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Differences in electrophysiological properties between neurones of the dorsal motor nucleus of the vagus in rat and guinea pig
Journal of the Autonomic Nervous System, 42 (1993) 89-98
89
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1838/93/$06.00 JANS 01349
Differences in electrophysiological properties between neurones of the dorsal motor nucleus of the vagus in rat and guinea pig Pankaj Sah and Elspeth M. McLachlan Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Australia (Received 9 June 1992) (Revision received 19 August 1992) (Accepted 25 August 1992)
Key words: P r e g a n g l i o n i c ; N o r a d r e n a l i n e ; A f t e r h y p e r p o l a r i s a t i o n ; S e r o t o n i n
Abstract We have examined the electrophysiological properties of neurones in the dorsal motor nucleus of the vagus (DMV) in rats and guinea pigs in transverse medullary slices maintained in vitro. There were only minor differences in the morphology of the neurones between the species, and their passive electrical properties were very similar. However, action potentials in guinea pig neurones had larger amplitudes and longer half-widths than did those in rat neurones. In both species, action potentials were followed by prolonged afterhyperpolarisations (AHPs). In the majority of guinea pig neurones, two calcium-activated potassium currents underlying the AHP could be separated into an early apamin-sensitive component and a late apamin-insensitive component. In rat neurones, the current underlying the AHP was briefer and entirely apamin-sensitive. In response to a step of depolarising current, neurones in the guinea pig only discharged once or twice and then ceased firing. In rat neurones, this manoeuvre produced repetitive firing. An inward rectifier was larger in neurones of the guinea pig than in those in the rat. The effects of 5-hydroxytryptamine and noradrenaline also differed between neurones of each species. We conclude that, despite many similarities of size and electrical properties, DMV neurones in the two species differ in terms of several voltage- and calcium-dependent conductances which determine their active electrical behaviour.
Introduction T h e cells of t h e dorsal m o t o r n u c le u s o f t h e vagus ( D M V ) lie in a c o l u m n e x t e n d i n g f r o m th e rostral to t h e c a u d a l m e d u l l a o b l o n g a t a in its d o r s o m e d i a l part. T h e s e n e u r o n e s a r e th e m a i n s o u r c e o f t h e p a r a s y m p a t h e t i c i n n e r v a t i o n o f th e s u b d i a p h r a g m a t i c visceral organs. T h e m a j o r physiological roles o f t h e s e n e u r o n e s are in t h e
Correspondence to: P. Sah, Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Queensland 4072, Australia.
c o n t r o l o f g a s t r o i n t e s t i n a l and p a n c r e a t i c secretion as well as s t o m a c h an d small intestinal motility. This n u c l e u s receives a diverse array of afferen t inputs f r o m m a n y b r a i n s t e m and h i g h e r centres [8]. M o s t studies of the physiology and a n a t o m y of b r a i n s t e m a u t o n o m i c c e n t r e s have focussed on rat or cat. T h e r e is little i n f o r m a t i o n r e g a r d i n g the a n a t o m y or f u n c t i o n a l significance o f t h ese c e n t r e s in t h e g u i n e a pig, a l t h o u g h species differen ces b e t w e e n c o m p a r a b l e cell g r o u p s in t h e v e n t r o l a t e r a l m e d u l l a have b e e n d e s c r i b e d [6,7]. S o m e m a r k e d d i f f e r e n c e s have also b e e n n o t e d in the D M V b e t w e e n rat and g u i n e a pig. In the
90
guinea pig, the D M V contains large numbers of oxytocin- and ACTH-containing terminals which make synapses with the soma and dendrites of D M V neurones; these inputs are absent in the rat [19]. On the other hand it has been shown that oxytocin excites neurones in the rat but not in the guinea pig D M V [15]. We have recently described the pharmacology of the two calcium-activated potassium conductances which underlie the afterhyperpolarisation (AHP) in guinea pig vagal neurones [17]. These two types of Ca2+-activated potassium conductance, named Gkc~.l and Gkca,2 respectively, can be separated both kinetically and pharmacologically (see also Cassell and McLachlan [3]). Activation of the slower time course conductance was shown to be dependent on calcium-induced calcium release [17]. This has been confirmed by immunohistochemical evidence for the presence of ryanodine receptor-like immunoreactivity in guinea pig D M V at considerably higher concentrations than in the rat (Sah, Francis, McLachlan and Junankar, unpublished observations), consistent with the distribution of Gkc~,, 2 in the two species. A calcium-induced calcium release mechanism is therefore likely to be rare or absent in rat D M V neurones [16]. As neurones in the D M V probably perform similar functional roles, it is important to establish whether other differences exist in the passive and active electrical properties of vagal neurones between species. In this paper we compare the morphological and electrophysiological properties of neurones in the dorsal motor nucleus of the vagus in rats and guinea pigs in transverse medullary slices maintained in vitro.
Materials and Methods
All experiments were done on transverse slices of medulla from young adult rats or guinea pigs maintained in vitro. Methods for preparation of slices from rat medulla were identical to those previously described for the guinea pig [17]. The animals were deeply anaesthetised with intraperitoneal pentobarbital (50 m g / k g ) and decapitated. The brainstem was quickly removed and ira-
mersed in cold Ringer of the following composition (raM): NaC1, 115; KCI, 5; MgSO4, 1.2; CaCI 2, 2.5; NaH2PO4, 1.2; NaHCO~, 25; glucose, 10, gassed with 95% 0 2 / 5 % CO 2. Three or four transverse slices (400 /~m thick) containing the D M V were cut with a Vibratome rostrally from approximately 1 mm caudal to the obex; these were allowed to recover for 1 h in a humidified chamber before recording was attempted. Slices were studied fully submerged in the recording chamber and perfused with Ringer warmed to 28-30°C. Picrotoxin (100 ~ M ) was routinely added to block the spontaneous inhibitory potentials which invariably appeared in neurones of both species when KCl-filled electrodes were used. Microelectrodes were fabricated on a BrownFlaming puller and filled with 0.5 M KCI (resistance 70-140 M~Q). By means of a combination of trans- and epi-illumination, the D M V was readily identified as a translucent area immediately dorsal to the hypoglossal nucleus. Most recordings were made from slices containing the obex or the region immediately rostral to it. Signals were recorded using a single electrode voltage clamp amplifier (Axo Clamp 2a, Axon Instruments), sampled at 0.5-10 kHz and stored and analysed on an IBM compatible computer. During voltage clamp experiments the headstage was continuously monitored to ensure complete settling of the voltage transient between samples and switching frequencies of 1.5-3 kHz were used. Electrode tips were coated with silicone oil in order to reduce the electrode capacitance. Action potentials were elicited from a given holding potential by injecting a 10 ms depolarising current pulse through the recording microelectrode. To measure the currents underlying the AHP, the cell was clamped at the resting m e m b r a n e potential ( - 6 0 to - 7 0 mV) and a brief (10 ms) depolarising voltage command step (usually 10-15 mV) was delivered. The magnitude of this pulse was increased until a single active response was elicited. The sodium current underlying the action potential cannot be controlled by the voltage clamp and results in a poorly controlled 'action current'. However, good voltage control is always obtained within 50 ms
91
RAT 1 mm
G U I N E A PIG /,
E Fig. 1. Morphology of biocytin-filled neurones in the dorsal motor nucleus of the vagus (DMV) in the rat and guinea pig. The schematic diagrams show a transverse section of the medulla with the dashed area representing the region illustrated at low magnification to the right. The calibration in the upper right-hand figure refers to both species. The calibration at the lower right corner refers to the enlarged views of the filled neurones to the left. Arrow indicates swollen axon ending at the surface of the slice.
