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The effects of QX-314 on medullary respiratory neurones
22
Brain Re~ear~h, 421)(1987) 22 -~1 Elsevicl
BRE 12829
The effects of QX-314 on medullary respiratory neurones Steven Mifflin* and Diethelm W. Richter 1. Physiologisches Institut, Universittit Heidelberg, Heidelberg (F. R. G.)
(Accepted 3 February 1987) Key words: Respiratory neuron; Repetitive discharge; Calcium current; QX-314
The synaptic and current-evoked responses of respiratory neurones located in the nucleus of the tractus solitarius, the para- and retroambigual regions and the nucleus ambiguus, were examined after voltage-dependent sodium currents were blocked by intracellular application of the quaternary lidocaine derivative QX-314. (1) QX-314 abolished orthodromically and antidromically evoked action potential discharge. Only antidromic action potentials recovered during negative DC current injection. (2) QX-314 did not alter the amplitude or duration of small and short excitatory and inhibitory postsynaptic potentials evoked by vagus or superior laryngeal nerve stimulation. Larger and longer waves of spontaneous membrane depolarizations, however, were slightly diminished. (3) The repetitive discharge evoked by depolarizing current pulses was blocked by QX-314. Positive current pulses produced less membrane depolarization than under control and often evoked only a single action potential at the beginning of the pulse, indicating that Q X-314 interferes with the processes responsible for repetitive firing. (4) When fast spike discharges were completely blocked, positive current pulses occasionally evoked depolarizing 'spikes' and potentials which were followed by a hyperpolarization. We conclude that a noninactivating sodium inward current and calcium currents contribute to the electroresponsiveness of respiratory neurones.
INTRODUCTION
input conductance of neurones studied in vitro is reduced by an o r d e r of magnitude 16'31 c o m p a r e d with
A network of inspiratory, postinspiratory and expiratory neurones within the b r a i n s t e m generates the respiratory rhythm 25'26. Synaptic interaction be-
those m e a s u r e d in in vivo studies. This is probably due to the fact that the dendritic tree of neurones studied in brainstem slices has been cut to less than 400 ~ m and their synaptic inputs are severed 6-8n. M e m b r a n e conductances d e m o n s t r a b l e in vitro might, therefore, be shunted in the in vivo situation. In addition, the m e m b r a n e potential of M R N s rhythmically oscillates during the respiratory cycle by 10-15 mV as a result of alternating synaptic depolarization and hyperpolarization and this might also influence the functional significance of voltage-dependent m e m b r a n e conductances 7'~2'%3° M e m b r a n e conductances of M R N s have been little studied in vivo due to technical difficulties, i.e. ongoing synaptic activity and inability to m a n i p u l a t e the extraceilular environment. In recent experiments 2° 21, however, we have found evidence that an increase in intracellular calcium has important consequences
tween m e d u l l a r y respiratory neurones ( M R N s ) within this network is an essential function and, therefore, the m e m b r a n e p r o p e r t i e s of M R N s are critically i m p o r t a n t because they ultimately d e t e r m i n e the conversion of synaptic depolarization into the discharge of action potentials 27'2s'3°. Studies in vitro using brain slice techniques have examined specific m e m b r a n e p r o p e r t i e s of n e u r o n e s within the b r a i n s t e m nuclei in which respiratory neurones are located 6-sA1. H o w e v e r , this a p p r o a c h to the p r o b l e m of r e s p i r a t o r y rhythmogenesis has several limitations: firstly, one cannot functionally identify neurones solely on the basis of their anatomical location. Secondly, the functional significance of a given m e m b r a n e conductance remains speculative, as the
* Present address: Dept. Internal Medicine, University of Iowa, Iowa City, IA 52242, U.S.A. Correspondence: D.W. Richter, I. Physiologisches Institut der Universit~it Heidelberg, Im Neuenheimer Feld 326, D-6900 Heidelberg, F.R.G. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
23 for the function of MRNs in vivo. We, therefore, wanted to gain a better insight into the nature of this calcium influx and used the quaternary |idocaine derivative QX-314, to block the transient inward sodium current which generates the action potential in axons 12'143638 and neuronal s o m a t a 5'9'22'33'35 and, if present, to block non-inactivating sodium currents 5"9'22"33-35. This enabled us to examine MRNs in the absence of action potential discharge carried by sodium ions. QX-314 has the advantage of binding to sodium channels from inside the membrane 14'24'36, so that it can be applied intracellularly leaving the synaptic inputs to the neurones intact. Since we examined the effects of QX-314 on MRNs in vivo, we could functionally identify the neurones, and therefore ascertain the functional significance of the effects. MATERIALS AND METHODS Experiments were performed on 18 cats of either sex (2.2-3.6 kg) anaesthetized with pentobarbitone (35-40 mg/kg i.p., supplemented as needed). The animals were artificially ventilated, paralyzed (gallamine triethiodide, 4-8 mg/h i.v.) and a pneumothorax was performed bilaterally. Arterial pressure was monitored via a cannula in the femoral artery. The animals' temperature was monitored via a rectal thermoprobe and maintained at 36-37 °C by a heating pad and an infrared lamp. Both phrenic nerves were cut, their central ends desheathed and placed on bipolar electrodes. Phrenic nerve activity was taken as an index of central respiratory rhythm. Respiratory neurones of the dorsal group were located 0.5-1.5 mm rostral to the obex, 1-2 mm lateral to the midline and 1.2-1.5 mm below the dorsal surface (ventral regions of the solitary tract) and the neurones of the ventral group were located - 3 mm caudal to +2 mm rostral to the obex, 2.5-3.5 mm lateral to the midline and 1.6-3 mm below the dorsal surface (para- and retroambigual regions) o r - 1 mm caudal to +2 mm rostral to the obex, 3.5-4 mm lateral to the midline and 3.5-4.2 mm below the dorsal surface (ambigual region). The axonal projections of neurones were determined by spinal cord stimulation at the C 2 level (bulbospinal neurones, BS) and stimulation of the cervical vagus and superior laryngeal nerves (vagal motoneurones, V). Neurones are
referred to as 'not antidromically activated' (NAA), if their axon projection could not be determined by these means. Intracellularly recorded potentials were led off to a voltage follower equipped with capacitance compensation and a bridge circuit for injecting current through the recording microelectrode. Recording electrodes were filled with 4 M potassium acetate and 50-100 mM N-(2,6-dimethylphenylcarbamoylmethyl-)triethylammonium bromide (QX-314; Astra Pharmaceutical Products Inc., Worcester, MA) with pH adjusted to 7.1. With the tips broken, electrodes had DC resistances of 30-80 Mr2. Only those neurones in which maximal membrane potential exceeded -40 mV after blockage of orthodromic discharge were analyzed. This arbitrary limit was set as it was the minimum value accepted in the in vitro studies of the membrane properties of neurones within these brainstem nuclei 7'~11. The records presented here were plotted on an x - y plotter after storage on a digital oscilloscope. Following antidromic identification of a neurone, we applied positive current pulses (2(I-50 ms duration at a frequency of 0.3-1.0 Hz) throughout the respiratory cycle. These pulses served several purposes: (A) Although the assumed transport number of the cation QX-314 is very low~, the pulses, in association with passive diffusion, facilitate the ejection of QX-314 from the microelectrode. (B) The action potential discharge evoked by the depolarizing pulses provides an index of the status of the fast, inward sodium current and the degree of blockage of this conductance by QX-314. (C) The evoked action potential discharge should also enhance the rate of QX-314 blockage of sodium channels as this process is use-dependent 9-36,3s. (D) Hyperpolarization removes QX-314 bound to the sodium channels 9.36.3s and as MRNs are hyperpolarized by spontaneous inhibitory postsynaptic potentials (IPSPs) during a portion of the respiratory cycle25, the positive current pulse may help to maintain the QX-314 block during these periods. When the QX-314 induced cessation of ~spontaneous' discharge of neurones was associated with a larger than 5 mV decrease in maximal negative DC membrane potential, the neurone was discarded from further analysis as similar changes can result from cell damage. An additional condition for acceptance was that the QX-314 treated neurones retained
24 their capability of discharging fast action potentials of normal amplitudes when negative DC current was injected.
