- Email: [email protected]
Slow IPSP and the noradrenaline-induced inhibition of the cat sympathetic preganglionic neuron in vitro
Brain Research, 419 (1987) 383-386 Elsevier
383
BRE 22461
Slow IPSP and the noradrenaline-induced inhibition of the cat sympathetic preganglionic neuron in vitro M. Yoshimura 1, C.
Polosa 2 a n d S. Nishi 1
l Department of Physiology, Kurume University School of Medicine, Kurume (Japan) and 2Department of Physiology, McGill University, Montreal, Que. (Canada) (Accepted 26 May 1987)
Key words: Catecholamine; Sympathetic neuron; Slow synaptic potential; Spinal cord; Potassium conductance
Focal electrical stimulation of the slice of the cat thoracic cord evoked in sympathetic preganglionic neurons a slow inhibitory postsynaptic potential (IPSP) associated with decreased neuronal input resistance. The slow IPSP decreased in amplitude with membrane hyperpolarization and reversed at about -90 mV. It increased in amplitude in low potassium and decreased in high potassium. Noradrenaline (NA) at doses of 10-50 #M caused in some of these cells a hyperpolarization with properties similar to those of the slow IPSP. Both the slow IPSP and the NA-evoked hyperpolarization were abolished by yohimbine, but not by prazosin or propranolol. These data suggest that both responses are due to an increase in potassium conductance and that NA may be the mediator of the slow IPSP evoked by focal stimulation.
The intermediolateral nucleus (IML) of the thoracic cord contains a rich network of catecholaminecontaining fibers of supraspinal origin 1,4. The results of iontophoretic application of catecholamines (CA) to sympathetic preganglionic neurons (SPNs) 2,9,1°A3, as well as of administration of adrenergic blockers 3'6, suggest an inhibitory action of C A on SPNs. This inhibitory action has been shown to be mediated by a 2 receptors 9'1°. Binding sites for the a 2 antagonist, yohimbine in the I M L of the cat have been demonstrated 5. Two questions concerning the function of the a 2 adrenoceptors on the SPN membrane need to be answered: (i) what is the mechanism by which these receptors inhibit SPN firing; and (ii) can these receptors be activated synaptically? The observations reported in the present paper, obtained in the in vitro slice of the cat thoracic spinal cord, show that: (i) the mechanism of the NA-evoked inhibition of the SPN mediated by a 2 receptors is a membrane hyperpolarization due to an increase in K-conductance; and (ii) that focal stimulation of the slice evokes synapticallymediated hyperpolarizations of very long time course (slow IPSP) which have the same ionic mechanism as
the NA-evoked hyperpolarization and are blocked by the a 2 antagonist, yohimbine. The experiments were performed on transverse slices of cat upper thoracic spinal cord. Intracellular recordings were made from SPNs of the IML, identified by antidromic stimulation as previously described 14. In cats anesthetized with chloralose and pentobarbital (60 and 10 mg/kg, respectively, i.p.), the second and third thoracic segments were excised and cut into 500/~m thick slices. These were transferred to a recording chamber and superfused with Krebs solution, equilibrated with 95% 0 2 and 5% CO 2, containing (in mM): NaC1 117, KC1 3.6, NaH2PO4 1.2, CaC12 2.5, MgC12 1.2, glucose 11.0, and N a H C O 3 25.0. Temperature was maintained at 37 °C. Cells were impaled using glass micropipettes filled with 3 M KC1 or 2 M potassium acetate or citrate. Electrode resistance varied between 40 and 120 Mr2. Intracellular recordings were obtained using a high input impedance amplifier with an active bridge circuit, enabling simultaneous measurement of membrane potential and intracellular current injection. The amplifier output was monitored on a digital oscil-
Correspondence: C. Polosa, Department of Physiology, McGill University, 3655 Drummond St., Montreal, Que. Canada H3G 1Y6. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
384 loscope. A DC pen-recorder was used to record membrane potential continuously. NA (10-50 #M) was added to the superfusing Krebs solution. Focal stimulation was performed with a monopolar, 50/~m diameter, silver wire electrode, insulated except at the tip. Stimuli were square pulses of 0.2 ms duration and variable voltage. Resting potential was - 6 0 + 2.7 mV (n = 31). Focal stimulation of the ipsilateral dorsolateral funiculus with single shocks or short trains of stimuli caused in 31 out of 82 cells tested a long-lasting, graded hyperpolarization. The response was preceded by a fast EPSP which, in some cases, evoked a spike. In such cases the slow hyperpolarization was superimposed on the afterhyperpolarization (AHP) which follows the spike. An example of slow hyperpolarization, recorded in the absence of a preceding spike (n = 10), is shown in Fig. 1A. Time-to-peak and half-decay time of maximal responses, in this
A st
st
ls
~
-
-
.