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following the action current [2,17] so that the tail current underlying the A H P can be measured. All traces shown (except action potentials) represent the average of 5 - 1 0 trials. For intracellular staining, electrode tips were loaded with biocytin by capillary action (4% in 0.5 M KC1 in 0.05 M Tris buffer, p H 7.4) and then backfilled with 0.5 M KC1. After the experiment, the slice was fixed in 4% buffered paraformaldehyde for 1 - 2 h, then rinsed and permeabilised by exposure to 0.5% Triton overnight. After washing, it was incubated in biotinylated H R P overnight, washed again and reacted using diaminobenzidine. Data are presented as mean + 1 S.E.M., and significant differences have been determined using Student's t-test.
Results
The data in this p a p e r are drawn from recordings made from 130 rat D M V neurones and 145 guinea pig D M V neurones.
Morphology The morphology of representative biocytinfilled neurones in the rat and guinea pig are shown in Fig. 1. Although some dendritic trees were lost at the edges of the slices, neuronal morphology and dimensions showed several consistent differences between the species. Cell bodies were smooth and ovoid with either a mediolateral or a rostrocaudal orientation. They were somewhat larger in the guinea pig, ranging from 22 to 4 5 / z m in the long diameter and from 14 to 25 p.m in the short diameter, compared with those in the rat, with ranges of 18-35 p~m and 10-22 > m respectively. Neuronal shape was distinctive, being stellate in the guinea pig, but in many cases bipolar in the rat, resulting in a smaller mean number of primary dendrites. However, dendritic branching was more extensive in the rat (see Table I). Dendrites of neurones lying above the obex typically branched dorsally, whereas those below the obex radiated from the soma. In most cases one or two dendrites terminated near the central canal
TABLE l
Comparison of dimensional jgatures oJ" rat and guinea pig D M V neurones
Long diameter ( ~ m ) Short diameter (~ m) Number of primary dendrites Number of terminating branches Longest dendrite ( ~ m ) Length of axon in slice (tzm)
Rat (n = 7)
Guinea pig (n=6)
28.3 + 2.6 16.0 + 1.6
34.0 _+3.5 17.3 + 1.8
2.9 + 0.4
4.3 _+0.6
11.9 + 2.0 336_+ 18
9.7 -+ 1.6 520_+56 *
650 _+223
300 _+86
* P<0.01.
(aqueduct) for neurones lying below the obex, or near the floor of the IVth ventricle for neurones lying above the obex. The relatively simple smooth dendrites were 2 - 4 / ~ m in diameter at their origin (generally thicker in guinea pig than in rat) and branched within 200 Fzm of the soma into two or more branches. There was marked tapering so that the distal dendrites were < 0.5 p~m in diameter and sometimes bore varicosities. Overall, the dendritic trees were finer and shorter in the rat than in the guinea pig. An axon was readily identified arising from each filled neurone by its termination in a clublike swelling at the surface of the slice (arrow in Fig. 1). Most axons originated from the soma, but their projections differed between species; they ran relatively straight and ventrolaterally in the rat, but were more coiled and tortuous in the guinea pig, and rarely extended as far laterally in the slice as in the rat. Guinea pig D M V axons were finer than rat D M V axons, often becoming < 1 p~m at about 30-50 /.tm from the soma, so that they were difficult to follow.
Passive electrical properties In parallel with the differences in structural features, input resistance was slightly lower and the capacitance slightly higher in guinea pig than in rat D M V neurones, with similar mean values for time constant (Table II).
93 TABLE II Comparison between passive membrane properties of guinea pig and rat DMV neurones
Rat (n = 44)
Guinea pig (n = 28)
Resting potential (mV) -59.8 5:1.4 -59.1 + 1.1 Input resistance (MI2) 293_+23 214_+27* Membrane time constant (ms) 32_+3 34 _+5 Input capacitance (pF) 115 _+5 137+ 9 * • P < 0.05.
A c t i o n potential Action potentials and the AHPs following them are shown in Fig. 2. Peak amplitude of the action potential was 85 ± 3 mV (n = 11) in the rat and
A
Rat
Gulnes Pig
L
/2o 2 mV ms
B
~ 1 0
mV
C
D lOOpA
1........... ~ JIJJ L
........
apamln
Fig. 2. Action potential and afterhyperpolarisation recorded from rat and guinea pig DMV neurones. A: action potentials elicited by injection of depolarising current from a holding potential of -60 mV. B: the AHP following a single action potential. Action potential peaks have been truncated. C: the tail current following the action current (see Materials and Methods for details). D: superimposed traces showing the effect of 100 nM apamin on the tail current.