In some neurones (16 of 42) subtle changes in the slow membrane potential fluctuations occurring during the respiratory cycle became visible as illustrated in Fig. 1. These changes were confined to the period of membrane depolarization. In the expiratory neurone depicted in the left panel, maximal membrane potential during inspiration remained constant but there was a decrease in the rate and maximal level of membrane depolarization during expiration after QX-314 was injected. Similarly, in the postinspiratory neurone shown on the right panel, the membrane depolarization during the expiratory interval was smaller compared to control conditions. The changes of membrane depolarization ~ccurred although phrenic burst discharges remained unaltered. However, there were variations in the duration of the stage I or stage II phase of the expiratory interval
RESULTS
Orthodromic discharge The membrane potential of MRNs alternates with the respiratory cycle between periods of synaptically induced depolarization (EPSPs), during which action potential discharge occurs, and synaptically induced hyperpolarization by IPSPs. These 'spontaneously' evoked orthodromic action potentials disappeared to reveal the underlying rhythmic swings of membrane potential (Fig. 1), following intracellular QX-314 application over a time period which varied from neurone to neurone (ranging from 4-20 min).
control
-68
PNA
i
PNA i 3sec
Q X - 314
i
/!
~ri~d
Fig. 1. Membrane potential pattern of a bulbospinai expiratory (left) and vagal postinspiratory (right) neurone :before (upper traces) and after intracellular injection of QX-314 (middle traces). The membrane potential patterns of each neurone are superimposed in the bottom traces. This is a pen recorder display, therefore the action potentials in the control records are attenuated. PNA, integrated phrenic nerve activity.
25
,A ( ontrol ~
QX-314
lqhns
B
Fig. 2. In A the response of an inspiratory, bulbospinal fl-neurone in the nucleus of the tractus solitarius to vagal nerve stimulation (filled circle) followed by a 3 nA positive current pulse before and after injection of QX-314 is illustrated. Each record contains two sweeps superimposed during inspiration (membrane potential = -54 mV). The intensity of vagal nerve stimulation was adjusted so that, under control conditions, it did not evoke action potential discharge. The duration, but not the intensity of the current pulse was increased after injection of QX314. Note that the voltage drop across the membrane during the current pulse was much smaller after QX-314 injection. In B the EPSP evoked by vagal nerve stimulation is displayed at higher gain and faster sweep speed (stimulus artifacts are the vertical bars). The responses before and after injection of QX314 are superimposed separately for stimuli applied during inspiration or expiration.
which indicates that the central r e s p i r a t o r y activity
Short-lasting synaptic p o t e n t i a l s w e r e not a l t e r e d
may not have r e m a i n e d a b s o l u t e l y constant. T h e ob-
by Q X - 3 1 4 injections. This was e x a m i n e d by stimu-
servations on p h r e n i c n e r v e activity, t h e r e f o r e , m a k e
lating v a r i o u s p e r i p h e r a l n e r v e s k n o w n to h a v e syn-
if difficult to r e f e r these changes in the m e m b r a n e po-
aptic c o n n e c t i o n s with M R N s . E l e c t r i c a l s t i m u l a t i o n
tential p a t t e r n of M R N s solely to the effect of Q X -
of the cervical vagus n e r v e p r o d u c e d a short latency,
314.
c o m p o u n d E P S P in inspiratory b e t a n e u r o n e s of the
A 1
E__[
lOmV
i
2ms
i
,omv[ '2ms
i
C
|ms
I
~lqqs ~
Fig. 3. Temporal decline in action potential amplitude following QX-314 injection and its recovery following hyperpolarizing DC current injection. A: expiratory vagal neurone A1 (membrane potential = -66 mV) - - decline in action potential amplitude evoked by 4 separate 5 nA depolarizing pulses delivered at increasing times after initial impalement (only the initial 10 ms of the 30 ms current pulses are shown). A2 - - recovery of the antidromic action potential in the same cell as hyperpolarizing DC current was applied. The afterhyperpolarizations following the action potentials were reduced in A2 compared to A1 because negative DC current was injected into the neurone. B, C: decline of amplitude of antidromic action potential as QX-314 was injected in 2 bulbospinal inspiratory neurones (membrane potential: B = -61 mV, C = -50 mV).
26 ventral nucleus of the tractus solitarius due to their synaptic input from slowly adapting lung stretch receptor afferents 2. Fig. 2 illustrates that after the action potential discharge evoked by a positive current pulse was blocked by QX-314, the EPSP evoked by vagal nerve stimulation was equal in amplitude and duration to that observed under control (n = 4). Similarly,there was no change in the amplitude or duration of the IPSP evoked by superior laryngeal nerve stimulation in bulbospinal expiratory neurones (n -
ing impalement with a QX-314-fiUed electrode (Figs. 2, 4 and 5B). This is in striking contrast to the discharge properties of M R N s observed using non-QX314-filled electrodes 2~,27. As repetitive discharge started to fail, occasionally a broad depolarizing wave, followed by a hyperpolarization, was observed (Fig. 4). After repetitive discharge had been abolished, typically a single action potential persisted
3, not illustrated).
Antidromic discharge Concomitant with the blockade of orthodromic discharge, we observed a reduction in the amplitude of antidromicaily evoked action potentials (Fig. 3). Antidromically evoked action potentials were more resistant to QX-314 than the orthodromically elicited discharge, requiring an additional 2-11 min of QX314 application to be abolished. There was a decrease in spike amplitude, an increase in spike duration and an increase in the initial s e g m e n t - s o m a t o dendritic (IS-SD) delay (Fig. 3C). After blockade of the SD-component of the antidromic action potential the IS-spike also decrease when the QX-314 injection was continued (Fig. 3B, C). We used the antidromic spike to examine the reversibility of the QX-314 blockade, it has been shown that a hyperpolarizing current will remove QX-314 bound to the sodium channel 9'36'38, therefore we examined the effects of a hyperpolarizing D C current on the antidromic spike after its SD-component had been abolished by QX-314. Hyperpolarizing current (2-11 hA) restored the SD-component of the antidromic action potential (Fig. 3A2). This recovery developed over 4 - 1 0 rain. Once the SD-component had reappeared, further application of hyperpolarizing current decreased the IS-SD delay and the amplitude of action potentials was further increased to nearly control levels. Recovery of the orthodromic discharge during the respiratory cycle was not observed.
Current evoked discharge We next examined the effects of QX-314 on the repetitive discharge of M R N s evoked by positive current pulses. The repetitive discharge evoked by a positive current pulse was quickly eliminated follow-
/
f
I0 m VI
d
!
4 ms
Fig. 4. Response of a bulbospinal, inspiratory neurone to positive current pulse of 3.5 nA as QX-314 begins to block the repetitive discharge. The pulses were given during the first 3 min following impalement of the neurone. Note the decrease in action potential amplitude, the inerease in action potential duration and increase in the duration of the interspike interval. In the bottom trace as action potential discharge failed a broad depolarizing 'wave' followed by a hyperpolarization was revealed (membrane potential = -56 mV).