.
.
.
.
10.3nA
st
1
'i ls
Fig. 1. A: slow IPSP increases in amplitude and duration by increasing number of stimuli (left column, 1-3 stimuli at 20 Hz) or intensity of stimulus (right column, 2 stimuli at 20 Hz). Each stimulus also causes a subthreshold fast EPSP. Resting potential, -65 inV. B: neuronal input resistance decreases during slow IPSP (2 stimuli at 20 Hz, two fast EPSPs precede the slow IPSP). Hyperpolarizing current pulses of 200 ms duration at 1 Hz (upper trace). Insert shows that hyperpolarization to the same level attained at the peak of the slow IPSP does not decrease input resistance. Resting potential, -68 inV.
group, were in the range of 0.3-0.8 and 3 - 6 s, respectively. In the more numerous cases (n = 21) in which the slow, stimulus-evoked, hyperpolarization occurred in the wake of the spike caused by the preceding fast EPSP, the slow response could be differentiated from the A H P on the basis of 3 criteria. The first is the relation to stimulus intensity, the slow hyperpolarization being graded while the A H P is all-ornone. The second criterion is the time course: timeto-peak and duration are longer for the slow hyperpolarization than for the AHP. The third is the fact that yohimbine (see below) attenuated the slow hyperpolarization but had no effect on the AHP. In some of the cells the slow hyperpolarization was followed by a slow depolarization (slow EPSP) the properties of which are described elsewhere t6. Superfusion of the slice with tetrodotoxin (TTX; 0.6 IzM) or with low Ca (0.25 mM) and high Mg (5.0 mM) solution abolished the response. This response will be referred to here as a slow IPSP. The slow IPSP was associated with a decrease in neuronal input resistance (Fig. 1B). The slow IPSP was recorded with KCI, potassium acetate or potassium citrate-filled electrodes. During cell impalement with KCI electrodes the slow IPSP had a relatively stable amplitude and time course over prolonged periods of time (e.g. 30 min-1 h) suggesting that this response was not due to a change in Cl-conductance of the SPN membrane. The response amplitude increased with membrane depolarization and decreased with hyperpolarization. The response was nullified at membrane potential of approximately -90 mY. Hyperpolarization beyond this value resulted in a reversal of the polarity of the response (n = 5). Increasing and decreasing Kout to 10.0 and 0.36 mM, respectively, decreased and increased, respectively, the slow IPSP amplitude (n = 5). The slow IPSP was blocked by yohimbine (0.5-1.0/~M, n = 5) but not by prazosin (1 /~M, n = 4) or propranolol (1-5 uM, n = 4). In all cases yohimbine unmasked a slow EPSP (Fig. 2). This suggests that the slow IPSP occurs concomitantly with a slow EPSP, and that the observed time course is the resultant of the summation of these two simultaneously occurring events. Of 28 neurons tested with concentrations between 10 and 50 pM, N A caused hyperpolarization in 11 and hyperpolarization followed by depolarization in 4. The remaining 12 neurons were depolarized by
385 N A 16. The hyperpolarization was associated with a decrease in neuronal input resistance. Its amplitude decreased with h y p e r p o l a r i z a t i o n and increased with depolarization of the m e m b r a n e and reversed in polarity at around - 9 0 mV. The a m p l i t u d e increased by decreasing Kout to 0.36 m M and decreased by increasing Kout to 10 m M (n = 4). The response persisted in T T X (0.6 p M n = 4) and during perfusion with low Ca (0.25 m M ) high Mg (5 raM) solution (n -4). This hyperpolarizing response e v o k e d by N A was
A
blocked by yohimbine ( 0 . 5 - 1 . 0 p M , n = 5) but not by prazosin ( l p M , n = 5), or p r o p r a n o l o l ( 3 p M , n = 4). Some of the p r o p e r t i e s of the N A - e v o k e d h y p e r p o larization are illustrated in Fig. 2 for a cell showing both a slow IPSP in response to focal stimulation and a hyperpolarization response to N A . A s for the slow IPSP, in the case of the N A - e v o k e d hyperpolarization yohimbine u n m a s k e d a depolarization which was blocked by prazosin. This is the first description of a slow IPSP in SPNs.
control
A1
As
,
A3 NA
B
yohimbine
gl
I~M B2
~.:
'~ .7 ~
.