9 8 + 3 mV ( n = 1 2 ) in the guinea pig. These values are significantly different ( P < 0.01). Peak rate of rise of the action potential was 189 + 18 V / s (n = 10) in the rat and 284 + 24 V / s (n --- 12) in the guinea pig. These values are significantly different ( P < 0.02). Action potential half-widths were 1.3 ± 0.07 ms (n = 11) in the rat and 1.4 + 0.09 (n = 12) in the guinea pig, and were not significantly different. A hump was clearly detectable on the decay phase of the action potential in guinea pig but not rat neurones (Fig. 2). As described elsewhere for the rat [17], repolarisation of the action potential in guinea pig D M V neurones was slower in the presence of tetraethylammonium (TEA; 400 /xM, n = 3) and charybdotoxin (CTX; 30 nM, n = 2) indicating that a calcium-activated potassium current (I c, 1) contributes to the initial repolarisation. In addition, again as in the rat [18], the action potential in guinea pig D M V neurones had a faster rate of repolarisation when elicited from a hyperpolarised membrane potential indicating that I A also contributes to action potential repolarisation in both species. Afterhyperpolarisation The ensuing afterhyperpolarisation in D M V neurones of both species lasted several seconds (Fig. 2B) and was dependent on Ca 2+ influx being blocked in the presence of cobalt (2 mM) or cadmium (100 izM) [17,18]. However the traces in Fig. 2C show that the currents underlying the AHPs were quite different. Although an early component (Gkca,1) was blocked by the toxin apamin in both species (Fig. 2D), the slower Gkca,2 (which was apamin-insensitive; Fig. 2D) was present in 96% of guinea pig D M V neurones, but in only 9% of the rat neurones; when present in the latter, it was always of much smaller amplitude than in guinea pig neurones. The electrophysiological consequence of the different type of A H P is shown in Fig. 3. In the guinea pig DMV, as increasing amounts of depolarising current were injected, the neurone fired one or at most two action potentials, and then ceased firing because the membrane rectified. It became impossible to evoke additional discharge. In contrast, in the rat DMV, repetitive activity
94
_
Guineapig
, J
-
i
J
-'
Ra~
~ -'
[
,~r
t.._
~"'
,
,
20mY II
~
,o0ma ,2 nA
J ....
l_
Fig. 3. Action potentials elicited by 400 ms depolarising current injections of 0.05, 0.1 and 0.16 nA (rat) and 0.1, 0.2 and 0.3 nA (guinea pig). The holding potential was - 6 2 mV and - 58 mV in the rat and guinea pig neurones respectively.
was readily elicited by depolarising current steps from the same levels of resting m e m b r a n e potential.
Voltage-dependent conductances (i) Transient outward current.
In rat D M V neurones, there is a transient potassium current similar to the A-currents seen in many other kinds of neurones [18]. This current was activated when the cell was depolarised from m e m b r a n e potentials more negative than the resting membrane potential and inactivated with a mean time constant of 420 ms [18]. A similar current was present in guinea pig D M V neurones (Fig. 4). The average time constant of I A in the guinea pig was 573 _+ 18 ms (n = 9). The voltage dependence of this current in the guinea pig was similar to that reported for the rat, so that the current was almost fully inactivated at the resting m e m b r a n e potential.
(ii) Inward rectification. Upon hyperpolarisation of guinea pig D M V neurones, there was a sag in the m e m b r a n e potential and upon repolarisation a rebound depolarisation was generated (Fig. 5A, arrow). With large hyperpolarisations, this rebound excitation was large enough to reach threshold for action potential generation (Fig. 5A). Under voltage clamp, hyperpolarisation of neurones past about - 6 0 mV resulted in a slowly activating inward current (Fig. 5B). This current was completely blocked by adding 1 mM Cs + to the perfusing Ringer (n = 3) but it was insensitive to Ba 2+ (1 mM, n = 2). This inward rectifier was seen in 28 (80%) of 35 neurones tested. In the rat DMV, 44 (57%) of 77 neurones displayed a similar current (Fig. 5B) with the same pharmacological characteristics. However, the current was always of smaller amplitude in the rat neurones and in no case was a rebound depolarisation apparent (e.g., Fig. 5A). This difference in current amplitude is more clearly seen in Fig. 5C where the current-voltage relationship is plotted after normalisation of the steady-state current to the cell capacitance. It should be noted that the guinea pig cell illustrated is typical, whereas the rat cell illustrated was picked because it had a particularly large inward current. Effects of transmitters Application of 5-hydroxytryptamine (5-HT, 20 ~ M ) led to a small depolarisation (5-10 mV) and
GuineaPig
Rat
lsec
Fig. 4. Transient outward current generated on return from a brief hyperpolarising voltage step from a holding potential of - 6 3 mV.
95
an increase in input resistance in both rat and guinea pig neurones. In guinea pig neurones, the slow component of the A H P was selectively abolished in the presence of 5-HT (Fig. 6; n = 4). Under voltage clamp it was confirmed that this was due to a selective blockade of Gkca,2 (not shown). In contrast, 5-HT had no effect on the A H P in rat neurones (Fig. 6; n = 4).
A
u i n e a Pig
I
~
~
t-
i 0 . 4 nA
rl.m!,Omv
100 m s
1 S~
Fig. 6. 5-HT selectively blocks the slow component of the afterhyperpolarisation in guinea pig DMV neurones. The AHP recorded in the rat (upper traces) and guinea pig (lower traces) in control solution and in the presence of 20 /~M 5-HT. The holding potential in each case was - 60 mV.
nA
--"-"----'J
Guinea Pig
mV
[ I°-=n"
~
5-HT
Rat
J
I,OmV
],
Rat
We have previously reported that noradrenaline selectively blocks Gkca,2 in guinea pig neurones [17]. Application of noradrenaline in rat cells had variable effects. Noradrenaline (100 ~ M ) was tested on five neurones. In each case there was a small depolarisation ( < 5 mV) and an increase in input resistance. In two cells there was a reduction ( > 50%) of the AHP amplitude while the AHP was unaffected in the other three cells.
C Discussion • Gplg
/r / ,
• - 110
, -95
-
, • , -80 -65 Vm ( m V )
•
, -50
Fig. 5. Properties of the inward rectifier. A: current clamp. Voltage response (upper traces) to a hyperpolarising current injection (lower traces) from a holding potential of - 6 0 mV. Note the rebound depolarisation (arrow) present in the guinea pig but not in the rat. B: voltage clamp. Current generated (upper traces) in response to hyperpolarising voltage steps (lower traces) from a holding potential of - 6 0 mV. Note the different scale bars between traces from guinea pig ~Ind rat neurones. C: steady-state current-voltage relation for the two neurones normalised to the cell capacitance. Leak currents have been subtracted by scaling the current response to a small voltage step.
