3801

Journal of Physiology (1995), 486.2, pp.439-451

439

A-current modifies the spike of C-type neurones in the rabbit nodose ganglion
Christian Ducreux and Jean-Jacques Puizillout
Laboratoire de Neurobiologie, Neuroregulations Cellulaires, BP. 71, 13402 Marseille Cedex 20, France
1.

2. 3.

4.

5.

6.

7.

In the rabbit nodose ganglion, C-type fibre neurones (C neurones) can be divided into two subtypes according to their after-hyperpolarizing potential (AHP) i.e. those with a fast AHP only and those with a fast AHP and a subsequent slow AHP produced by a slow calcium-dependent potassium current. In addition we have shown that some C neurones can be divided into two groups according to the effect of membrane hyperpolarization on their spikes i.e. type 1 in which duration and amplitude do not change and type 2 in which duration and amplitude decrease significantly. In the present report we studied the effect of A-current (IA) on spike duration, amplitude and slow AHP using intracellular recording techniques. To detect the presence of IA, we first applied a series of increasing rectangular hyperpolarizing pulses to remove IA inactivation and then a short depolarizing pulse to trigger a spike. In type 1 C neurones the lag time of the spike in relation to hyperpolarization remains constant whereas in type 2 C neurones the spike only appears after IA inactivation and lag time in relation to hyperpolarization is lengthened. Thus, type 2 C neurones have an IA while type 1 C neurones do not. The fact that addition of cadmium did not change the lag time in type 2 C neurones shows that the IA is not calcium dependent. Nodose neurones can be orthodromically activated by stimulation of the vagal peripheral process. In this way, after a hyperpolarizing pulse, IA can be fully activated by the orthodromic spike itself. Under these conditions it is possible to analyse the effects of IA on the spike. This was done by increasing either the hyperpolarizing potential, pulse duration, or the delay of the spike after the end of the pulse. We observed that maximum IA inactivation removal was always associated with the lowest duration and amplitude of the spike. When IA inhibitors, 4-aminopyridine (4-AP) or catechol, were applied to type 2 C neurones, the delay of the spike after the hyperpolarization-depolarization test was no longer observed. In addition 4-AP abolished the shortening of the duration of the spike induced by steady hyperpolarization. In type 2 C neurones with slow AHP, the IA-related decrease in spike duration was associated with a disappearence of the slow AHP. This indicates that 'A decreases the calcium influx during the spike. In conclusion, since IA regulates the amplitude and duration of the spike in prehyperpolarized cells, it is logical to assume that IA is able to regulate the calcium influx into the cell. If this mechanism occurs at axon terminals, it could interfere with transmitter release and thus modulate synaptic transmission.

A-current, first observed by Hagiwara et al. (1961) in molluscan neurones and later termed IA by Connor & Stevens (1971a), is a fast transient outward voltagedependent potassium current. At resting potential, IA is inactivated but this inactivation can be removed by hyper-

polarization (de-inactivation). IA has been implicated in several processes. It slows repetitive discharges by decreasing the rate of decay of the fast after-hyperpolarizing potential (Connor & Stevens, 1971 b; Hille, 1992). It modulates the efficacy of synaptic transmission (Rogawski, 1985). It has

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Table 1. Membrane and spike properties of C neurones in the rabbit nodose ganglion

Resting potential (mV)

C neurones without slow AHP (n= 51) -53 6 + 7-4 C neurones with slow AHP (n= 41) -57-4 + 8-3

Spike amplitude (mV)

96-0 + 7-3

Spike duration (ms)

4-2 +1P2

Conduction velocity Peripheral Central process process (m s'l) (m sl)

Slow AHP Slow AHP amplitude duration (mV) (s)