.
,
..... : ~ -
>
is
B3
~C C
.
.
.
.
NA
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
:,,:.~.,.:.,.i
.....
: .....
::,,;il;:;.i::i,,.i'.,i...:;;
J~ o
lOs Fig. 2. Yohimbine blocks the slow IPSP and the hyperpolarizing response to NA. All records obtained in the same cell. A: control. Al: slow IPSP evoked by two stimuli at 20 Hz. Two fast EPSPs precede the slow IPSP. A2: AHP following spike evoked by intracellular current pulse of 5 ms duration. Spike truncated by low frequency response of pen-recorder. A3: hyperpolarization produced by superfusion with NA 40/~M for 34 s. Neuronal input resistance (measured with 200 ms hyperpolarizing pulses at 0.7 Hz) decreases during the course of the NA-evoked hyperpolarization. B: 10 min after onset of superfusion with yohimbine 1/~M. BI: the slow IPSP evoked as in A 1 is considerably attenuated and replaced in part by a slow EPSP. B2: AHP evoked as in A 2 is not affected. B3- NA 40/tM now produces a depolarization associated with an increase in input resistance16. Sag in repolarizing trajectories during NA depolarization presumably due to activation of A-current15. Time scale in B3 applies also to A 3. Time scale in B E applies to all other panels. Resting potential,-71 inV.
386 Graded hyperpolarizing synaptic responses, which can be labelled as fast IPSPs (time course of less than 100 ms), have been observed both in vivo 7'8 and in vitro 11A4. These fast IPSPs are a relatively infrequent finding and seem due to a C1 current 7. In addition, hyperpolarizations of several hundred milliseconds duration, always preceded by an E P S P and insensitive to internal CI concentration changes, have been described 7 in response to stimulation of the dorsolateral funiculus or of spinal afferents in vivo: the mechanism of these longer-lasting responses is not known. The slow IPSP described here has duration at least an order of magnitude longer than that of the just-mentioned responses. The reversal potential of the slow IPSP and its dependence on external K suggests that it is due to a K current. A similar conclusion can be reached for the hyperpolarizing response to NA. In addition, the fact that both the slow IPSP and the N A - e v o k e d hyperpolarization are abolished by the same adrenergic antagonist, yohimbine, suggests that the same adrenergic receptor mediates both responses. It is possible, therefore, that N A (or some other catecholamine activating a 2 receptors) is the
1 Carlsson, A., Falck, B., Fuxe, K. and Hillarp, N.A., Cellular localization of monoamines in the spinal cord, Acta Physiol. Scand., 60 (1964) 112-119. 2 Coote, J.H., Macleod, V.H., Fleetwood-Walker, S. and Gilbey, M.P., The response of individual sympathetic preganglionic neurons to microelectrophoretically applied endogenous monoamines, Brain Research, 215 (1981) 135-145. 3 Coote, J.H., Mcleod, V.H., Fleetwood-Walker, S.M. and Gilbey, M.P., B aroreceptor inhibition of sympathetic activity at the spinal site, Brain Research, 220 (1981) 81-93. 4 Dahlstrom, A. and Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Acta Physiol. Scand., Suppl. 247, 64 (1965) 1-36. 5 Dashwood, M.R., Gilbey, M.P. and Spyer, K.M., The localization of adrenoeeptors and opiate receptors in regions of the cat central nervous system involved in cardiovascular control, Neuroscience, 15 (1985) 537-551. 6 Dembowsky, K., Lackner, K., Czachurski, J. and Seller, H., Tonic cateeholaminergic inhibition of the spinal somato-sympathetic reflexes originating in the ventrolateral meduUa oblongata, J. Auton. Nerv. Syst., 3 (1981) 277-290. 7 Dembowsky, K., Czachurski, J. and Seller, H., An intracellular study of the synaptic input to sympathetic preganglionic neurons of the third thoracic segment of the cat, J. Auton. Nerv. Syst., 13 (1985) 201-244. 8 Fedorko, L., Lioy, F. and Trzebski, A., Synaptic inhibition of sympathetic preganglionic neurones induced by stimulation of the aortic nerve in the cat, J. Physiol. (London), 360
mediator of the slow IPSP of SPNs. The fact that N A causes both depolarizing and hyperpolarizing responses in SPNs (sometimes the same cell shows both effects, see Fig. 2) which are mediated by different adrenoceptors, and that the slow IPSP and the slow EPSP t6 have ionic mechanisms and pharmacology similar to those of the NAevoked hyper- and depolarization, respectively, suggests that C A may be mediators of both excitatory and inhibitory synaptic pathways to the SPN. Suggestions to this effect have been already made. Thus, the C 1 neuron group of the V L M has been suggested as source of excitatory input to the SPN 12. Some yet undefined C A neuron group in the brainstem has been postulated as the source of the supraspinal tonic inhibition of the early somato-sympathetic reflex 6 and of baroreceptor inhibition of sympathetic activity 3 This work was supported by a Grant-in-Aid for Scientific Research by the Ministry of Education, Science and Culture of Japan to S.N. and to M.Y., as well as by grants to C.P. by the Q u e b e c Heart Foundation and the Medical Research Council of Canada.