In this study we have examined the membrane properties of neurones of the dorsal motor nucleus of the vagus in the rat and guinea pig. The main finding of this study is that, whereas the passive membrane properties of the neurones in the two species are very similar and the small differences can be accounted for by their morphological differences, the active properties of these cells (in terms of voltage- and calcium-dependent conductances) are quite different (Table III). In both species both sodium and calcium currents contributed to the action potential. The peak amplitude was larger and the duration
96 TABLE llI
Summa~ of differences in active properties of DMV neurones in rat and guinea pig Rat
Guinea pig
Action potential amplitude (mV) rate of rise ( V / s ) half-width (ms)
85 + 3 (11) 189 _+ 18 (10) 1.3 + 0.07 (10)
98 + 3 * (12) 284 + 24 * * (12) 1.4 + 0.09 (12)
Current underlying AHP
Gkc,~, ~
Gkc,,. I + Gk(~.,
Noradrenaline 5-HT
depolarisation only depolarisation only
depol. + block Gk (,,.e depol. + block Gkc,.2
All values are given as mean _+ S.E.M.; values of n in parentheses. * P < 0 . 0 1 ; ** P < 0.02.
somewhat longer in the guinea pig. This difference in the action potential could be due to a difference in the inward currents (e.g., larger and longer lasting calcium current in the guinea pig) or due to a difference in a repolarising current (e.g., a larger potassium current active during repolarisation in the rat). From the maximum rate of rise of the action potential and the input capacitance, the inward current during the upstroke of the action potential can be calculated to be about 39 nA in the guinea pig but only 29 nA in the rat. This difference may explain the difference in peak amplitude of the action potential between the two species. In both the rat [18] and the guinea pig, a calcium-activated potassium current (I c) and a voltage-dependent potassium current (I A) are active during repolarisation. However, our data do not allow us to quantitate the relative magnitudes of these currents in the two species. The mechanism underlying the later parts of the A H P was quite different in the two species. All rat and 96% of guinea pig neurones had an apamin-sensitive calcium-activated potassium current (Gkca,1). In the guinea pig, however, the majority of neurones also had another calciumactivated potassium current which had slower kinetics and was apamin-insensitive (Gkca.2; see Sah and McLachlan [17]). The action potential in guinea pig neurones is larger and broader than in the rat which means that more calcium may enter during the action potential in guinea pig neutones. However, the absence of Gkca,2 in most
rat DMV neurones is not due to insufficient calcium influx because increasing calcium influx in these neurones (either by addition of T E A or firing multiple action potentials) does not reveal Gk c~,,2 [16]. Because Gkca,2 is so prolonged in guinea pig neurones, the functional consequence is that during prolonged injection of depolarising current neurones in the guinea pig only fire one or two action potentials and then stop whereas the neurones of the rat fire repetitively. This difference in the firing properties of the neurones between the two species is similar to the difference in firing properties between tonic and L A H cells in the guinea pig coeliac ganglion which have Gkca, l and both Gkca,l and Gkca,2 respectively [2,3]. When most D M V neurones, particularly those of the guinea pig, were hyperpolarised beyond - 6 0 mV, an inward current was activated which was blocked by extracellular Cs + but not by Ba 2 +. This current has the voltage dependence and pharmacological profile of the current called 11 o r I h in other neurones [10,11,22]. If this current is relatively large, there is a rebound depolarisation when the m e m b r a n e repolarises following a period of hyperpolarisation [e.g., 10,11]. In the rat DMV, this current was detectable in fewer neurones than in the guinea pig DMV, and in those cases when it could be measured it was smaller, and there was no rebound depolarisation. Both noradrenergic [21] and serotonergic [20] afferents are known to innervate the rat DMV. Comparable data are however lacking for the
97
guinea pig. We have shown that both substances depolarise neurones in both species. The depolarising action of noradrenaline in the rat has previously been described by Fukuda et al. [5] and is confirmed here. In the guinea pig there is an additional blockade of Gkca,2. Functionally, the effect of this is to increase the firing frequency of neurones in response to prolonged depolarisations [e.g., 4,9]. Blockade of Gkca,2 has a similar effect in DMV neurones as well (Sah, unpublished observations). Our data on the morphology and action potential characteristics of guinea pig DMV neurones are in good agreement with results obtained by Yarom et al. [23; see also 14] in a previous study of these cells. Yarom et al., however, did not discuss the different types of neurones in the DMV and did not identify the two different types of calcium-activated conductance. In addition, the I A described by them appears to inactivate faster than those we recorded. Examination of their Fig. 13 [23] suggests a time constant of decay of about 100 ms. However, the voltage-dependent characteristics of the current they described are similar to our findings. Comparable data have not been previously published for rat DMV neurones. Why are there these differences between the two species? It is possible that they reflect a species difference in gene expression. If so, it is of interest that a similar long duration AHP is achieved by two entirely different mechanisms. For comparison, in sympathetic lumbar ganglia in the same two species, the time course of the excitatory synaptic potential is similar despite a two-fold difference in membrane time constant; this results from comparable differences in the time course of the synaptic currents [12]. On the other hand, the DMV is a large nucleus with different functions and the neurones are organised viscerotopically. Another possibility therefore arises because, although recordings were made at similar levels relative to the obex in both species, the rostrocaudal extent of the DMV is located somewhat more caudally relative to the obex in the rat than in the guinea pig (E. McLachlan, unpublished observations). Thus recordings may not have been made from functionally equivalent populations. Although this
could be checked using neuroanatomical tracing techniques, the present experiments have sampled neurones over nearly 2 mm without detecting any topographic differences within either species. Calcium-activated potassium conductances have been shown to be modulated by many transmitters in a variety of cell types [13]. We have shown that Gkca,2 can be selectively blocked by application of noradrenaline [17] or serotonin (Fig. 6). The fact that rat neurones do not display this current implies that modulation of the integrative properties of DMV neurones by at least these two transmitters must be quite different in the two species.
Acknowledgements This research was supported by grants from the National Health and Medical Research Council of Australia. We thank Mandy Bauer for assistance with the histology.