0'6 + 0 1 0 3 + 0 1
0-6 + 0 1
03+01 4-7 + 2-3

95 0 + 8-7

3-7 + 1P3

7-6 + 2-5

been implicated in spike repolarization in rat motor sympathetic neurones by Belluzzi, Sacchi & Wanke (1985a, b) and Belluzzi & Sacchi (1988). The nodose ganglion contains most of the sensory cell bodies of the vagus nerve. From these cell bodies emerges a short stem axon that divides inside the ganglion into two branches i.e. the peripheral process and the central process. The central process projects into the nucleus of the solitary tract in the medulla. Since there are no synaptic contacts, the rabbit nodose ganglion provides a convenient model for studying basic electrical membrane properties. Moreover IA can be activated directly by an orthrodromic spike elicited by peripheral process stimulation. Intracellular recordings can be performed on unmyelinated C-type fibre neurones (C neurones) that can be readily identified by conduction velocity. Most C neurones initiate Nae and Ca2+ spikes (Ito, 1982) followed by a transient fast AHP but a few display an additional slow AHP produced by a slow calcium-dependent potassium current, slow IK,ca (Higashi, Morita & North, 1984). The amplitudes of both fast and slow AHP, are dependent on membrane potential. However, unlike fast AHP which is reversed near -70 mV by hyperpolarization in all neurones, slow AHP is either reversed or only cancelled

out according to the neurones (Fowler, Greene & Weinreich, 1985). We previously showed that when slow AHP was only cancelled out, spike duration decreased and that, as a result, slow IK,ca was not activated (Ducreux, Reynaud & Puizillout, 1992). Since some nodose neurones develop IA (McFarlane & Cooper, 1991), we speculated that this process is dependent on IA. The purpose of this study was to test

the validity of this hypothesis.

METHODS
Adult male rabbits, weighting 1 5-2-5 kg, were anaesthetized with halothane and then killed by cervical dislocation. Nodose ganglia and their vagal processes were removed and pinned to the bottom of a 3 ml recording chamber covered with 3 mm Sylgard (Dow Corning), at room temperature. The peripheral process was stimulated by two vaseline-coated platinum wire electrodes, spaced 2 mm apart. The cathode was placed 1 cm from the centre of the ganglion. Pulses of 0 5 ms and 1-2 mA were used to activate C neurones. Under a stereomicroscope (Nikon, SMZ-U) the tissue around the ganglion was removed and its capsule was carefully opened so as to allow access to individual neurones. The control solution was composed of (mM): NaCl, 119; KCl, 5; CaCl2, 2X5; MgSO4, 1; NaH2PO4, 1; NaHCO3, 25; glucose, 11.1 equilibrated with 5% CO2 and 95% 02. When CdCl2 (2 mM) was

Table 2. Incidence of membrane hyperpolarization on duration (ms) of type 1 and type 2 C neurone spikes

C neurone duration (ms)

Without slow AHP n= 20 4-3 + 1P2 3-9 + 1P0
n= 11

With slow AHP

Type 1 (60 8%) Control Experimental

3.7 + 1'1 3*0 + 1P0

n=9 3-7 + 1P3 1-6 + 0-4

n= 11

Type 2 (39 2 %)
Control

3.9 + 1-1
1.9 + 0.5

Experimental

A decrease in spike duration was observed in both neurones with or without slow AHP. This decrease was highly significant in type 2 neurones (P < 0'001, Student's t test). Control, resting potential; experimental, hyperpolarization at -90 mV.

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A -current modifies the spike of C neurones

441

used, the bicarbonate buffer was replaced by 25 mm Tris at pH 7-4. The flow rate was 1 ml min-'. In some experiments catechol or 4-aminopyridine (4-AP; Sigma), was pressure ejected (2 je1, 5 mM) from a pipette with a 20 ,um tip diameter fixed near the recorded neurone. When 4-AP was used, NaH2PO4 was also replaced by Tris buffer. Cells were impaled with borosilicate glass micropipettes filled with 3 M KCl (20-50 M.Q) and connected to an electrometer Biologic VF 180. Steady currents or rectangular pulse currents (0 1-1 nA) were applied intracellularly to the cell body. Membrane current and voltage were displayed on a Tektronix oscilloscope 5111A, digitized by a pulse code modulation unit (Sony) and stored on a video recorder (JVC HR-D750S). For off-line analysis, the signals were transferred via an analog-digital interface CED 1401 (Cambridge Electronic Design, Cambridge, UK) to a PC computer and processed using SIGAVG 5.20 software from CED. Spike amplitudes and durations were measured peak to peak and at half amplitude, respectively. The data points in Tables 1 and 2 are means + S.E.M. of n observations.