27 at the onset of the current pulse. Within the time of intracellular recording, it was impossible to eliminate a presumed sodium spike in 25 of 42 neurones. The repetitive discharge characteristics of neurones were immediately affected by QX-314. In 18 of 25 neurones, blockade of repetitive discharge was associated with a decrease in the membrane depolarization induced by the current pulse (Figs. 2A and 5). The decrease in the amplitude of the voltage response to a positive current pulse was essentially complete by the time that repetitive discharge (evoked by a positive current pulse) had ceased and only a single sodium action potential persisted at the onset of the current pulse. At the speed with which QX-314 affected mem-
brane properties, we could not measure the current-voltage relationship under control conditions. We, therefore, used a constant positive current pulse as a test. Prior to the QX-314 block, the depolarization in response to this test current pulse was difficult to quantify because of the repetitive discharge. We, therefore, estimated the 'control' level of membrane depolarization as the threshold level of the second or fourth action potentials evoked by the test current pulse. Using this approximation, we measured a reduction of the input resistance in response to a positive current pulse ranging from 23 to 43% in 18 cells. In neurones in which it was possible to eliminate the first action potential at the onset of a positive current pulse (n = 25), several patterns of response were
A
IOnA[ t IOmV[,~ B
10nA[
I
I
4m'~ 4hA
[
41rlb
C l[]nA[ .............
I
5
nl\
4ms Fig. 5. Response of an expiratory vagal neurone to antidromic stimulus followed by positive current pulse. A: responses recorded within the first min after impalement with the QX-314-filled electrode (membrane potential = -58 mV). B 1: responses of the same cell to identical stimuli recorded after 8 min. B2: effect of increasing the amplitude of the current pulse. The trace was taken app. 30 s after that depicted in B1. C: recovery of both antidromic and current evoked action potential discharge after and during hyperpolarizing DC current injection. The hyperpolarizing current injection started 4 min before this recording was made. The zero level of DC current is indicated by the dashed line. (Note the decrease in the afterhyperpolarization following the action potentials.)
28 evoked by an equivalent amplitude current pulse. Typically (n = 22) a rounded depolarization 'wave' was present at the onset of the current pulse (Figs. 5B1, 7Aa, Bb). In 10 neurones, a small 'spike' was generated at the peak of the depolarizing 'wave', a clear inflection marking its origin (Fig. 3AI, arrows in Fig. 6A, B). The spikes either arose spontaneously from the rounded depolarizing 'wave' at the onset of the current pulse (Figs. 3A1 and 6A, B) or could be evoked by increasing the intensity of the positive current pulse (Fig. 7A, B). An increase in the intensity of the positive current pulse was required to generate large and broad 'depolarizations'. (Figs. 5B2 and 6C, D). They arose from either the rounded depolarizing 'wave' (Fig. 5B2) or with no obvious transition from a preceding depolarizing 'wave' (Figs. 6D and 7C). Currents that were subthreshold for these depolarizing 'potentials' revealed smooth electrotonic charging of the membrane capacitance (Figs. 6D and 7C). The depolarizing 'potentials' did not exhibit any obvious inflection
A
on their rising phase. The relative distribution of the 'waves', 'spikes' and depolarizing 'potentials' observed in the various classes of MRNs are summarized in Table I. At a constant amplitude positive current pulse, the waveform of the depolarizing 'wave', the 'spike' or the depolarizing 'potential' was consistent in shape, duration and time to peak in any given neurone. The 'spikes' and depolarizing 'potentials' were followed by a hyperpolarization the durations and amplitudes of which ranged between 4.3-8.8 ms and 2-5 mV. respectively. There was no obvious difference in the depolarizing 'waves', 'spikes' and depolarizing 'potentials' observed in V-neurones as compared to BSor NAA-neurones and no difference whether they were observed in inspiratory (I) or expiratory (E) neurones of a given class. Increasing the intensity of the positive current pulse decreased the time to peak of the 'spikes' and depolarizing 'potentials', but did not alter their amplitudes (Fig. 7). Depolarizing 'waves', 'spikes' and
B E-V
l-BS
!
F---'-I 2ms
!
2ms
C
i-v
b
-
~-
-
D
~nA 2nA
....
a[
~ C
lore V[
20rn V [ •
I 4ms 4ms Fig. 6. The responses to positive current pulses are illustrated before and after QX-314 blockade of action potential discharge (A-C). Low-thresholdsodium action potentials were blocked in D. The current pulses are illustrated when their intensity was varied. The onset and end of the positive current pulses are indicated by the capacitative transient artifacts in the traces A and B. I-BS, inspiratory bulbospinal neurones (A + D); I-V, inspiratoryvagal motoneurone; E-V, expiratory vagal motoneurone. Membrane potentials were -56 mV (A), -64 mV (B), -57 mV (C) and -53 mV (D).
29 TABLE I
A
S~A[ ~bI
'
'
[l-ls
For a description of waves, spikes and depolarizing potentials see text.
2ms B
d
S
1,, i
2ms
3n
lOmV[
'
l - -
4ms
'
Effect of QX-314 on the responses to positive current pulses in medullary respiratory neurones
'
'
I-V
-I ~
Fig. 7. Depolarizing 'waves', 'spikes" and 'potentials' observed after blockage of low-threshold sodium action potentials. The responses were evoked by positive current pulses of increasing intensity. I-BS, inspiratory bulbospinal neurone; E-V, expiratory vagal motoneurone; I-V, inspiratory vagal motoneurone. Membrane potentials were -54 mV (A), -64 mV (B) and -57 mV (C). 'potentials' were never observed to occur repetitively, regardless of the amplitude or duration of the positive current pulse. This was most likely because we did not block potassium conductances (recall that the depolarizing responses were followed by hyperpolarizations). H o w e v e r , the current pulses which we had to use due to the p o o r current carrying abilities of QX-314 electrodes, may have been too weak and to short to reveal such properties. We have previously r e p o r t e d that following the burst of action potential discharge evoked by a positive current pulse, there is a hyperpolarization which is markedly reduced following intracellular injection of the calcium chelator E G T A 2°. Such 'post-pulse hyperpolarizations' following the positive current pulses became progressively smaller in the present study as QX-314 abolished repetitive action potential discharge. DISCUSSION This study illustrates the limitations and advantages of examining the m e m b r a n e properties of
Neurones (n) Vagal Inspiratory (14) Expiratory (11) Postinsp. (8) Bulbospinal Inspiratory* (19) Expiratory (9)
First A Ps
Waves
Spikes
Depolarizing potentials
7
7
3
1
4
3
2
1
3
3
1
-
7
6
2
1
2
1
1
-
1
-
Not antidromically activated Inspiratory* (8) 1 I Expiratory (3)
Postinsp. (4)
-
1
1
-
-
-
-
* Combined populations from nuclei of the tractus solitarius, the para- and rctro-ambigual region.