(1985) 45P. 9 Guyenet, P.G. and Cabot, J.B., Inhibition of sympathetic preganglionic neurons by catecholamines and clonidine: mediation by an alpha-adrenergic receptor, J. Neurosci., 1 (1981) 908-917. 10 Kadzielawa, K., Inhibition of the activity of sympathetic preganglionic neurons and neurons activated by visceral afferents by alpha-methyl-noradrenaline and endogenous catecholamines, Neuropharmacology, 22 (1983) 3-17. 11 Mo, N. and Dun, N.J., Is glycine an inhibitory transmitter in rat lateral horn cells?, Brain Research, 400 (1987) 139-144. 12 Ross, C.A., Ruggiero. D.A.. Park. D.H.. Hoh, T.H., Sved, A.F., Fernandez-Pardal. J.. Saavedra. J.M. and Reis, D.J., Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing CI adrenaline neurons on arterial pressure, heart rate and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 474-494. 13 Ryall, R.W., The effect of monoamines on sympathetic preganglionic neurons. Circ. Res.. Suppl. 3. 21 (1967) 83-87. 14 Yoshimura, M., Polosa, C. and Nishi, S., Electrophysiological properties of sympathetic pregangtionic neurons in the cat spinal cord in vitro, Pflagers Arch., 406 (1986) 91-98. 15 Yoshimura, M., Polosa, C. and Nishi, S., A transient outward rectification in the cat sympathetic preganglionic neuron, PflfigersArch., 408 (1987) 207-208. 16 Yoshimura, M., Polosa, C. and Nishi, S., Slow EPSP and the depolarizing action of noradrenaline on sympathetic neurons, Brain Research, in press.
383
BRE 22461
Slow IPSP and the noradrenaline-induced inhibition of the cat sympathetic preganglionic neuron in vitro M. Yoshimura 1, C.
Polosa 2 a n d S. Nishi 1
l Department of Physiology, Kurume University School of Medicine, Kurume (Japan) and 2Department of Physiology, McGill University, Montreal, Que. (Canada) (Accepted 26 May 1987)
Key words: Catecholamine; Sympathetic neuron; Slow synaptic potential; Spinal cord; Potassium conductance
Focal electrical stimulation of the slice of the cat thoracic cord evoked in sympathetic preganglionic neurons a slow inhibitory postsynaptic potential (IPSP) associated with decreased neuronal input resistance. The slow IPSP decreased in amplitude with membrane hyperpolarization and reversed at about -90 mV. It increased in amplitude in low potassium and decreased in high potassium. Noradrenaline (NA) at doses of 10-50 #M caused in some of these cells a hyperpolarization with properties similar to those of the slow IPSP. Both the slow IPSP and the NA-evoked hyperpolarization were abolished by yohimbine, but not by prazosin or propranolol. These data suggest that both responses are due to an increase in potassium conductance and that NA may be the mediator of the slow IPSP evoked by focal stimulation.