RESULTS
Electrophysiological properties of C neurones In ninety-two neurones with a resting potential of greater negativity than -45 mV, stimulation of the peripheral process elicited overshooting spikes that will be referred to in this paper as 'orthodromic spikes'. The estimated conduction velocity of C neurones was 0-6 m s-'. Failure to produce spikes, observed in some neurones, was probably due either to damage of axons during the preparation of the ganglion or to the fact that the axon belonged to the laryngeal or aortic nerves that were not co-stimulated with the vagus nerve. However, these neurones can always produce spikes by a transmembrane depolarizing current pulse (0 3-0 4 nA). Fast AHP was observed in all C neurones. Fast AHP was followed by a slowly developing and long-lasting slow AHP (5-10 s) in forty-one neurones (44 5%). No slow AHP was

1-5

2-5
"A -9

3-5 4.5 Spike duration (ms)

C

5.5

65

Type 2
7

N
N

1-5

2-5

3S 5 4d 5

5-5

6(5

Spike duration (ms)

Spike duration (ms)

Figure 1. Distribution of type 1 and type 2 C neurones A, distribution and frequency of spike duration (measured at half amplitude) of 51 C neurones (with or without slow AHP) recorded at resting potential (E) and after hyperpolarization (around -90 mV; C1). Two subsets of C neurones can be distinguished according to spike duration during hyperpolarization. In type 1 neurones (60'8%, n = 31, B), spike duration did not change significantly after hyperpolarization. In type 2 neurones (39 2%, n = 20, C), spike duration decreased after hyperpolarization. Spikes were evoked by stimulation of the peripheral process of the vagus nerve. Bin width, 0S5 ms.

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observed in fifty-one neurones (555 %). The resting potentials, amplitudes and durations of the spikes and slow AHP, as well as the conduction velocities of the peripheral and central processes, are summarized in Table 1. When the peripheral process was stimulated during steady hyperpolarization, two subsets, including both C neurones A
5
ms
MV

with and without slow AHP (n = 51), were found. In the first subset called type 1 neurones (n = 31, 608 %), spike duration decreased less than 20%. In the second subset, called type 2 neurones (n = 20, 39 2 %), spike duration decreased dramatically (around 50%) (Fig. 1 and Table 2).

B
5 ms
-

0mv-

IAA

270
-

ftjto0 mvI
-p

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oswwbuomp

1-

I20mV
Ca

2s

20OmV
Da

1 s

120 mV
1

20 ms

5 mV

2s

-g

Figure 2. Hyperpolarizing currents applied to C neurones with a slow AHP

A and B, steady hyperpolarizing currents applied to type 1 (A) or type 2 (B) neurones. In type 1 (membrane potentials = -55, -77 and -94 mV), spike duration did not change (inset) but slow AHP was readily reversed (lower traces; reversal potential, -72 mV). In type 2 (membrane potentials = -55, -70 and -91 mV), spike duration decreased (inset) and slow AHP was cancelled out (at -76 mV) but not reversed. Spikes were evoked by peripheral process stimulation as described in Fig. 1. C-D, hyperpolarizing current pulses applied to type 1 (C) or type 2 (D) neurones. The peripheral process stimulus was applied before the end of the pulse in order to obtain slow AHP at the resting potential of the cell i.e. just after the end of the pulse. C, in type 1 neurones (membrane potentials = -60 (1), -78, -92, -112 and -126 mV (5)), no change was noted in either spike duration (Ca) or slow AHP, shown here at the resting potential (Cb). D, in type 2 neurones (membrane potentials = -60 (1), -81, -95, -112 and -126 mV (5)), spike duration decreased (Da) and slow AHP was cancelled out (D b) as in B.