MRNs in vivo. The principal limitation is that one cannot perform pharmacological manipulations of the extracellular environment necessary to definitively ascertain the ionic mechanism underlying a particular m e m b r a n e event, for example the depolarizing potentials which we observed after blockade of sodium electrogenesis. H o w e v e r , in vivo studies have the advantage that one can ascertain specific physiologic characteristics of the recorded neurone, e.g. it is definitely respiratory. In addition, it is possible to investigate whether a response observed in vitro has a functional relevance under normal circumstances. This question is especially critical in M R N s in vivo where, due to tonic synaptic b o m b a r d m e n t , input conductances are an o r d e r of magnitude higher than in neurones in the same brainstem nuclei studied in vitro (see ref. 30). As observed in other neurones 5.9,22,33,35, QX-314 blocked the transient inward sodium current generating repetitive discharge of action potentials in MRNs. As intracellular injection of QX-314 produced no change in the amplitude or duration of short-lasting synaptic potentials of small amplitudes,
30 it seems reasonable to conclude that the rapidly inactivating sodium current which gives rise to action potential discharge does not play any significant role in the rhythmic membrane potential fluctuations associated with the respiratory cycle, i.e., the ion channels activated by synaptic processes are distinct from those which generate the action potential. QX-314 has been also shown to effectively block depolarizing inward rectification 5'9'22"33-35. The QX-314 induced reduction of membrane depolarization evoked by current pulses (Figs. 2 + 5) indicates that a similar inward rectifying current, carried by sodium ions, exists in MRNs. The reduction in the rate of rise of 'spontaneous' membrane depolarizations (Fig. 1) may be explained similarly and points to the functional significance of the persistent sodium current. A persistent sodium conductance facilitates repetitive firing in neocortical neurones 34. The failure of MRNs to discharge repetitively following injections of QX-314 (Figs. 2, 4, 5), therefore could in part result from a QX-314 block of such a conductance 5 9,35. We conclude that depolarizing inward rectification, produced by a sodium current, is an important factor contributing to the repetitive discharge of MRNs. Calcium spikes have been demonstrated in virtually every neurone studied (see refs. 1 and 10 for refs.). In vitro recordings from neurones within medullary nuclei known to contain respiratory neurones have revealed depolarizing potentials which were abolished when extracellular calcium ions were replaced by cobalt ions 7'8. Under the in vivo conditions of the present experiments we were also able to completely eliminate fast action potential discharge in 25 of 42 neurones. Although we cannot eliminate the possibility that the depolarizing 'waves', 'potentials" and 'spikes' represent residual patches of membrane capable of sodium electrogenesis, there is resemblance between the depolarizing responses we observed (Fig. 5, 6C, D and 7C) and the calcium induced potential changes demonstrated in in vitro preparations 7,8,15,3°,32,35. Depolarizing 'waves' may result from activation of a low-threshold, transient calcium current and there are several explanations for the short duration of the depolarizing 'potentials'
REFERENCES 1 Adams, P.R., Voltage-dependent conductances of vertebrate neurones, Trends Neurosci.. 5 (1982) 116-119.
and 'spikes" compared to the calcium spikes which are supposed to originate from high-threshold, persistent calcium currents ~3'15"~'>,3:~-~5. Firstly, in the present study, we did not attempt to block potassium conductances. Several sorts of potassium conductances were, no doubt, activated as indicated by the fact that the 'spikes" and depolarizing 'potentials' were invariably followed by hyperpolarizations. Persistence of potassium conductances may also explain the fact that we never observed repetitive firing of the high threshold potentials. Indeed. repetitive calcium spikes were not observed unless potassium blockers were applied to neocortical neurones ~5 Block of repetitive discharge reduces calcium influx which explains the disappearance of the 'post-pulse hyperpolarization' seen in the present study. Secondly, calcium potentials are generated in the dendrites of a variety of neurones;'S ~e t:-i,~. ~:.>,~7 The low input resistance and short electrotonic length of the dendrites of MRNs ~', due to repetitive activation of distributed excitatory and inhibitory synapses 3''~252<293t , limit the dendritic spread of current injected into the soma >. If, in MRNs. a proportion of the ion channels producing the depolarizing potential have a dendritic locus, they could be partly shunted and not fully activated in vivo. In conclusion, this study provides an insight into the membrane properties of MRNs in vivo. It indicates that a depolarizing inward rectification, produced by a non-inactivating sodium current, and calcium currents contribute to the electroresponsiveness of respiratory neurones. We assume that these represent important processes whereby MRNs convert synaptic depolarization into action potential discharge to generate the respiratory rhythm. ACKNOWLEDGEMENTS The generous gift of QX-314 by Astra Pharmaceutical Products, Inc., is gratefully acknowledged. We are indebted to Mrs. Annemarie Bischoff and Mrs. Anita Ktihner for their technical assistance and we thank Drs, D. Ballantyne and E. Lawson for valuable comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft and the Alexander yon Humboldt Stiftung.
2 Backman, S.B., Anders, C., Ballantync, D., Rohrig, N., Camerer, H., Mifflin, S., Jordan, D., Dickhaus, H., Spyer, K.M. and Richter, D.W., Evidence for a monosynaptic connection between slowly adaptin~ stretch receptor after-
31 ents and inspiratory beta neurones, Pfliigers Arch., 402 (1984) 129-136. 3 Ballantyne, D. and Richter, D.W., Postsynaptic inhibition of bulbar inspiratory neurones in the cat, J. Physiol. (London), 348 (1984) 67-87. 4 Ballantyne, D. and Richter, D.W., The non-uniform character of expiratory synaptic activity in expiratory bulbospinal neurones of the cat, J. Physiol. (London), 370 (1986) 433-456. 5 Bernardo, L.S., Masukawa, L.M. and Prince, D.A., Electrophysiology of isolated hippocampal pyramidal dendrites, J. Neurosci., 2 (1982) 1614-1622. 6 Champagnat, J., Jacquin, T. and Richter, D.W., Voltagedependent currents in neurones of the nuclei of the solitary tract of rat brainstem slices, Pfliigers Arch., 406 (1986) 372-379. 7 Champagnat, J., Richter, D.W., Jacquin, T. and DenavitSaubie, M., Voltage-dependent conductances in neurones of the ventrolateral NTS in rat brainstem slices. In C. von Euler and H. Lagercrantz (Eds.), Neurobiology of the Control of Breathing, Raven press, New York, 1986, pp. 217-221. 8 Champagnat, J., Siggins, G.R., Koda, L.Y. and DenavitSaubie, M., Synaptic response of neurons of the nucleus tractus solitarius in vitro, Brain Research, 325 (1985) 49-56. 