The intermediolateral nucleus (IML) of the thoracic cord contains a rich network of catecholaminecontaining fibers of supraspinal origin 1,4. The results of iontophoretic application of catecholamines (CA) to sympathetic preganglionic neurons (SPNs) 2,9,1°A3, as well as of administration of adrenergic blockers 3'6, suggest an inhibitory action of C A on SPNs. This inhibitory action has been shown to be mediated by a 2 receptors 9'1°. Binding sites for the a 2 antagonist, yohimbine in the I M L of the cat have been demonstrated 5. Two questions concerning the function of the a 2 adrenoceptors on the SPN membrane need to be answered: (i) what is the mechanism by which these receptors inhibit SPN firing; and (ii) can these receptors be activated synaptically? The observations reported in the present paper, obtained in the in vitro slice of the cat thoracic spinal cord, show that: (i) the mechanism of the NA-evoked inhibition of the SPN mediated by a 2 receptors is a membrane hyperpolarization due to an increase in K-conductance; and (ii) that focal stimulation of the slice evokes synapticallymediated hyperpolarizations of very long time course (slow IPSP) which have the same ionic mechanism as
the NA-evoked hyperpolarization and are blocked by the a 2 antagonist, yohimbine. The experiments were performed on transverse slices of cat upper thoracic spinal cord. Intracellular recordings were made from SPNs of the IML, identified by antidromic stimulation as previously described 14. In cats anesthetized with chloralose and pentobarbital (60 and 10 mg/kg, respectively, i.p.), the second and third thoracic segments were excised and cut into 500/~m thick slices. These were transferred to a recording chamber and superfused with Krebs solution, equilibrated with 95% 0 2 and 5% CO 2, containing (in mM): NaC1 117, KC1 3.6, NaH2PO4 1.2, CaC12 2.5, MgC12 1.2, glucose 11.0, and N a H C O 3 25.0. Temperature was maintained at 37 °C. Cells were impaled using glass micropipettes filled with 3 M KC1 or 2 M potassium acetate or citrate. Electrode resistance varied between 40 and 120 Mr2. Intracellular recordings were obtained using a high input impedance amplifier with an active bridge circuit, enabling simultaneous measurement of membrane potential and intracellular current injection. The amplifier output was monitored on a digital oscil-
Correspondence: C. Polosa, Department of Physiology, McGill University, 3655 Drummond St., Montreal, Que. Canada H3G 1Y6. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
384 loscope. A DC pen-recorder was used to record membrane potential continuously. NA (10-50 #M) was added to the superfusing Krebs solution. Focal stimulation was performed with a monopolar, 50/~m diameter, silver wire electrode, insulated except at the tip. Stimuli were square pulses of 0.2 ms duration and variable voltage. Resting potential was - 6 0 + 2.7 mV (n = 31). Focal stimulation of the ipsilateral dorsolateral funiculus with single shocks or short trains of stimuli caused in 31 out of 82 cells tested a long-lasting, graded hyperpolarization. The response was preceded by a fast EPSP which, in some cases, evoked a spike. In such cases the slow hyperpolarization was superimposed on the afterhyperpolarization (AHP) which follows the spike. An example of slow hyperpolarization, recorded in the absence of a preceding spike (n = 10), is shown in Fig. 1A. Time-to-peak and half-decay time of maximal responses, in this
A st
st
ls
~
-
-
.
.
.
.
.
10.3nA
st
1
'i ls
Fig. 1. A: slow IPSP increases in amplitude and duration by increasing number of stimuli (left column, 1-3 stimuli at 20 Hz) or intensity of stimulus (right column, 2 stimuli at 20 Hz). Each stimulus also causes a subthreshold fast EPSP. Resting potential, -65 inV. B: neuronal input resistance decreases during slow IPSP (2 stimuli at 20 Hz, two fast EPSPs precede the slow IPSP). Hyperpolarizing current pulses of 200 ms duration at 1 Hz (upper trace). Insert shows that hyperpolarization to the same level attained at the peak of the slow IPSP does not decrease input resistance. Resting potential, -68 inV.