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J Physiol.486.2

A -current modifies the spike of C neurones

A
0 mV 20 mV
5 ms

443
Type 2

Type 1

C
\\

120 mV
2 ms

B
0mV20 mV |
- -

E
-

0-5 s

20

mVl

0-2 s

-I
F

C
0

120 mV
5 ms

G
160'

H
160

E
Cu

a, 120'
9.

co

120'

0 cJ D

80'
.

c.) C

a)

80'
40 '

cn

40

CO)

-110

-90

-70

-50

Membrane potential (mV)

Membrane potential (mV)

Figure 3. Evidence for IA in type 2 but not type 1 C neurones A-C, type 1 neurones. A, spikes were triggered by peripheral process stimulation. Spike duration remained constant after steady hyperpolarizing currents (membrane potentials = -50, -66, -83 and -90 mV). B, after hyperpolarizing pulses, a short depolarization pulse triggered firing in the same neurone. C, an expanded view shows the latency of spikes in relation to hyperpolarization (membrane potentials = -53 (1), -66, -75 and -89 mV (4)). Note that neither spike duration nor latency change much with hyperpolarization. D-H, type 2 neurones. D, spike duration decreased after steady hyperpolarization (membrane potentials = -50, -60, -69 and -90 mV). E and F, spike lag time increased as a function of hyperpolarization (membrane potentials = -52 (1), -71, -77 and -83 mV (4); note the difference in calibration between F and C). 0 and H, depending on membrane hyperpolarization, the increase in spike latency in type 2 neurones could be progressive (G) or occur abruptly (H).
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Effect of membrane hyperpolarization on spike duration and slow AHP amplitude in type 1 versus
type 2 neurones Membrane hyperpolarization was performed using either a steady current or a 90 ms current pulse. When the current pulse was used, the spike was triggered before the end of the pulse in order to obtain the slow AHP just after the end of the pulse i.e. at the resting potential of the cell. This sequence avoids interference between the current injected and the current produced by the increase in the slow K+ conductance that leads to the slow AHP.

When steady currents (resulting in membrane potentials up to -120 mV) were applied to type 1 neurones, the slow AHP was cancelled out at -80 mV and reversed beyond -80 mV (Fig. 2A). In type 2 neurones, slow AHP was cancelled out but never reversed (Fig. 2B). When pulse currents were applied to type 1 neurones, slow AHP at the resting potential did not change at membrane potentials as hyperpolarized as -120 mV (Fig. 2Ca and Cb). When the pulses were applied to type 2 neurones, slow AHP decreased and disappeared (Fig. 2Da and Db) with the spike duration decrease.

A
0mV- -

I\

20 mV

120 mV

120 mV \ 2 ms

200 ms

2 ms

rI

Negative peak (fast AHP)

D
-

100-

EX co

90 4

E 5~ 30-j
e co
0

+1

cn) 804-0 1
0

J0
10j

a.

E
a

Cl
'O
Q
cn

3-5

ES -650

a-70. e)
O

,J

oa

in

_7

-100

-70 -60 -80 -90 Membrane potential (mV)

-50

Z -100

-60 -90 -80 -70 Membrane potential (mV)

-50

Figure 4. Effect of hyperpolarization level on duration and amplitude of orthodromic spikes A, decrease in the duration of orthodromic spike triggered after steady hyperpolarization. Identification of a type 2 neurone (spike duration decrease, 42 %). B-E, the orthodromic spike is triggered 40 ms after the hyperpolarizing pulses (membrane potentials = -53, -66 and -105 mV) in such a way that its depolarizing phase activates IA (B). Under these conditions, a decrease in the duration (X; C and D), amplitude (O; C and D) and positive peak (+; C and E) of the spike was observed. The fast AHP (0) did not change (E). Spike amplitude was measured peak to peak and spike duration at half amplitude.

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A -current modifies the spike of C neurones

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Evidence of IA in type 2 neurones After removal of IA inactivation by hyperpolarization using increasing current pulses, IA can be activated by a short depolarizing pulse adjusted to also trigger a spike. The IA is evaluated according to the lag time of the spike which occurs after full IA inactivation. The longer the lag time, the stronger the IA current is (Gola & Romey, 1971). We tested both C neurones with and without slow AHP and compared type 1 and type 2 neurones that were identified by measuring spike duration after steady hyperpolarizations (Fig. 3A and D). For type 1 neurones (Fig. 3A), the lag time of the spike triggered by the depolarizing pulse was practically the same (Fig. 3B and C) regardless of the level of hyperpolarization (variations in spike latencies, 2-3 ms).