9 Connors, B.W. and Prince, D.A., Effects of local anesthetic QX-314 on the membrane properties of hippocampal pyramidal neurons, J. Pharmacol. Exp. Ther., 220 (1982) 476-481. 10 Crill, W.E. and Schwindt, P.C., Active currents in mammalian central neurons, Trends Neurosci., 6 (1983) 236-240. 11 Dekin, M.S. and Getting, P.A., Firing pattern of neurons in the nucleus tractus solitarius: modulation by membrane hyperpolarization, Brain Research, 324 (1984) 180-184. 12 Frazier, D.T., Narahashi, T. and Yamada, M., The site of action and form of local anesthetics. II. Experiments with quaternary compounds, J. Pharrnacol. Exp. Ther., 171 (1970) 45-51. 13 Harada, Y. and Takahashi, T., The calcium component of the action potential in spinal motoneurones of the rat, J. Physiol. (London), 335 (1983) 89-100. 14 Hille, B., Local anesthetic: hydrophilic and hydrophobic pathways for the drug receptor reaction, J. Gen. Physiol., 69 (1977) 497-515. 15 Kita, H., Kita, T. and Kitai, S.T., Active membrane properties of rat neostriatal neurons in an in vitro slice preparation, Exp. Brain Res., 60 (1985) 54-62. 16 Kreuter. F., Richter, D.W., Camerer, H. and Senekowitsch, R., Studies on the synaptic interconnection between bulbar respiratory neurones of cats, Pfliiger Arch., 380 (1979) 245-257. 17 Llinas, R. and Sugimori, M., Calcium conductances in Purkinje cell dendrites: their role in development and integration, Prog. Brain Res., 51 (1979) 323-334. 18 Llinas, R. and Sugimori, M., Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices, J. Physiol. (London), 305 (1980) 197-213. 19 Llinas, R. and Yarom, Y., Properties and distribution of ionic conductances generating electroresponsiveness of mammalian inferior olivary neurones in vitro, J. Physiol. (London), 315 (1981) 569-584. 20 Mifflin, S., Ballantyne, D., Backman, S. and Richter, D.W., Evidence for a calcium activated potassium conductance in medullary respiratory neurones. In A.L. Bianchi and M. Denavit-Saubie (Eds.), Neurogenesis of Central Re-
spiratory Rhythm, MTP Press, Lancaster, 1985, pp. 179-182. 21 Mifflin, S.W. and Richter, D.W., Repetitive firing properties of medullary respiratory neurones in the cat, J. Physiol. (London), 37 (1986) 116P. 22 Mulle, C., Steriade, M. and Deschenes, M., The effects of QX-314 on thalamic neurons, Brain Research, 333 (1985) 350-354. 23 Murase, K. and Randic, M., Electrophysiological properties of rat spinal dorsal horn neurones in vitro: calciumdependent action potentials, J. Physiol. (London), 334 (1983) 141-153. 24 Narahashi, T., Frazier, D.T. and Yamada, M., The site of action and active form of local anesthetics. I. Theory and pH experiments with tertiary compounds, J. Pharmacol. Exp. Ther., 171 (1970) 32-44. 25 Richter, D.W., Generation of maintenance of the respiratory rhythm, J. Exp. Biol., 100 (1982) 93-107. 26 Richter, D.W. and Ballantyne, D., A three phase theory about the basic respiratory pattern generator. In M.E. Schl~ifke, H.P. Koepchen and W.R. See (Eds.), Central
Neurone Environment and the Control Systems of Breathing and Circulation, Springer, Berlin, 1983, pp. 164-174. 27 Richter, D:W. and Heyde, F., Accommodative reactions of medullary respiratory neurons of the cat, J. Neurophysiol., 38 (1975) 1172-1180. 28 Richter, D.W., Ballantyne, D. and Mifflin, S., Interaction between postsynaptic activities and membrane properties of medullary respiratory neurones. In A.L. Bianchi and M. Denavit-Saubie (Eds.), Neurogenesis of Central Respiratory Rhythm, MTP Press, Lancaster, 1985, pp. 172-178. 29 Richter, D.W., Camerer, H., Meesmann, M. and R6hrig, N., Studies on the synaptic interconnection between bulbar respiratory neurones of cats, Pflfigers Arch., 380 (1979) 245-257. 30 Richter, D.W., Champagnat, J. and Mifflin, S., Membrane properties involved in respiratory rhythm generation. In C. von Euler and H. Lagercrantz (Eds.), Neurobiology of the Control of Breathing, Raven Press, New York, 1986, pp. 141-147. 31 Richter, D.W., Heyde, F. and Gabriel, M., Intracellular recordings from different types of medullary respiratory neurons of the cat, J. Neurophysiol., 38 (1975) 1162-1171. 32 Schwartzkroin, P.A. and Slawsky, M., Probable calcium spikes in hippocampal neurons, Brain Research, 135 (1977) 157-161. 33 Schwindt, P.C. and Crill, W.E., Properties of a persistent inward current in normal and TEA-injected motoneurons, J. Neurophysiol., 43 (1980) 1700-1724. 34 Stafstrom, C.E., Schwindt, P.C. and Crill, W.E., Repetitive firing in Layer V neurons from cat neocortex in vitro, J. Neurophysiol., 52 (1984) 264-277. 35 Stafstrom, C.E., Schwindt, P.C., Chubb, M.C. and Crill, W.E., Properties of persistent sodium conductance and calcium conductance of Layer V neurons from cat sensorimotor cortex in vitro, J. Neurophysiol., 53 (1985) 153-170. 36 Strichartz, G.R., The inhibition of sodium currents in myelinated nerve by quaternary derivatives of lidocaine, J. Gen. Physiol., 62 (1973) 37-57. 37- Wong, R.K., Prince, D.A. and Basbaum, A.L., Intradendritic recordings from hippocampal neurons, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 986-990. 38 Yeh, J., Sodium inactivation mechanism modulates QX314 block of sodium channels in squid axons, Biophys. J., 24 (1978) 568-574.
Brain Re~ear~h, 421)(1987) 22 -~1 Elsevicl
BRE 12829
The effects of QX-314 on medullary respiratory neurones Steven Mifflin* and Diethelm W. Richter 1. Physiologisches Institut, Universittit Heidelberg, Heidelberg (F. R. G.)
(Accepted 3 February 1987) Key words: Respiratory neuron; Repetitive discharge; Calcium current; QX-314
The synaptic and current-evoked responses of respiratory neurones located in the nucleus of the tractus solitarius, the para- and retroambigual regions and the nucleus ambiguus, were examined after voltage-dependent sodium currents were blocked by intracellular application of the quaternary lidocaine derivative QX-314. (1) QX-314 abolished orthodromically and antidromically evoked action potential discharge. Only antidromic action potentials recovered during negative DC current injection. (2) QX-314 did not alter the amplitude or duration of small and short excitatory and inhibitory postsynaptic potentials evoked by vagus or superior laryngeal nerve stimulation. Larger and longer waves of spontaneous membrane depolarizations, however, were slightly diminished. (3) The repetitive discharge evoked by depolarizing current pulses was blocked by QX-314. Positive current pulses produced less membrane depolarization than under control and often evoked only a single action potential at the beginning of the pulse, indicating that Q X-314 interferes with the processes responsible for repetitive firing. (4) When fast spike discharges were completely blocked, positive current pulses occasionally evoked depolarizing 'spikes' and potentials which were followed by a hyperpolarization. We conclude that a noninactivating sodium inward current and calcium currents contribute to the electroresponsiveness of respiratory neurones.