group, were in the range of 0.3-0.8 and 3 - 6 s, respectively. In the more numerous cases (n = 21) in which the slow, stimulus-evoked, hyperpolarization occurred in the wake of the spike caused by the preceding fast EPSP, the slow response could be differentiated from the A H P on the basis of 3 criteria. The first is the relation to stimulus intensity, the slow hyperpolarization being graded while the A H P is all-ornone. The second criterion is the time course: timeto-peak and duration are longer for the slow hyperpolarization than for the AHP. The third is the fact that yohimbine (see below) attenuated the slow hyperpolarization but had no effect on the AHP. In some of the cells the slow hyperpolarization was followed by a slow depolarization (slow EPSP) the properties of which are described elsewhere t6. Superfusion of the slice with tetrodotoxin (TTX; 0.6 IzM) or with low Ca (0.25 mM) and high Mg (5.0 mM) solution abolished the response. This response will be referred to here as a slow IPSP. The slow IPSP was associated with a decrease in neuronal input resistance (Fig. 1B). The slow IPSP was recorded with KCI, potassium acetate or potassium citrate-filled electrodes. During cell impalement with KCI electrodes the slow IPSP had a relatively stable amplitude and time course over prolonged periods of time (e.g. 30 min-1 h) suggesting that this response was not due to a change in Cl-conductance of the SPN membrane. The response amplitude increased with membrane depolarization and decreased with hyperpolarization. The response was nullified at membrane potential of approximately -90 mY. Hyperpolarization beyond this value resulted in a reversal of the polarity of the response (n = 5). Increasing and decreasing Kout to 10.0 and 0.36 mM, respectively, decreased and increased, respectively, the slow IPSP amplitude (n = 5). The slow IPSP was blocked by yohimbine (0.5-1.0/~M, n = 5) but not by prazosin (1 /~M, n = 4) or propranolol (1-5 uM, n = 4). In all cases yohimbine unmasked a slow EPSP (Fig. 2). This suggests that the slow IPSP occurs concomitantly with a slow EPSP, and that the observed time course is the resultant of the summation of these two simultaneously occurring events. Of 28 neurons tested with concentrations between 10 and 50 pM, N A caused hyperpolarization in 11 and hyperpolarization followed by depolarization in 4. The remaining 12 neurons were depolarized by
385 N A 16. The hyperpolarization was associated with a decrease in neuronal input resistance. Its amplitude decreased with h y p e r p o l a r i z a t i o n and increased with depolarization of the m e m b r a n e and reversed in polarity at around - 9 0 mV. The a m p l i t u d e increased by decreasing Kout to 0.36 m M and decreased by increasing Kout to 10 m M (n = 4). The response persisted in T T X (0.6 p M n = 4) and during perfusion with low Ca (0.25 m M ) high Mg (5 raM) solution (n -4). This hyperpolarizing response e v o k e d by N A was
A
blocked by yohimbine ( 0 . 5 - 1 . 0 p M , n = 5) but not by prazosin ( l p M , n = 5), or p r o p r a n o l o l ( 3 p M , n = 4). Some of the p r o p e r t i e s of the N A - e v o k e d h y p e r p o larization are illustrated in Fig. 2 for a cell showing both a slow IPSP in response to focal stimulation and a hyperpolarization response to N A . A s for the slow IPSP, in the case of the N A - e v o k e d hyperpolarization yohimbine u n m a s k e d a depolarization which was blocked by prazosin. This is the first description of a slow IPSP in SPNs.
control
A1
As
,
A3 NA
B
yohimbine
gl
I~M B2
~.:
'~ .7 ~
.
.
,
..... : ~ -
>
is
B3
~C C
.
.
.
.
NA
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
:,,:.~.,.:.,.i
.....
: .....
::,,;il;:;.i::i,,.i'.,i...:;;
J~ o
lOs Fig. 2. Yohimbine blocks the slow IPSP and the hyperpolarizing response to NA. All records obtained in the same cell. A: control. Al: slow IPSP evoked by two stimuli at 20 Hz. Two fast EPSPs precede the slow IPSP. A2: AHP following spike evoked by intracellular current pulse of 5 ms duration. Spike truncated by low frequency response of pen-recorder. A3: hyperpolarization produced by superfusion with NA 40/~M for 34 s. Neuronal input resistance (measured with 200 ms hyperpolarizing pulses at 0.7 Hz) decreases during the course of the NA-evoked hyperpolarization. B: 10 min after onset of superfusion with yohimbine 1/~M. BI: the slow IPSP evoked as in A 1 is considerably attenuated and replaced in part by a slow EPSP. B2: AHP evoked as in A 2 is not affected. B3- NA 40/tM now produces a depolarization associated with an increase in input resistance16. Sag in repolarizing trajectories during NA depolarization presumably due to activation of A-current15. Time scale in B3 applies also to A 3. Time scale in B E applies to all other panels. Resting potential,-71 inV.