For type 2 neurones (Fig. 3D), the lag time increased with the level of hyperpolarization (variations in spike latencies, 100-200 ms; Fig. 3E and F). Similar observations were made after addition of CdCl2 (no change in lag time but decrease in spike amplitude). Depending on the neurone, the lag time of the spike either increased progressively with the level of hyperpolarization or exhibited a sharp drop (Fig. 30 and H). The shape of the spike was not affected whatever the hyperpolarizing step values. To simplify, our findings can be summarized as follows: IA was observed in type 2 neurones but not in type 1 neurones. Incidence of the A-current on the duration and amplitude of the orthodromic spike We studied the incidence of 'A on the shape of the spike in type 2 neurones with and without slow AHP. Neurones

A
0 mV-

B
- -

-1 14-1

20 mV 10 ms

l1
C
E a) 0 ~0 E
X
a

-F..
95-

*0
75 J 3-5

D
> 25-

(n

R 20

:J
a.

+4

15

X 2-5
'0

-60'
_

-s5 2-0 (.1)

a) m 0i&
-

-65
,
I

1 *5

100

300 Spike latency (ms)

260

400

100

200 300 Spike latency (ms)

400

Figure 5. Effect of lag time between the end of the pulse and orthodromic spike on spike duration and amplitude A, peripheral process stimulation was delivered to type 2 C neurones to obtain spikes at resting potential but at different lag times (10-200 ms) after the same hyperpolarizing pulse (membrane potential = -85 mV). B, expanded view of A, showing spike duration and positive peak increase with spike latency. C, spike amplitude (0) and duration (X) versus spike latency. D, positive (+) and negative (0; fast AHP) peaks of the spike versus spike latency.

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J Physiol 486.2

were identified as type 2 if a decrease in spike duration was observed after steady hyperpolarization (Fig. 4A). Then hyperpolarizing pulses were applied to remove IA inactivation. The peripheral process was stimulated in such a way that the orthodromic spike occurred near the resting potential of the cell (10-500 ms after the end of the pulse) and thus contributed to IA activation. This method avoided interference due to current injection during the spike and

allowed more precise measurement of positive and negative (fast AHP) peaks.

Influence of hyperpolarization level. Hyperpolarizing pulses resulting in membrane potential variations ranging from 0 to -70 mV (0l5-1P5 s; 0 3 Hz) were applied to the neurones. The orthodromic spike fired a few milliseconds after the end of the pulse (Fig. 4B). A progressive decrease

A
200 ms
0 mV- - _ _ _

B
_ _ _

20

mVI

0mv-

trr
C
20 mV
0 mV-

rr
200 ms

IL-L -t
-

1r r F
D
> 110E
0 -6-:

F r

r
E
50-

X 105E
0)

E
(a

Q 45.

cD 100
1-

++
--Q~~~~I _o
+

co
C

4-51

0L

40J -551
4-0

i0 'D 0
U,

*' E
zo
1.0 0-5 Pulse duration (s)
1 -5
0) 5

o-60
N

1.0 0-5 Pulse duration (s)

1*5

Figure 6. Effect of pulse duration on duration and amplitude of orthodromic spikes

A, hyperpolarizing pulses (membrane potential = -75 mV) of increasing duration were applied to a type 2 C neurone. Spike firing was triggered 30 ms after the end of the pulse. B, increasing hyperpolarization duration resulted in a decrease in spike amplitude and duration. C, in some neurones increasing hyperpolarization duration impaired somatic spike so that only an 'A spike' remained. D, spike duration (X) and amplitude (0) versus pulse duration. E, potentials of positive (+) and negative (0; fast AHP) peaks versus pulse duration.