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22
23
vagus nerve are excited by oxytocin in the rat but not in the guinea pig, Proc. Natl. Acad. Sci. USA, 84 (1987) 3926-3930. Sah, P., The role of calcium influx and calcium buffering in the kinetics of a Ca 2+ activated K + current in rat vagal motoneurons, J. Neurophysiol., 1992 (in press). Sah, P. and McLachlan, E.M., Ca 2+ activated K ÷ currents underlying the afterhyperpolarization in guinea pig vagal neurons: A role for Ca 2+ activated Ca 2+ release, Neuron, 7 (1991) 257-264. Sah, P. and McLachlan, E.M., Potassium currents contributing to action potential repolarization and the afterhyperpolarization in rat vagal motoneurons, J. Neurophysiol., (1992) (in press). Siaud, P., Denoroy, L., Assenmacher, 1. and Alonso, G., Comparative immunocytochemical study of the catecholamine and peptidergic afferent innervation of the dorsal vagal complex in rat and guinea pig, J. Comp. Neurol., 290 (1989) 323-335. Steinbusch, H.W.M., Distribution of serotonin-immunoreactivity in the central nervous system of the rat. Cell bodies and terminals, Neuroscience, 6 (1981) 557-618. Ter Horst, G.J., Toes, G.J. and Van Willigen, J.D., Locus coeruleus projections to the dorsal motor vagus nucleus in the rat, Neuroscience, 45 (1991) 153-160. Tokimasa, T. and Akasu, T., Cyclic AMP regulates an inward rectifying sodium-potassium current in dissociated bull-frog sympathetic neurones, J. Physiol., 420 (1990) 409-429. Yarom, Y., Sugimori, M. and Llinas, R.R., Ionic currents and firing patterns of mammalian vagal motoneurones in vitro, Neuroscience, 16 (1985) 719-737.
89
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1838/93/$06.00 JANS 01349
Differences in electrophysiological properties between neurones of the dorsal motor nucleus of the vagus in rat and guinea pig Pankaj Sah and Elspeth M. McLachlan Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Australia (Received 9 June 1992) (Revision received 19 August 1992) (Accepted 25 August 1992)
Key words: P r e g a n g l i o n i c ; N o r a d r e n a l i n e ; A f t e r h y p e r p o l a r i s a t i o n ; S e r o t o n i n
Abstract We have examined the electrophysiological properties of neurones in the dorsal motor nucleus of the vagus (DMV) in rats and guinea pigs in transverse medullary slices maintained in vitro. There were only minor differences in the morphology of the neurones between the species, and their passive electrical properties were very similar. However, action potentials in guinea pig neurones had larger amplitudes and longer half-widths than did those in rat neurones. In both species, action potentials were followed by prolonged afterhyperpolarisations (AHPs). In the majority of guinea pig neurones, two calcium-activated potassium currents underlying the AHP could be separated into an early apamin-sensitive component and a late apamin-insensitive component. In rat neurones, the current underlying the AHP was briefer and entirely apamin-sensitive. In response to a step of depolarising current, neurones in the guinea pig only discharged once or twice and then ceased firing. In rat neurones, this manoeuvre produced repetitive firing. An inward rectifier was larger in neurones of the guinea pig than in those in the rat. The effects of 5-hydroxytryptamine and noradrenaline also differed between neurones of each species. We conclude that, despite many similarities of size and electrical properties, DMV neurones in the two species differ in terms of several voltage- and calcium-dependent conductances which determine their active electrical behaviour.
Introduction T h e cells of t h e dorsal m o t o r n u c le u s o f t h e vagus ( D M V ) lie in a c o l u m n e x t e n d i n g f r o m th e rostral to t h e c a u d a l m e d u l l a o b l o n g a t a in its d o r s o m e d i a l part. T h e s e n e u r o n e s a r e th e m a i n s o u r c e o f t h e p a r a s y m p a t h e t i c i n n e r v a t i o n o f th e s u b d i a p h r a g m a t i c visceral organs. T h e m a j o r physiological roles o f t h e s e n e u r o n e s are in t h e
Correspondence to: P. Sah, Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Queensland 4072, Australia.
c o n t r o l o f g a s t r o i n t e s t i n a l and p a n c r e a t i c secretion as well as s t o m a c h an d small intestinal motility. This n u c l e u s receives a diverse array of afferen t inputs f r o m m a n y b r a i n s t e m and h i g h e r centres [8]. M o s t studies of the physiology and a n a t o m y of b r a i n s t e m a u t o n o m i c c e n t r e s have focussed on rat or cat. T h e r e is little i n f o r m a t i o n r e g a r d i n g the a n a t o m y or f u n c t i o n a l significance o f t h ese c e n t r e s in t h e g u i n e a pig, a l t h o u g h species differen ces b e t w e e n c o m p a r a b l e cell g r o u p s in t h e v e n t r o l a t e r a l m e d u l l a have b e e n d e s c r i b e d [6,7]. S o m e m a r k e d d i f f e r e n c e s have also b e e n n o t e d in the D M V b e t w e e n rat and g u i n e a pig. In the
90
guinea pig, the D M V contains large numbers of oxytocin- and ACTH-containing terminals which make synapses with the soma and dendrites of D M V neurones; these inputs are absent in the rat [19]. On the other hand it has been shown that oxytocin excites neurones in the rat but not in the guinea pig D M V [15]. We have recently described the pharmacology of the two calcium-activated potassium conductances which underlie the afterhyperpolarisation (AHP) in guinea pig vagal neurones [17]. These two types of Ca2+-activated potassium conductance, named Gkc~.l and Gkca,2 respectively, can be separated both kinetically and pharmacologically (see also Cassell and McLachlan [3]). Activation of the slower time course conductance was shown to be dependent on calcium-induced calcium release [17]. This has been confirmed by immunohistochemical evidence for the presence of ryanodine receptor-like immunoreactivity in guinea pig D M V at considerably higher concentrations than in the rat (Sah, Francis, McLachlan and Junankar, unpublished observations), consistent with the distribution of Gkc~,, 2 in the two species. A calcium-induced calcium release mechanism is therefore likely to be rare or absent in rat D M V neurones [16]. As neurones in the D M V probably perform similar functional roles, it is important to establish whether other differences exist in the passive and active electrical properties of vagal neurones between species. In this paper we compare the morphological and electrophysiological properties of neurones in the dorsal motor nucleus of the vagus in rats and guinea pigs in transverse medullary slices maintained in vitro.