INTRODUCTION
input conductance of neurones studied in vitro is reduced by an o r d e r of magnitude 16'31 c o m p a r e d with
A network of inspiratory, postinspiratory and expiratory neurones within the b r a i n s t e m generates the respiratory rhythm 25'26. Synaptic interaction be-
those m e a s u r e d in in vivo studies. This is probably due to the fact that the dendritic tree of neurones studied in brainstem slices has been cut to less than 400 ~ m and their synaptic inputs are severed 6-8n. M e m b r a n e conductances d e m o n s t r a b l e in vitro might, therefore, be shunted in the in vivo situation. In addition, the m e m b r a n e potential of M R N s rhythmically oscillates during the respiratory cycle by 10-15 mV as a result of alternating synaptic depolarization and hyperpolarization and this might also influence the functional significance of voltage-dependent m e m b r a n e conductances 7'~2'%3° M e m b r a n e conductances of M R N s have been little studied in vivo due to technical difficulties, i.e. ongoing synaptic activity and inability to m a n i p u l a t e the extraceilular environment. In recent experiments 2° 21, however, we have found evidence that an increase in intracellular calcium has important consequences
tween m e d u l l a r y respiratory neurones ( M R N s ) within this network is an essential function and, therefore, the m e m b r a n e p r o p e r t i e s of M R N s are critically i m p o r t a n t because they ultimately d e t e r m i n e the conversion of synaptic depolarization into the discharge of action potentials 27'2s'3°. Studies in vitro using brain slice techniques have examined specific m e m b r a n e p r o p e r t i e s of n e u r o n e s within the b r a i n s t e m nuclei in which respiratory neurones are located 6-sA1. H o w e v e r , this a p p r o a c h to the p r o b l e m of r e s p i r a t o r y rhythmogenesis has several limitations: firstly, one cannot functionally identify neurones solely on the basis of their anatomical location. Secondly, the functional significance of a given m e m b r a n e conductance remains speculative, as the
* Present address: Dept. Internal Medicine, University of Iowa, Iowa City, IA 52242, U.S.A. Correspondence: D.W. Richter, I. Physiologisches Institut der Universit~it Heidelberg, Im Neuenheimer Feld 326, D-6900 Heidelberg, F.R.G. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
23 for the function of MRNs in vivo. We, therefore, wanted to gain a better insight into the nature of this calcium influx and used the quaternary |idocaine derivative QX-314, to block the transient inward sodium current which generates the action potential in axons 12'143638 and neuronal s o m a t a 5'9'22'33'35 and, if present, to block non-inactivating sodium currents 5"9'22"33-35. This enabled us to examine MRNs in the absence of action potential discharge carried by sodium ions. QX-314 has the advantage of binding to sodium channels from inside the membrane 14'24'36, so that it can be applied intracellularly leaving the synaptic inputs to the neurones intact. Since we examined the effects of QX-314 on MRNs in vivo, we could functionally identify the neurones, and therefore ascertain the functional significance of the effects. MATERIALS AND METHODS Experiments were performed on 18 cats of either sex (2.2-3.6 kg) anaesthetized with pentobarbitone (35-40 mg/kg i.p., supplemented as needed). The animals were artificially ventilated, paralyzed (gallamine triethiodide, 4-8 mg/h i.v.) and a pneumothorax was performed bilaterally. Arterial pressure was monitored via a cannula in the femoral artery. The animals' temperature was monitored via a rectal thermoprobe and maintained at 36-37 °C by a heating pad and an infrared lamp. Both phrenic nerves were cut, their central ends desheathed and placed on bipolar electrodes. Phrenic nerve activity was taken as an index of central respiratory rhythm. Respiratory neurones of the dorsal group were located 0.5-1.5 mm rostral to the obex, 1-2 mm lateral to the midline and 1.2-1.5 mm below the dorsal surface (ventral regions of the solitary tract) and the neurones of the ventral group were located - 3 mm caudal to +2 mm rostral to the obex, 2.5-3.5 mm lateral to the midline and 1.6-3 mm below the dorsal surface (para- and retroambigual regions) o r - 1 mm caudal to +2 mm rostral to the obex, 3.5-4 mm lateral to the midline and 3.5-4.2 mm below the dorsal surface (ambigual region). The axonal projections of neurones were determined by spinal cord stimulation at the C 2 level (bulbospinal neurones, BS) and stimulation of the cervical vagus and superior laryngeal nerves (vagal motoneurones, V). Neurones are
referred to as 'not antidromically activated' (NAA), if their axon projection could not be determined by these means. Intracellularly recorded potentials were led off to a voltage follower equipped with capacitance compensation and a bridge circuit for injecting current through the recording microelectrode. Recording electrodes were filled with 4 M potassium acetate and 50-100 mM N-(2,6-dimethylphenylcarbamoylmethyl-)triethylammonium bromide (QX-314; Astra Pharmaceutical Products Inc., Worcester, MA) with pH adjusted to 7.1. With the tips broken, electrodes had DC resistances of 30-80 Mr2. Only those neurones in which maximal membrane potential exceeded -40 mV after blockage of orthodromic discharge were analyzed. This arbitrary limit was set as it was the minimum value accepted in the in vitro studies of the membrane properties of neurones within these brainstem nuclei 7'~11. The records presented here were plotted on an x - y plotter after storage on a digital oscilloscope. Following antidromic identification of a neurone, we applied positive current pulses (2(I-50 ms duration at a frequency of 0.3-1.0 Hz) throughout the respiratory cycle. These pulses served several purposes: (A) Although the assumed transport number of the cation QX-314 is very low~, the pulses, in association with passive diffusion, facilitate the ejection of QX-314 from the microelectrode. (B) The action potential discharge evoked by the depolarizing pulses provides an index of the status of the fast, inward sodium current and the degree of blockage of this conductance by QX-314. (C) The evoked action potential discharge should also enhance the rate of QX-314 blockage of sodium channels as this process is use-dependent 9-36,3s. (D) Hyperpolarization removes QX-314 bound to the sodium channels 9.36.3s and as MRNs are hyperpolarized by spontaneous inhibitory postsynaptic potentials (IPSPs) during a portion of the respiratory cycle25, the positive current pulse may help to maintain the QX-314 block during these periods. When the QX-314 induced cessation of ~spontaneous' discharge of neurones was associated with a larger than 5 mV decrease in maximal negative DC membrane potential, the neurone was discarded from further analysis as similar changes can result from cell damage. An additional condition for acceptance was that the QX-314 treated neurones retained
24 their capability of discharging fast action potentials of normal amplitudes when negative DC current was injected.
In some neurones (16 of 42) subtle changes in the slow membrane potential fluctuations occurring during the respiratory cycle became visible as illustrated in Fig. 1. These changes were confined to the period of membrane depolarization. In the expiratory neurone depicted in the left panel, maximal membrane potential during inspiration remained constant but there was a decrease in the rate and maximal level of membrane depolarization during expiration after QX-314 was injected. Similarly, in the postinspiratory neurone shown on the right panel, the membrane depolarization during the expiratory interval was smaller compared to control conditions. The changes of membrane depolarization ~ccurred although phrenic burst discharges remained unaltered. However, there were variations in the duration of the stage I or stage II phase of the expiratory interval
RESULTS
Orthodromic discharge The membrane potential of MRNs alternates with the respiratory cycle between periods of synaptically induced depolarization (EPSPs), during which action potential discharge occurs, and synaptically induced hyperpolarization by IPSPs. These 'spontaneously' evoked orthodromic action potentials disappeared to reveal the underlying rhythmic swings of membrane potential (Fig. 1), following intracellular QX-314 application over a time period which varied from neurone to neurone (ranging from 4-20 min).
control
-68
PNA
i
PNA i 3sec
Q X - 314
i
/!
~ri~d
Fig. 1. Membrane potential pattern of a bulbospinai expiratory (left) and vagal postinspiratory (right) neurone :before (upper traces) and after intracellular injection of QX-314 (middle traces). The membrane potential patterns of each neurone are superimposed in the bottom traces. This is a pen recorder display, therefore the action potentials in the control records are attenuated. PNA, integrated phrenic nerve activity.
25
,A ( ontrol ~
QX-314
lqhns
B
Fig. 2. In A the response of an inspiratory, bulbospinal fl-neurone in the nucleus of the tractus solitarius to vagal nerve stimulation (filled circle) followed by a 3 nA positive current pulse before and after injection of QX-314 is illustrated. Each record contains two sweeps superimposed during inspiration (membrane potential = -54 mV). The intensity of vagal nerve stimulation was adjusted so that, under control conditions, it did not evoke action potential discharge. The duration, but not the intensity of the current pulse was increased after injection of QX314. Note that the voltage drop across the membrane during the current pulse was much smaller after QX-314 injection. In B the EPSP evoked by vagal nerve stimulation is displayed at higher gain and faster sweep speed (stimulus artifacts are the vertical bars). The responses before and after injection of QX314 are superimposed separately for stimuli applied during inspiration or expiration.
which indicates that the central r e s p i r a t o r y activity
Short-lasting synaptic p o t e n t i a l s w e r e not a l t e r e d
may not have r e m a i n e d a b s o l u t e l y constant. T h e ob-
by Q X - 3 1 4 injections. This was e x a m i n e d by stimu-
servations on p h r e n i c n e r v e activity, t h e r e f o r e , m a k e
lating v a r i o u s p e r i p h e r a l n e r v e s k n o w n to h a v e syn-
if difficult to r e f e r these changes in the m e m b r a n e po-
aptic c o n n e c t i o n s with M R N s . E l e c t r i c a l s t i m u l a t i o n
tential p a t t e r n of M R N s solely to the effect of Q X -
of the cervical vagus n e r v e p r o d u c e d a short latency,
314.
c o m p o u n d E P S P in inspiratory b e t a n e u r o n e s of the
A 1
E__[
lOmV
i
2ms
i
,omv[ '2ms
i
C
|ms
I
~lqqs ~
Fig. 3. Temporal decline in action potential amplitude following QX-314 injection and its recovery following hyperpolarizing DC current injection. A: expiratory vagal neurone A1 (membrane potential = -66 mV) - - decline in action potential amplitude evoked by 4 separate 5 nA depolarizing pulses delivered at increasing times after initial impalement (only the initial 10 ms of the 30 ms current pulses are shown). A2 - - recovery of the antidromic action potential in the same cell as hyperpolarizing DC current was applied. The afterhyperpolarizations following the action potentials were reduced in A2 compared to A1 because negative DC current was injected into the neurone. B, C: decline of amplitude of antidromic action potential as QX-314 was injected in 2 bulbospinal inspiratory neurones (membrane potential: B = -61 mV, C = -50 mV).