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447

in the duration and amplitude of the spike, proportional to the hyperpolarization level, was observed (Fig. 4C and D). Decreases in amplitude (measured peak to peak) involved only positive peaks. Fast AHP was not affected (Fig. 4E). The maximal effect was observed at levels of -90 mV or more. Influence of lag time between the end of the pulse and orthodromic spike. To analyse the inactivation of IA' we applied the hyperpolarizing pulse producing the maximal IA (-90 mV, 0 7 s) and timed peripheral process stimulation so that the spikeoccurred 10-500 ms after the end of the pulse (Fig. 5A). Maximal effects (44 % decrease in duration, 9% decrease in amplitude) were obtained when the orthodromic spike occurred immediately after the pulse (Fig. 5A, B and C). These effects progressively disappeared 100-200 ms after the pulse corresponding to the time

required to inactivate IA. As mentioned previously the decrease in amplitude involved only the positive peak (Fig. 5D).
Influence of the pulse duration. Hyperpolarizing pulses and the timing of the orthodromic spike were maintained at -75 mV and 30 ms, respectively while pulse duration was varied from 0.01 to 1'5 s (Fig. 6A). As the duration of the pulse increased, some neurones showed a progressive decrease in duration, amplitude and positive peak (Fig. 6A, B, D and E). At pulse durations between 100 to 500 ms, other neurones showed a transitory period (lasting 400-500 ms) during which only A spikes were observed i.e. axonal spikes unable to invade the soma (Fig. 6C). In all neurones the maximal decrease in the orthodromic spike duration was obtained with pulse duration values between 0-2 and 1-2 s.
c

Aa

b
NUR%WOMMdFA
%,

b r
Ba

IAAV

b
s-AHP

0 mV

120 mV
25 ms

110 mV
1 s

b
0 mV s-AHP
0-

120 mV
25 ms

110 mV
1 s

Ca
0 mV

|20 mV
1 s

10 ms

Figure 7. Relationships between IA and slow AHP (sAHP) in type 2 C neurones with sAHP Three different experiments were carried out to obtain maximum IA activation i.e. A a, increasing the hyperpolarizing pulse (membrane potentials = -60 and -90 mV); Ba, decreasing the delay of the spike after the pulse (20 and 280 ms); C, increasing the duration of the hyperpolarizing pulse from 0 1 s in a to 1 1 s in b. Action potentials were obtained by vagal stimulation. As shown previously in type 2 C neurones without slow AHP, spike duration and amplitude decreased with activation of IA in all three experiments (A b, A c, Bb, Bc, Ca and Cb) (the traces labelled b and c in A a and Ba are recorded on a faster time scale). Here, in addition, slow AHP disappeared (A a, Ba and Cb).
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Incidence of IA on slow AHP The three experimental procedures used in the section, 'Incidence of the A-current on the duration and amplitude of the orthodromic spike' were repeated with type 2 neurones, focusing special attention on slow AHP. As previously noted, a decrease in the duration and amplitude of orthodromic spikes was observed. In all three experiments this decrease was associated with a disappearence of slow AHP (Fig. 7).

IA pharmacological inhibition
Neurones exhibiting IA were identified as described in the section 'Evidence of IA in type 2 neurones' (hyperpolarizing pulse followed by a short depolarizing pulse). Before addition of 4-AP, a test hyperpolarizing pulse of -100 mV (trace 4 in Fig. 8A) triggered a spike 70 ms after the end of the pulse (Fig. 8B, traces a and a'). After addition of 4-AP, a slight increase in the membrane resistance was observed leading to a weak increase of the conditioning hyper-

A
O mV
-

20 mV

200 ms
1

-I

r L-1D
4-AP

c
0 mV
-

E
4

F
4AP

32

~0

._o

0 mV-

CD

Control

x 9
-110

-90 -70 Membrane potential (mV)

-50

20 ms

Figure 8. Effects of pharmacological inhibitors on IA A, IA is detected as described in Fig. 3E and F. B, after addition of 4-AP (2 mM), spike latency progressively decreases (b' is obtained 15 s, and c', 30 s after addition of 4-AP). C, in control experiment, spikes were triggered by peripheral process stimulation at different hyperpolarization levels. Spike duration was 66% lower after hyperpolarization. D, after 4-AP the decrease was much smaller (20%). E, spike duration versus membrane potential in control (X) and 4-AP-treated neurones (o). F, decrease in the latency of the spike after application of catechol (2 mM) under the same conditions as in B.