Materials and Methods
All experiments were done on transverse slices of medulla from young adult rats or guinea pigs maintained in vitro. Methods for preparation of slices from rat medulla were identical to those previously described for the guinea pig [17]. The animals were deeply anaesthetised with intraperitoneal pentobarbital (50 m g / k g ) and decapitated. The brainstem was quickly removed and ira-
mersed in cold Ringer of the following composition (raM): NaC1, 115; KCI, 5; MgSO4, 1.2; CaCI 2, 2.5; NaH2PO4, 1.2; NaHCO~, 25; glucose, 10, gassed with 95% 0 2 / 5 % CO 2. Three or four transverse slices (400 /~m thick) containing the D M V were cut with a Vibratome rostrally from approximately 1 mm caudal to the obex; these were allowed to recover for 1 h in a humidified chamber before recording was attempted. Slices were studied fully submerged in the recording chamber and perfused with Ringer warmed to 28-30°C. Picrotoxin (100 ~ M ) was routinely added to block the spontaneous inhibitory potentials which invariably appeared in neurones of both species when KCl-filled electrodes were used. Microelectrodes were fabricated on a BrownFlaming puller and filled with 0.5 M KCI (resistance 70-140 M~Q). By means of a combination of trans- and epi-illumination, the D M V was readily identified as a translucent area immediately dorsal to the hypoglossal nucleus. Most recordings were made from slices containing the obex or the region immediately rostral to it. Signals were recorded using a single electrode voltage clamp amplifier (Axo Clamp 2a, Axon Instruments), sampled at 0.5-10 kHz and stored and analysed on an IBM compatible computer. During voltage clamp experiments the headstage was continuously monitored to ensure complete settling of the voltage transient between samples and switching frequencies of 1.5-3 kHz were used. Electrode tips were coated with silicone oil in order to reduce the electrode capacitance. Action potentials were elicited from a given holding potential by injecting a 10 ms depolarising current pulse through the recording microelectrode. To measure the currents underlying the AHP, the cell was clamped at the resting m e m b r a n e potential ( - 6 0 to - 7 0 mV) and a brief (10 ms) depolarising voltage command step (usually 10-15 mV) was delivered. The magnitude of this pulse was increased until a single active response was elicited. The sodium current underlying the action potential cannot be controlled by the voltage clamp and results in a poorly controlled 'action current'. However, good voltage control is always obtained within 50 ms
91
RAT 1 mm
G U I N E A PIG /,
E Fig. 1. Morphology of biocytin-filled neurones in the dorsal motor nucleus of the vagus (DMV) in the rat and guinea pig. The schematic diagrams show a transverse section of the medulla with the dashed area representing the region illustrated at low magnification to the right. The calibration in the upper right-hand figure refers to both species. The calibration at the lower right corner refers to the enlarged views of the filled neurones to the left. Arrow indicates swollen axon ending at the surface of the slice.
92
following the action current [2,17] so that the tail current underlying the A H P can be measured. All traces shown (except action potentials) represent the average of 5 - 1 0 trials. For intracellular staining, electrode tips were loaded with biocytin by capillary action (4% in 0.5 M KC1 in 0.05 M Tris buffer, p H 7.4) and then backfilled with 0.5 M KC1. After the experiment, the slice was fixed in 4% buffered paraformaldehyde for 1 - 2 h, then rinsed and permeabilised by exposure to 0.5% Triton overnight. After washing, it was incubated in biotinylated H R P overnight, washed again and reacted using diaminobenzidine. Data are presented as mean + 1 S.E.M., and significant differences have been determined using Student's t-test.
Results
The data in this p a p e r are drawn from recordings made from 130 rat D M V neurones and 145 guinea pig D M V neurones.
Morphology The morphology of representative biocytinfilled neurones in the rat and guinea pig are shown in Fig. 1. Although some dendritic trees were lost at the edges of the slices, neuronal morphology and dimensions showed several consistent differences between the species. Cell bodies were smooth and ovoid with either a mediolateral or a rostrocaudal orientation. They were somewhat larger in the guinea pig, ranging from 22 to 4 5 / z m in the long diameter and from 14 to 25 p.m in the short diameter, compared with those in the rat, with ranges of 18-35 p~m and 10-22 > m respectively. Neuronal shape was distinctive, being stellate in the guinea pig, but in many cases bipolar in the rat, resulting in a smaller mean number of primary dendrites. However, dendritic branching was more extensive in the rat (see Table I). Dendrites of neurones lying above the obex typically branched dorsally, whereas those below the obex radiated from the soma. In most cases one or two dendrites terminated near the central canal
TABLE l
Comparison of dimensional jgatures oJ" rat and guinea pig D M V neurones
Long diameter ( ~ m ) Short diameter (~ m) Number of primary dendrites Number of terminating branches Longest dendrite ( ~ m ) Length of axon in slice (tzm)
Rat (n = 7)
Guinea pig (n=6)
28.3 + 2.6 16.0 + 1.6
34.0 _+3.5 17.3 + 1.8
2.9 + 0.4
4.3 _+0.6
11.9 + 2.0 336_+ 18
9.7 -+ 1.6 520_+56 *
650 _+223
300 _+86
* P<0.01.
(aqueduct) for neurones lying below the obex, or near the floor of the IVth ventricle for neurones lying above the obex. The relatively simple smooth dendrites were 2 - 4 / ~ m in diameter at their origin (generally thicker in guinea pig than in rat) and branched within 200 Fzm of the soma into two or more branches. There was marked tapering so that the distal dendrites were < 0.5 p~m in diameter and sometimes bore varicosities. Overall, the dendritic trees were finer and shorter in the rat than in the guinea pig. An axon was readily identified arising from each filled neurone by its termination in a clublike swelling at the surface of the slice (arrow in Fig. 1). Most axons originated from the soma, but their projections differed between species; they ran relatively straight and ventrolaterally in the rat, but were more coiled and tortuous in the guinea pig, and rarely extended as far laterally in the slice as in the rat. Guinea pig D M V axons were finer than rat D M V axons, often becoming < 1 p~m at about 30-50 /.tm from the soma, so that they were difficult to follow.
Passive electrical properties In parallel with the differences in structural features, input resistance was slightly lower and the capacitance slightly higher in guinea pig than in rat D M V neurones, with similar mean values for time constant (Table II).
93 TABLE II Comparison between passive membrane properties of guinea pig and rat DMV neurones
Rat (n = 44)
Guinea pig (n = 28)
Resting potential (mV) -59.8 5:1.4 -59.1 + 1.1 Input resistance (MI2) 293_+23 214_+27* Membrane time constant (ms) 32_+3 34 _+5 Input capacitance (pF) 115 _+5 137+ 9 * • P < 0.05.
A c t i o n potential Action potentials and the AHPs following them are shown in Fig. 2. Peak amplitude of the action potential was 85 ± 3 mV (n = 11) in the rat and
A
Rat
Gulnes Pig
L
/2o 2 mV ms
B
~ 1 0
mV
C
D lOOpA
1........... ~ JIJJ L
........
apamln
Fig. 2. Action potential and afterhyperpolarisation recorded from rat and guinea pig DMV neurones. A: action potentials elicited by injection of depolarising current from a holding potential of -60 mV. B: the AHP following a single action potential. Action potential peaks have been truncated. C: the tail current following the action current (see Materials and Methods for details). D: superimposed traces showing the effect of 100 nM apamin on the tail current.
