor channels controllingrespiratory timing mechanisms (Foutz et al. 1988a). It istherefore likely that dizocilpine, diffusing within theextracellular space in the brainstem respiratory network,modified the activity of respiratory neurones by blockingthe NMDA receptor-related depolarization. Most of thechanges affecting the membrane potential reflect alterationsof the respiratory cycle: prolongation of inspiration,shortening of stage 1 expiration, unchanged stage 2expiration. We have found, however, some phases of therespiratory cycle during which the membrane input resistancewas particularly increased by NMDA receptor blockade,which indicates a selective action of dizocilpine on certainneural mechanisms.

Effect of dizocilpine during membrane depolarizationin I and PI neuronesNMDA and non-NMDA receptors exert co-operative effectsin producing the discharge activity of RNs (Pierrefiche et al.1991; Pierrefiche, Foutz, Champagnat & Denavit-Saubie,1994). It is expected that dizocilpine would be more activeat depolarized potentials because a voltage-dependent Mg2+block of these channels prevents binding of the drug athyperpolarized potentials (Nowak, Bregestovski, Ascher,Herbet & Prochiantz, 1984; Huettner & Bean, 1988). Aprevious study with iontophoretic application of NMDA andnon-NMDA agonists on RNs showed that the voltage-dependent properties of NMDA channels may be expressedwithin the range of active phase depolarization in each typeof RN (Pierrefiche et al. 1991). This can account for themuch larger increase of membrane resistance induced bydizocilpine in I and PI neurones during their active periods,than in other parts of the respiratory cycle.

Post-inhibitory rebound excitation has been proposed as amechanism contributing to depolarization in PI neurones(Richter et al. 1986a; Bianchi, Champagnat & Denavit-Saubie, 1995). Excitatory synaptic drives as well as voltage-dependent conductances are involved in this depolarization(Takeda & Haji, 1993; Richter, Champagnat, Jacquin &Benacka, 1993). Post-inhibitory rebound excitation inPI neurones might have been weaker after dizocilpine,because early-inspiratory inhibition observed in theseneurones was reduced. This mechanism of action isuncertain, however, since the most negative membranepotential (Hmax) during inspiration was unchanged bydizocilpine. Another possible mechanism of action would bea reduction of high voltage-activated Ca2+ conductance(Takeda & Haji, 1993; Richter et al. 1993), resulting fromthe effect of dizocilpine on excitatory synaptic potentials.

Disfacilitation of I neurones by dizocilpine did not result inthe elimination of the HFO waves of around 80 Hz thatwere recorded during inspiration in PI neurones. However,

we did not observe correlated waves oscillating at afrequency of 10-20 Hz, which were clearly demonstratedduring MK-801-induced apneusis in anaesthetized cats(Feldman, Windhorst, Anders & Richter, 1992). Because thefrequency of HFO waves was not significantly alteredduring dizocilpine-induced apneusis and because HFOwaves were not consistently present in all PI neurones, thiskind of oscillation is not likely to be the consequence ofNMDA receptor blockade, as was suggested in the previousstudy for the low frequency oscillation (Feldman et al. 1992).

Effects of dizocilpine resulting from an action atpresynaptic inhibitory early-inspiratory neuronesAn important result of the present study is that dizocilpinereduced the inhibition occurring during inspiration inPI neurones (75% increase in input resistance). Thedecreased amplitude (47 %) of the abrupt hyperpolarizationof PI neurones at the onset of inspiration is attributed toreduced inhibitory currents, which are known to originatefrom eI neurones (Richter et al. 1986a; Ezure, Manabe &Otake, 1989; Ezure, 1990; Champagnat & Richter, 1994).Strong inhibitory inputs from GABAergic and glycinergicneurones have been found to be responsible for the periodicmembrane hyperpolarizations and decreased membraneresistance during the respiratory cycle (Richter, 1982;Champagnat, Denavit-Saubie, Moyanova & Rondouin,1982; Haji, Remmers, Connelly & Takeda, 1990; Haji,Takeda & Remmers, 1992). The effect of dizocilpine oninspiratory inhibition of PI neurones clearly does not resultfrom a direct action of dizocilpine on the recorded neuronessince, to our knowledge, there is no evidence of an interactionof dizocilpine with GABAergic or glycinergic (strychnine-sensitive) sites, and the blockade of NMDA receptorchannels by dizocilpine is lower during membrane hyper-polarization (Nowak et al. 1984). Since the lengthening ofinspiration paralleled the blockade of NMDA receptors, itcan be argued that the reduced inspiratory inhibition was aconsequence of the altered timing of synaptic interactionsbrought about by the prolongation of inspiration. A studyby Feldman et al. (1992) indicates that this was not the case.In animals with intact vagus nerves, NMDA receptors werefirst blocked by dizocilpine, then a reversible apneusis wasobtained by withholding lung inflation during selectedrespiratory cycles. In the same conditions of NMDAreceptor blockade, short respiratory cycles obtained withlung inflation were compared with long respiratory cyclesobtained by withholding lung inflation. This revealedrelatively small differences in the input resistance and early-inspiratory membrane hyperpolarization of E2 neuronesbetween the conditions of eupnoea and apneusis. Therefore,in the present study, the large increase in input resistanceand the reduced membrane hyperpolarization of PI neuronesduring inspiration were not a consequence of theprolongation of this phase. They probably resulted mainlyfrom NMDA receptor blockade on presynaptic inhibitoryinspiratory interneurones.