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polarization value (Fig. 8B, traces a, b and c) and the spike occurred immediately after the depolarizing pulse. The maximal effect (c and c') was obtained 1 min after addition of 4-AP.

Before addition of 4-AP, successive steady hyperpolarization (up to -80 mV) and peripheral process stimulation (Fig. 8C) led to a decrease in spike duration of 66 %. After 4-AP, the decrease was only 20%, even with hyperpolarizing pulses as high as 100 mV (Fig. 8D). This level was applied to rule out a possible shift of the 1A inactivation curve to more negative potentials. At the resting potential, spike duration was the same before and after addition of 4-AP (Fig. 8E). The effect is reversible in 3-5 min.

Catechol, another inhibitor of IA (Erdelyi & Such, 1988; Sah & McLachlan, 1992) also caused a dramatic but reversible decrease in the spike latency (Fig. 8F).

DISCUSSION
Applying steady hyperpolarizing current to C neurones caused a decrease in the duration of resulting orthodromic spikes. In some neurones, herein designated as type 1, this decrease is weak (< 20%) and in others, designated as type 2, the decrease is strong ('50%). We hypothesized that the strong decrease is due to IA. To study this possibility, IA inactivation was removed by applying hyperpolarizing pulses. When the membrane returns to resting potential at the end of the pulse, only 5% of the IA is activated (see activation curve described by McFarlane & Cooper, 1991). Based on this finding, a full IA activation could be induced after the pulse by depolarization of the membrane either by an outward current injected in the soma or by the orthodromic spike itself. When an outward current is used, IA is activated before the inward Na+ and Ca2P currents of the spike. Thus, the spike arises after IA inactivation i.e. with a latency proportional to the duration of IA inactivation. Type 1 neurones have spike latencies that remain constant under hyperpolarization while type 2 neurones have spike latencies that increase. In some type 2 neurones spike latency increases progressively (slope, 3-8 ms mV-') up to 130 ms under hyperpolarization; in others latency jumps abruptly (slope, 13-3 ms mV-W) to 90 ms. These differences could be due to fast IA (10-30 ms) and slow IA (150-300 ms) inactivation time constants and to the slope factor of the inactivation curve (k = -8 and -14 mV, respectively), as described by McFarlane & Cooper (1991). In the case of activation by the orthodromic spike itself, IA arises simultaneously with the inward Na+ and Ca2+ currents. Under these conditions in type 2 neurones, IA may modify the shape of the spike which is consistently recorded at the resting potential. This assumption is

supported by three findings in our study. The first is that when the spike occurred immediately after a pulse, its duration and positive peak depended on the hyperpolarization level. The maximum effect was observed around -100 mV, which is in agreement with the IA inactivation curve described by McFarlane & Cooper (1991; halfinactivation of fast IA was found at -73 mV). The decrease in the positive peak means that IA must hinder full development of the inward currents of the spike. The second supporting finding is that when spike latencies are increased (10-300 ms) after a given hyperpolarizing pulse, the spike duration and positive peak progressively increased returning to initial values after 100-200 ms i.e. when IA is completely inactivated. The third is that when the hyperpolarizing pulse duration is increased, the duration and amplitude of the spike firing just after the pulse decreased progressively, probably according to the time necessary to de-inactivate IA. A similar test was described by Aibara, Ebihara & Akaike, 1992, in paraganglion cells, using voltage clamp technique: full de-inactivation needs 80-100 ms against 0-2-1-2 s found in nodose neurones (the present paper) or in synaptosomes (Edry-Chiller & Rahamimoff, 1993). These differences could be due to the experimental material or to the experimental protocol. We have no explanation for the transient spike block observed in some neurones (Fig. 6C) when we studied the influence of the pulse duration on IA. This could be associated with an undefined property of the fast K+ transient current or with a decrease of the inward currents which impair the development of the full spike. The IA inhibitors, 4-AP and catechol almost completely suppress the spike delay observed following the hyperpolarization-depolarization test. This effect has also been described for 4-AP by Dekin & Getting (1984) and Tell & Bradley (1994) in CNS slices and by Segal, Rogawski & Barker (1984) in neuronal cultures. We have also observed that 4-AP prevents the decrease in spike duration if the spike is recorded during hyperpolarization but has no effect at the resting potential. This finding demonstrates that 4-AP does not affect other K+ conductances of the spike in type 2 nodose cells and therefore is specific for IA. Some transient outward current has been shown to be calcium dependent (Bourque, 1988). This was not the case for the nodose cells since the addition of a Ca2+ channel blocker (cadmium) does not suppress IA. In these nodose ganglion neurones, membrane hyperpolarization is a prerequisite for regulation of spike duration, but these neurones have no synaptic inputs to modulate the membrane polarization and consequently, IA at resting potential, has no effect on their spike duration. However, we must keep in mind that membrane hyperpolarizations can be induced in nodose cells by hormonal signals (Higashi, Ueda, Nishi, Gallagher & Shinnick-Gallagher,