26 ventral nucleus of the tractus solitarius due to their synaptic input from slowly adapting lung stretch receptor afferents 2. Fig. 2 illustrates that after the action potential discharge evoked by a positive current pulse was blocked by QX-314, the EPSP evoked by vagal nerve stimulation was equal in amplitude and duration to that observed under control (n = 4). Similarly,there was no change in the amplitude or duration of the IPSP evoked by superior laryngeal nerve stimulation in bulbospinal expiratory neurones (n -
ing impalement with a QX-314-fiUed electrode (Figs. 2, 4 and 5B). This is in striking contrast to the discharge properties of M R N s observed using non-QX314-filled electrodes 2~,27. As repetitive discharge started to fail, occasionally a broad depolarizing wave, followed by a hyperpolarization, was observed (Fig. 4). After repetitive discharge had been abolished, typically a single action potential persisted
3, not illustrated).
Antidromic discharge Concomitant with the blockade of orthodromic discharge, we observed a reduction in the amplitude of antidromicaily evoked action potentials (Fig. 3). Antidromically evoked action potentials were more resistant to QX-314 than the orthodromically elicited discharge, requiring an additional 2-11 min of QX314 application to be abolished. There was a decrease in spike amplitude, an increase in spike duration and an increase in the initial s e g m e n t - s o m a t o dendritic (IS-SD) delay (Fig. 3C). After blockade of the SD-component of the antidromic action potential the IS-spike also decrease when the QX-314 injection was continued (Fig. 3B, C). We used the antidromic spike to examine the reversibility of the QX-314 blockade, it has been shown that a hyperpolarizing current will remove QX-314 bound to the sodium channel 9'36'38, therefore we examined the effects of a hyperpolarizing D C current on the antidromic spike after its SD-component had been abolished by QX-314. Hyperpolarizing current (2-11 hA) restored the SD-component of the antidromic action potential (Fig. 3A2). This recovery developed over 4 - 1 0 rain. Once the SD-component had reappeared, further application of hyperpolarizing current decreased the IS-SD delay and the amplitude of action potentials was further increased to nearly control levels. Recovery of the orthodromic discharge during the respiratory cycle was not observed.
Current evoked discharge We next examined the effects of QX-314 on the repetitive discharge of M R N s evoked by positive current pulses. The repetitive discharge evoked by a positive current pulse was quickly eliminated follow-
/
f
I0 m VI
d
!
4 ms
Fig. 4. Response of a bulbospinal, inspiratory neurone to positive current pulse of 3.5 nA as QX-314 begins to block the repetitive discharge. The pulses were given during the first 3 min following impalement of the neurone. Note the decrease in action potential amplitude, the inerease in action potential duration and increase in the duration of the interspike interval. In the bottom trace as action potential discharge failed a broad depolarizing 'wave' followed by a hyperpolarization was revealed (membrane potential = -56 mV).
27 at the onset of the current pulse. Within the time of intracellular recording, it was impossible to eliminate a presumed sodium spike in 25 of 42 neurones. The repetitive discharge characteristics of neurones were immediately affected by QX-314. In 18 of 25 neurones, blockade of repetitive discharge was associated with a decrease in the membrane depolarization induced by the current pulse (Figs. 2A and 5). The decrease in the amplitude of the voltage response to a positive current pulse was essentially complete by the time that repetitive discharge (evoked by a positive current pulse) had ceased and only a single sodium action potential persisted at the onset of the current pulse. At the speed with which QX-314 affected mem-
brane properties, we could not measure the current-voltage relationship under control conditions. We, therefore, used a constant positive current pulse as a test. Prior to the QX-314 block, the depolarization in response to this test current pulse was difficult to quantify because of the repetitive discharge. We, therefore, estimated the 'control' level of membrane depolarization as the threshold level of the second or fourth action potentials evoked by the test current pulse. Using this approximation, we measured a reduction of the input resistance in response to a positive current pulse ranging from 23 to 43% in 18 cells. In neurones in which it was possible to eliminate the first action potential at the onset of a positive current pulse (n = 25), several patterns of response were
A
IOnA[ t IOmV[,~ B
10nA[
I
I
4m'~ 4hA
[
41rlb
C l[]nA[ .............
I
5
nl\
4ms Fig. 5. Response of an expiratory vagal neurone to antidromic stimulus followed by positive current pulse. A: responses recorded within the first min after impalement with the QX-314-filled electrode (membrane potential = -58 mV). B 1: responses of the same cell to identical stimuli recorded after 8 min. B2: effect of increasing the amplitude of the current pulse. The trace was taken app. 30 s after that depicted in B1. C: recovery of both antidromic and current evoked action potential discharge after and during hyperpolarizing DC current injection. The hyperpolarizing current injection started 4 min before this recording was made. The zero level of DC current is indicated by the dashed line. (Note the decrease in the afterhyperpolarization following the action potentials.)
28 evoked by an equivalent amplitude current pulse. Typically (n = 22) a rounded depolarization 'wave' was present at the onset of the current pulse (Figs. 5B1, 7Aa, Bb). In 10 neurones, a small 'spike' was generated at the peak of the depolarizing 'wave', a clear inflection marking its origin (Fig. 3AI, arrows in Fig. 6A, B). The spikes either arose spontaneously from the rounded depolarizing 'wave' at the onset of the current pulse (Figs. 3A1 and 6A, B) or could be evoked by increasing the intensity of the positive current pulse (Fig. 7A, B). An increase in the intensity of the positive current pulse was required to generate large and broad 'depolarizations'. (Figs. 5B2 and 6C, D). They arose from either the rounded depolarizing 'wave' (Fig. 5B2) or with no obvious transition from a preceding depolarizing 'wave' (Figs. 6D and 7C). Currents that were subthreshold for these depolarizing 'potentials' revealed smooth electrotonic charging of the membrane capacitance (Figs. 6D and 7C). The depolarizing 'potentials' did not exhibit any obvious inflection
A
on their rising phase. The relative distribution of the 'waves', 'spikes' and depolarizing 'potentials' observed in the various classes of MRNs are summarized in Table I. At a constant amplitude positive current pulse, the waveform of the depolarizing 'wave', the 'spike' or the depolarizing 'potential' was consistent in shape, duration and time to peak in any given neurone. The 'spikes' and depolarizing 'potentials' were followed by a hyperpolarization the durations and amplitudes of which ranged between 4.3-8.8 ms and 2-5 mV. respectively. There was no obvious difference in the depolarizing 'waves', 'spikes' and depolarizing 'potentials' observed in V-neurones as compared to BSor NAA-neurones and no difference whether they were observed in inspiratory (I) or expiratory (E) neurones of a given class. Increasing the intensity of the positive current pulse decreased the time to peak of the 'spikes' and depolarizing 'potentials', but did not alter their amplitudes (Fig. 7). Depolarizing 'waves', 'spikes' and
B E-V
l-BS
!
F---'-I 2ms
!