859J Physiol.495.3



860 A. Haji and others J Physiol.495.3

Unperturbed stage 2 expiratory phaseFor both I and PI neurones, relatively small changes ofinput resistance occurred in the stage 2 expiratory phaseafter administration of dizocilpine. Since the activity of PIneurones was depressed and the stage 1 expiratoryinhibitions were shortened, the main inhibitory processespersisting in the expiratory phase during an apneusticbreathing originated from E2 neurones. These neurones arethe only ones which are not depressed during apneusticbreathing after i.v. injection of dizocilpine (Pierrefiche et al.1992). This obviously results from changes in therespiratory network during apneusis, possibly a decreasedinhibition from PI neurones, because during eupnoea, thedischarge of E2 cells is decreased, not increased, by NMDAantagonists applied either systemically (Feldman et al. 1992)or directly by iontophoresis (Pierrefiche et al. 1991).

Reduced stage 1 expiratory phaseAn important perturbation during dizocilpine-inducedapneusis was the depression of PI neuronal activity, and theconcomitantly shortened stage 1 expiratory hyper-polarization of I neurones. A possible involvement of PIneuronal depression in the genesis of apneusis musttherefore be examined. PI neurones are a major source ofinhibitions to other main types of bulbar RNs (Richter,1982; Ballantyne & Richter, 1984; Richter et al. 1986a), andit has been proposed that they produce the 'irreversible'phase of inspiratory off-switch by preventing a rebound ofinspiratory activity (von Euler, 1986; Richter, 1982).Nevertheless, the impaired activity of PI neurones may notbe directly involved in the production of apneusis, for tworeasons. Firstly, the small increase in membrane resistanceof I neurones during stage 1 expiration (17 %) was of thesame magnitude as the change observed during stage 2expiration and was much lower than the 75% increase in theinput resistance of PI neurones during inspiration. Thus, ofthe two major sources of inhibition within the respiratorynetwork, stage 1 expiratory inhibition was less stronglyaffected than early-inspiratory inhibition. Secondly, ageneralized blockade of non-NMDA receptors by a selectiveantagonist, also produces a potent depression of PI neuronesand of phrenic nerve after-discharge, but without anyconcomitent increase in the duration of inspiration(Pierrefiche et al. 1994).

ConclusionI and PI neurones have been recorded intracellularly beforeand after the administration of dizocilpine. The prolongationof inspiration during the induced apneusis was found toresult from a unique combination of dizocilpine effects uponthe different types of RNs. In this respect, the action ofdizocilpine differs from that of other agents that do notproduce apneusis, such as non-NMDA receptor antagonists,which depress excitatory synaptic activities and respiratorymotor output (Pierrefiche et al. 1994), and some anaestheticswhich produce a general weakening of excitatory and

inhibitory synaptic activity in many classes of RNs(Takeda, Haji & Hukuhara, 1990). In contrast, dizocilpinecauses a disfacilitation of el, I and PI neurones but not E2neurones. Therefore, a reduced activation of NMDAreceptors and a consequent reduction of the effectiveness ofsynaptic inhibitory pathways between early-inspiratory andother neurones are probably responsible for inspiratoryprolongation.

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AcknowledgementsWVe thank Dr K. Grant for stylistic correction of the manuscript.This work was supported by CNRS/MONBUSHO, DRET 95/091(France) and by a Grand-in-Aid for Scientific Research from theAIinistry of Education, Science and Culture of Japan (nos 05680672and 07044238).

Author's email addressAM. Denavit-Saubie: [email protected]

Received 12 October 1995; accepted 23 Mlay 1996.



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Membrane potentials of respiratory neurones during dizocilpine-induced apneusis in adult cats