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1982). It is possible that these effects also operate in vivo as the nodose ganglion structure is devoid of a blood-brain barrier. It has been shown in other neurone models (Thorn, Wang & Lemos, 1991; Lockery & Spitzer, 1992; Tell & Bradley, 1994) that hyperpolarization is not a prerequisite for regulation of spike duration as the inactivation curves are shifted to less negative potentials. IA is observed in such a variety of neuronal and nonneuronal cells that it is generally considered as a characteristic feature of excitable cells (Rogawski, 1985). However, our findings indicate that only 39 2% of C neurones in the nodose ganglion exhibit IA. Several physiological roles have been proposed for IA. The most widespread hypothesis is that IA is implicated in the regulation of the firing frequency of the cells (Connor & Stevens, 1971 b). It has also been suggested that IA modulates the efficacy of synaptic transmission by postsynaptic or presynaptic mechanisms (Rogawski, 1985). IA could also have an effect on spike repolarization (Belluzzi et al. 1985a,b; Belluzzi & Sacchi, 1988). The results of this study support the repolarization effect. In C neurones with slow AHP, the AHP is due to a slow calcium-dependent potassium current. We showed that IA de-inactivation by hyperpolarization decreases the duration and amplitude of the evoked spike and suppresses the slow AHP. This finding shows that the inward Ca2+ current, decreased by IA, is no longer sufficient to activate the slow IE Ca Thus, in type 2 neurones with slow AHP, IA has a modulatory role on the Ca2+ entry into the cell, which is detected by the slow AHP. Although generally detected in soma, IA has also been observed in the presynaptic terminals (Thorn et al. 1991; Bielefeldt, Rotter & Jackson, 1992). In Aplysia, Shimahara (1983) documented that a presynaptic hyperpolarization induced a decrease in the evoked EPSP ampitude due to a reduction in the presynaptic spike amplitude; this effect was abolished in the presence of 4-AP and thus, it could be attributed to IA. Thesleff (1980) stated that 4-AP improves the release of neurotransmitters. A 4-AP-sensitive potassium current has been demonstrated in peripheral terminals of sinusal baroceptor fibres, that have perikaryons in petrosal ganglion, a structure similar to nodose cells (Chapleau, Lu, Hajduczok & Abboud, 1993). If the channels of the vagal terminal membranes are similar to those of the soma, our electrophysiological studies, performed in nodose cell bodies, could be extrapolated to axon terminals located in the nucleus of the solitary tract. The prerequisite hyperpolarization to obtain IA activation could be achieved via axo-axonal presynaptic control in the nucleus of the solitary tract (Mrini & Jean, 1994). Thus, inhibitory inputs would be able to adjust the inward calcium current during the spike and consequently modulate, via IA the transmitter release in the nucleus of the solitary tract.

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Acknowledgements
We thank B. Besson and J.-C. Reynaud for expert technical assistance.
Received 13 September 1994; accepted 21 December 1994.

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