2ms
C
i-v
b
-
~-
-
D
~nA 2nA
....
a[
~ C
lore V[
20rn V [ •
I 4ms 4ms Fig. 6. The responses to positive current pulses are illustrated before and after QX-314 blockade of action potential discharge (A-C). Low-thresholdsodium action potentials were blocked in D. The current pulses are illustrated when their intensity was varied. The onset and end of the positive current pulses are indicated by the capacitative transient artifacts in the traces A and B. I-BS, inspiratory bulbospinal neurones (A + D); I-V, inspiratoryvagal motoneurone; E-V, expiratory vagal motoneurone. Membrane potentials were -56 mV (A), -64 mV (B), -57 mV (C) and -53 mV (D).
29 TABLE I
A
S~A[ ~bI
'
'
[l-ls
For a description of waves, spikes and depolarizing potentials see text.
2ms B
d
S
1,, i
2ms
3n
lOmV[
'
l - -
4ms
'
Effect of QX-314 on the responses to positive current pulses in medullary respiratory neurones
'
'
I-V
-I ~
Fig. 7. Depolarizing 'waves', 'spikes" and 'potentials' observed after blockage of low-threshold sodium action potentials. The responses were evoked by positive current pulses of increasing intensity. I-BS, inspiratory bulbospinal neurone; E-V, expiratory vagal motoneurone; I-V, inspiratory vagal motoneurone. Membrane potentials were -54 mV (A), -64 mV (B) and -57 mV (C). 'potentials' were never observed to occur repetitively, regardless of the amplitude or duration of the positive current pulse. This was most likely because we did not block potassium conductances (recall that the depolarizing responses were followed by hyperpolarizations). H o w e v e r , the current pulses which we had to use due to the p o o r current carrying abilities of QX-314 electrodes, may have been too weak and to short to reveal such properties. We have previously r e p o r t e d that following the burst of action potential discharge evoked by a positive current pulse, there is a hyperpolarization which is markedly reduced following intracellular injection of the calcium chelator E G T A 2°. Such 'post-pulse hyperpolarizations' following the positive current pulses became progressively smaller in the present study as QX-314 abolished repetitive action potential discharge. DISCUSSION This study illustrates the limitations and advantages of examining the m e m b r a n e properties of
Neurones (n) Vagal Inspiratory (14) Expiratory (11) Postinsp. (8) Bulbospinal Inspiratory* (19) Expiratory (9)
First A Ps
Waves
Spikes
Depolarizing potentials
7
7
3
1
4
3
2
1
3
3
1
-
7
6
2
1
2
1
1
-
1
-
Not antidromically activated Inspiratory* (8) 1 I Expiratory (3)
Postinsp. (4)
-
1
1
-
-
-
-
* Combined populations from nuclei of the tractus solitarius, the para- and rctro-ambigual region.
MRNs in vivo. The principal limitation is that one cannot perform pharmacological manipulations of the extracellular environment necessary to definitively ascertain the ionic mechanism underlying a particular m e m b r a n e event, for example the depolarizing potentials which we observed after blockade of sodium electrogenesis. H o w e v e r , in vivo studies have the advantage that one can ascertain specific physiologic characteristics of the recorded neurone, e.g. it is definitely respiratory. In addition, it is possible to investigate whether a response observed in vitro has a functional relevance under normal circumstances. This question is especially critical in M R N s in vivo where, due to tonic synaptic b o m b a r d m e n t , input conductances are an o r d e r of magnitude higher than in neurones in the same brainstem nuclei studied in vitro (see ref. 30). As observed in other neurones 5.9,22,33,35, QX-314 blocked the transient inward sodium current generating repetitive discharge of action potentials in MRNs. As intracellular injection of QX-314 produced no change in the amplitude or duration of short-lasting synaptic potentials of small amplitudes,
30 it seems reasonable to conclude that the rapidly inactivating sodium current which gives rise to action potential discharge does not play any significant role in the rhythmic membrane potential fluctuations associated with the respiratory cycle, i.e., the ion channels activated by synaptic processes are distinct from those which generate the action potential. QX-314 has been also shown to effectively block depolarizing inward rectification 5'9'22"33-35. The QX-314 induced reduction of membrane depolarization evoked by current pulses (Figs. 2 + 5) indicates that a similar inward rectifying current, carried by sodium ions, exists in MRNs. The reduction in the rate of rise of 'spontaneous' membrane depolarizations (Fig. 1) may be explained similarly and points to the functional significance of the persistent sodium current. A persistent sodium conductance facilitates repetitive firing in neocortical neurones 34. The failure of MRNs to discharge repetitively following injections of QX-314 (Figs. 2, 4, 5), therefore could in part result from a QX-314 block of such a conductance 5 9,35. We conclude that depolarizing inward rectification, produced by a sodium current, is an important factor contributing to the repetitive discharge of MRNs. Calcium spikes have been demonstrated in virtually every neurone studied (see refs. 1 and 10 for refs.). In vitro recordings from neurones within medullary nuclei known to contain respiratory neurones have revealed depolarizing potentials which were abolished when extracellular calcium ions were replaced by cobalt ions 7'8. Under the in vivo conditions of the present experiments we were also able to completely eliminate fast action potential discharge in 25 of 42 neurones. Although we cannot eliminate the possibility that the depolarizing 'waves', 'potentials" and 'spikes' represent residual patches of membrane capable of sodium electrogenesis, there is resemblance between the depolarizing responses we observed (Fig. 5, 6C, D and 7C) and the calcium induced potential changes demonstrated in in vitro preparations 7,8,15,3°,32,35. Depolarizing 'waves' may result from activation of a low-threshold, transient calcium current and there are several explanations for the short duration of the depolarizing 'potentials'
REFERENCES 1 Adams, P.R., Voltage-dependent conductances of vertebrate neurones, Trends Neurosci.. 5 (1982) 116-119.
and 'spikes" compared to the calcium spikes which are supposed to originate from high-threshold, persistent calcium currents ~3'15"~'>,3:~-~5. Firstly, in the present study, we did not attempt to block potassium conductances. Several sorts of potassium conductances were, no doubt, activated as indicated by the fact that the 'spikes" and depolarizing 'potentials' were invariably followed by hyperpolarizations. Persistence of potassium conductances may also explain the fact that we never observed repetitive firing of the high threshold potentials. Indeed. repetitive calcium spikes were not observed unless potassium blockers were applied to neocortical neurones ~5 Block of repetitive discharge reduces calcium influx which explains the disappearance of the 'post-pulse hyperpolarization' seen in the present study. Secondly, calcium potentials are generated in the dendrites of a variety of neurones;'S ~e t:-i,~. ~:.>,~7 The low input resistance and short electrotonic length of the dendrites of MRNs ~', due to repetitive activation of distributed excitatory and inhibitory synapses 3''~252<293t , limit the dendritic spread of current injected into the soma >. If, in MRNs. a proportion of the ion channels producing the depolarizing potential have a dendritic locus, they could be partly shunted and not fully activated in vivo. In conclusion, this study provides an insight into the membrane properties of MRNs in vivo. It indicates that a depolarizing inward rectification, produced by a non-inactivating sodium current, and calcium currents contribute to the electroresponsiveness of respiratory neurones. We assume that these represent important processes whereby MRNs convert synaptic depolarization into action potential discharge to generate the respiratory rhythm. ACKNOWLEDGEMENTS The generous gift of QX-314 by Astra Pharmaceutical Products, Inc., is gratefully acknowledged. We are indebted to Mrs. Annemarie Bischoff and Mrs. Anita Ktihner for their technical assistance and we thank Drs, D. Ballantyne and E. Lawson for valuable comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft and the Alexander yon Humboldt Stiftung.
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