9 8 + 3 mV ( n = 1 2 ) in the guinea pig. These values are significantly different ( P < 0.01). Peak rate of rise of the action potential was 189 + 18 V / s (n = 10) in the rat and 284 + 24 V / s (n --- 12) in the guinea pig. These values are significantly different ( P < 0.02). Action potential half-widths were 1.3 ± 0.07 ms (n = 11) in the rat and 1.4 + 0.09 (n = 12) in the guinea pig, and were not significantly different. A hump was clearly detectable on the decay phase of the action potential in guinea pig but not rat neurones (Fig. 2). As described elsewhere for the rat [17], repolarisation of the action potential in guinea pig D M V neurones was slower in the presence of tetraethylammonium (TEA; 400 /xM, n = 3) and charybdotoxin (CTX; 30 nM, n = 2) indicating that a calcium-activated potassium current (I c, 1) contributes to the initial repolarisation. In addition, again as in the rat [18], the action potential in guinea pig D M V neurones had a faster rate of repolarisation when elicited from a hyperpolarised membrane potential indicating that I A also contributes to action potential repolarisation in both species. Afterhyperpolarisation The ensuing afterhyperpolarisation in D M V neurones of both species lasted several seconds (Fig. 2B) and was dependent on Ca 2+ influx being blocked in the presence of cobalt (2 mM) or cadmium (100 izM) [17,18]. However the traces in Fig. 2C show that the currents underlying the AHPs were quite different. Although an early component (Gkca,1) was blocked by the toxin apamin in both species (Fig. 2D), the slower Gkca,2 (which was apamin-insensitive; Fig. 2D) was present in 96% of guinea pig D M V neurones, but in only 9% of the rat neurones; when present in the latter, it was always of much smaller amplitude than in guinea pig neurones. The electrophysiological consequence of the different type of A H P is shown in Fig. 3. In the guinea pig DMV, as increasing amounts of depolarising current were injected, the neurone fired one or at most two action potentials, and then ceased firing because the membrane rectified. It became impossible to evoke additional discharge. In contrast, in the rat DMV, repetitive activity
94
_
Guineapig
, J
-
i
J
-'
Ra~
~ -'
[
,~r
t.._
~"'
,
,
20mY II
~
,o0ma ,2 nA
J ....
l_
Fig. 3. Action potentials elicited by 400 ms depolarising current injections of 0.05, 0.1 and 0.16 nA (rat) and 0.1, 0.2 and 0.3 nA (guinea pig). The holding potential was - 6 2 mV and - 58 mV in the rat and guinea pig neurones respectively.
was readily elicited by depolarising current steps from the same levels of resting m e m b r a n e potential.
Voltage-dependent conductances (i) Transient outward current.
In rat D M V neurones, there is a transient potassium current similar to the A-currents seen in many other kinds of neurones [18]. This current was activated when the cell was depolarised from m e m b r a n e potentials more negative than the resting membrane potential and inactivated with a mean time constant of 420 ms [18]. A similar current was present in guinea pig D M V neurones (Fig. 4). The average time constant of I A in the guinea pig was 573 _+ 18 ms (n = 9). The voltage dependence of this current in the guinea pig was similar to that reported for the rat, so that the current was almost fully inactivated at the resting m e m b r a n e potential.
(ii) Inward rectification. Upon hyperpolarisation of guinea pig D M V neurones, there was a sag in the m e m b r a n e potential and upon repolarisation a rebound depolarisation was generated (Fig. 5A, arrow). With large hyperpolarisations, this rebound excitation was large enough to reach threshold for action potential generation (Fig. 5A). Under voltage clamp, hyperpolarisation of neurones past about - 6 0 mV resulted in a slowly activating inward current (Fig. 5B). This current was completely blocked by adding 1 mM Cs + to the perfusing Ringer (n = 3) but it was insensitive to Ba 2+ (1 mM, n = 2). This inward rectifier was seen in 28 (80%) of 35 neurones tested. In the rat DMV, 44 (57%) of 77 neurones displayed a similar current (Fig. 5B) with the same pharmacological characteristics. However, the current was always of smaller amplitude in the rat neurones and in no case was a rebound depolarisation apparent (e.g., Fig. 5A). This difference in current amplitude is more clearly seen in Fig. 5C where the current-voltage relationship is plotted after normalisation of the steady-state current to the cell capacitance. It should be noted that the guinea pig cell illustrated is typical, whereas the rat cell illustrated was picked because it had a particularly large inward current. Effects of transmitters Application of 5-hydroxytryptamine (5-HT, 20 ~ M ) led to a small depolarisation (5-10 mV) and
GuineaPig
Rat
lsec
Fig. 4. Transient outward current generated on return from a brief hyperpolarising voltage step from a holding potential of - 6 3 mV.
95
an increase in input resistance in both rat and guinea pig neurones. In guinea pig neurones, the slow component of the A H P was selectively abolished in the presence of 5-HT (Fig. 6; n = 4). Under voltage clamp it was confirmed that this was due to a selective blockade of Gkca,2 (not shown). In contrast, 5-HT had no effect on the A H P in rat neurones (Fig. 6; n = 4).
A
u i n e a Pig
I
~
~
t-
i 0 . 4 nA
rl.m!,Omv
100 m s
1 S~
Fig. 6. 5-HT selectively blocks the slow component of the afterhyperpolarisation in guinea pig DMV neurones. The AHP recorded in the rat (upper traces) and guinea pig (lower traces) in control solution and in the presence of 20 /~M 5-HT. The holding potential in each case was - 60 mV.
nA
--"-"----'J
Guinea Pig
mV
[ I°-=n"
~
5-HT
Rat
J
I,OmV
],
Rat
We have previously reported that noradrenaline selectively blocks Gkca,2 in guinea pig neurones [17]. Application of noradrenaline in rat cells had variable effects. Noradrenaline (100 ~ M ) was tested on five neurones. In each case there was a small depolarisation ( < 5 mV) and an increase in input resistance. In two cells there was a reduction ( > 50%) of the AHP amplitude while the AHP was unaffected in the other three cells.
C Discussion • Gplg
/r / ,
• - 110
, -95
-
, • , -80 -65 Vm ( m V )
•
, -50
Fig. 5. Properties of the inward rectifier. A: current clamp. Voltage response (upper traces) to a hyperpolarising current injection (lower traces) from a holding potential of - 6 0 mV. Note the rebound depolarisation (arrow) present in the guinea pig but not in the rat. B: voltage clamp. Current generated (upper traces) in response to hyperpolarising voltage steps (lower traces) from a holding potential of - 6 0 mV. Note the different scale bars between traces from guinea pig ~Ind rat neurones. C: steady-state current-voltage relation for the two neurones normalised to the cell capacitance. Leak currents have been subtracted by scaling the current response to a small voltage step.
In this study we have examined the membrane properties of neurones of the dorsal motor nucleus of the vagus in the rat and guinea pig. The main finding of this study is that, whereas the passive membrane properties of the neurones in the two species are very similar and the small differences can be accounted for by their morphological differences, the active properties of these cells (in terms of voltage- and calcium-dependent conductances) are quite different (Table III). In both species both sodium and calcium currents contributed to the action potential. The peak amplitude was larger and the duration
96 TABLE llI
Summa~ of differences in active properties of DMV neurones in rat and guinea pig Rat
Guinea pig
Action potential amplitude (mV) rate of rise ( V / s ) half-width (ms)
85 + 3 (11) 189 _+ 18 (10) 1.3 + 0.07 (10)
98 + 3 * (12) 284 + 24 * * (12) 1.4 + 0.09 (12)
Current underlying AHP
Gkc,~, ~
Gkc,,. I + Gk(~.,
Noradrenaline 5-HT
depolarisation only depolarisation only
depol. + block Gk (,,.e depol. + block Gkc,.2
All values are given as mean _+ S.E.M.; values of n in parentheses. * P < 0 . 0 1 ; ** P < 0.02.
somewhat longer in the guinea pig. This difference in the action potential could be due to a difference in the inward currents (e.g., larger and longer lasting calcium current in the guinea pig) or due to a difference in a repolarising current (e.g., a larger potassium current active during repolarisation in the rat). From the maximum rate of rise of the action potential and the input capacitance, the inward current during the upstroke of the action potential can be calculated to be about 39 nA in the guinea pig but only 29 nA in the rat. This difference may explain the difference in peak amplitude of the action potential between the two species. In both the rat [18] and the guinea pig, a calcium-activated potassium current (I c) and a voltage-dependent potassium current (I A) are active during repolarisation. However, our data do not allow us to quantitate the relative magnitudes of these currents in the two species. The mechanism underlying the later parts of the A H P was quite different in the two species. All rat and 96% of guinea pig neurones had an apamin-sensitive calcium-activated potassium current (Gkca,1). In the guinea pig, however, the majority of neurones also had another calciumactivated potassium current which had slower kinetics and was apamin-insensitive (Gkca.2; see Sah and McLachlan [17]). The action potential in guinea pig neurones is larger and broader than in the rat which means that more calcium may enter during the action potential in guinea pig neutones. However, the absence of Gkca,2 in most
rat DMV neurones is not due to insufficient calcium influx because increasing calcium influx in these neurones (either by addition of T E A or firing multiple action potentials) does not reveal Gk c~,,2 [16]. Because Gkca,2 is so prolonged in guinea pig neurones, the functional consequence is that during prolonged injection of depolarising current neurones in the guinea pig only fire one or two action potentials and then stop whereas the neurones of the rat fire repetitively. This difference in the firing properties of the neurones between the two species is similar to the difference in firing properties between tonic and L A H cells in the guinea pig coeliac ganglion which have Gkca, l and both Gkca,l and Gkca,2 respectively [2,3]. When most D M V neurones, particularly those of the guinea pig, were hyperpolarised beyond - 6 0 mV, an inward current was activated which was blocked by extracellular Cs + but not by Ba 2 +. This current has the voltage dependence and pharmacological profile of the current called 11 o r I h in other neurones [10,11,22]. If this current is relatively large, there is a rebound depolarisation when the m e m b r a n e repolarises following a period of hyperpolarisation [e.g., 10,11]. In the rat DMV, this current was detectable in fewer neurones than in the guinea pig DMV, and in those cases when it could be measured it was smaller, and there was no rebound depolarisation. Both noradrenergic [21] and serotonergic [20] afferents are known to innervate the rat DMV. Comparable data are however lacking for the
97
guinea pig. We have shown that both substances depolarise neurones in both species. The depolarising action of noradrenaline in the rat has previously been described by Fukuda et al. [5] and is confirmed here. In the guinea pig there is an additional blockade of Gkca,2. Functionally, the effect of this is to increase the firing frequency of neurones in response to prolonged depolarisations [e.g., 4,9]. Blockade of Gkca,2 has a similar effect in DMV neurones as well (Sah, unpublished observations). Our data on the morphology and action potential characteristics of guinea pig DMV neurones are in good agreement with results obtained by Yarom et al. [23; see also 14] in a previous study of these cells. Yarom et al., however, did not discuss the different types of neurones in the DMV and did not identify the two different types of calcium-activated conductance. In addition, the I A described by them appears to inactivate faster than those we recorded. Examination of their Fig. 13 [23] suggests a time constant of decay of about 100 ms. However, the voltage-dependent characteristics of the current they described are similar to our findings. Comparable data have not been previously published for rat DMV neurones. Why are there these differences between the two species? It is possible that they reflect a species difference in gene expression. If so, it is of interest that a similar long duration AHP is achieved by two entirely different mechanisms. For comparison, in sympathetic lumbar ganglia in the same two species, the time course of the excitatory synaptic potential is similar despite a two-fold difference in membrane time constant; this results from comparable differences in the time course of the synaptic currents [12]. On the other hand, the DMV is a large nucleus with different functions and the neurones are organised viscerotopically. Another possibility therefore arises because, although recordings were made at similar levels relative to the obex in both species, the rostrocaudal extent of the DMV is located somewhat more caudally relative to the obex in the rat than in the guinea pig (E. McLachlan, unpublished observations). Thus recordings may not have been made from functionally equivalent populations. Although this
could be checked using neuroanatomical tracing techniques, the present experiments have sampled neurones over nearly 2 mm without detecting any topographic differences within either species. Calcium-activated potassium conductances have been shown to be modulated by many transmitters in a variety of cell types [13]. We have shown that Gkca,2 can be selectively blocked by application of noradrenaline [17] or serotonin (Fig. 6). The fact that rat neurones do not display this current implies that modulation of the integrative properties of DMV neurones by at least these two transmitters must be quite different in the two species.
Acknowledgements This research was supported by grants from the National Health and Medical Research Council of Australia. We thank Mandy Bauer for assistance with the histology.
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