386 Graded hyperpolarizing synaptic responses, which can be labelled as fast IPSPs (time course of less than 100 ms), have been observed both in vivo 7'8 and in vitro 11A4. These fast IPSPs are a relatively infrequent finding and seem due to a C1 current 7. In addition, hyperpolarizations of several hundred milliseconds duration, always preceded by an E P S P and insensitive to internal CI concentration changes, have been described 7 in response to stimulation of the dorsolateral funiculus or of spinal afferents in vivo: the mechanism of these longer-lasting responses is not known. The slow IPSP described here has duration at least an order of magnitude longer than that of the just-mentioned responses. The reversal potential of the slow IPSP and its dependence on external K suggests that it is due to a K current. A similar conclusion can be reached for the hyperpolarizing response to NA. In addition, the fact that both the slow IPSP and the N A - e v o k e d hyperpolarization are abolished by the same adrenergic antagonist, yohimbine, suggests that the same adrenergic receptor mediates both responses. It is possible, therefore, that N A (or some other catecholamine activating a 2 receptors) is the
1 Carlsson, A., Falck, B., Fuxe, K. and Hillarp, N.A., Cellular localization of monoamines in the spinal cord, Acta Physiol. Scand., 60 (1964) 112-119. 2 Coote, J.H., Macleod, V.H., Fleetwood-Walker, S. and Gilbey, M.P., The response of individual sympathetic preganglionic neurons to microelectrophoretically applied endogenous monoamines, Brain Research, 215 (1981) 135-145. 3 Coote, J.H., Mcleod, V.H., Fleetwood-Walker, S.M. and Gilbey, M.P., B aroreceptor inhibition of sympathetic activity at the spinal site, Brain Research, 220 (1981) 81-93. 4 Dahlstrom, A. and Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system. II. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems, Acta Physiol. Scand., Suppl. 247, 64 (1965) 1-36. 5 Dashwood, M.R., Gilbey, M.P. and Spyer, K.M., The localization of adrenoeeptors and opiate receptors in regions of the cat central nervous system involved in cardiovascular control, Neuroscience, 15 (1985) 537-551. 6 Dembowsky, K., Lackner, K., Czachurski, J. and Seller, H., Tonic cateeholaminergic inhibition of the spinal somato-sympathetic reflexes originating in the ventrolateral meduUa oblongata, J. Auton. Nerv. Syst., 3 (1981) 277-290. 7 Dembowsky, K., Czachurski, J. and Seller, H., An intracellular study of the synaptic input to sympathetic preganglionic neurons of the third thoracic segment of the cat, J. Auton. Nerv. Syst., 13 (1985) 201-244. 8 Fedorko, L., Lioy, F. and Trzebski, A., Synaptic inhibition of sympathetic preganglionic neurones induced by stimulation of the aortic nerve in the cat, J. Physiol. (London), 360
mediator of the slow IPSP of SPNs. The fact that N A causes both depolarizing and hyperpolarizing responses in SPNs (sometimes the same cell shows both effects, see Fig. 2) which are mediated by different adrenoceptors, and that the slow IPSP and the slow EPSP t6 have ionic mechanisms and pharmacology similar to those of the NAevoked hyper- and depolarization, respectively, suggests that C A may be mediators of both excitatory and inhibitory synaptic pathways to the SPN. Suggestions to this effect have been already made. Thus, the C 1 neuron group of the V L M has been suggested as source of excitatory input to the SPN 12. Some yet undefined C A neuron group in the brainstem has been postulated as the source of the supraspinal tonic inhibition of the early somato-sympathetic reflex 6 and of baroreceptor inhibition of sympathetic activity 3 This work was supported by a Grant-in-Aid for Scientific Research by the Ministry of Education, Science and Culture of Japan to S.N. and to M.Y., as well as by grants to C.P. by the Q u e b e c Heart Foundation and the Medical Research Council of Canada.
(1985) 45P. 9 Guyenet, P.G. and Cabot, J.B., Inhibition of sympathetic preganglionic neurons by catecholamines and clonidine: mediation by an alpha-adrenergic receptor, J. Neurosci., 1 (1981) 908-917. 10 Kadzielawa, K., Inhibition of the activity of sympathetic preganglionic neurons and neurons activated by visceral afferents by alpha-methyl-noradrenaline and endogenous catecholamines, Neuropharmacology, 22 (1983) 3-17. 11 Mo, N. and Dun, N.J., Is glycine an inhibitory transmitter in rat lateral horn cells?, Brain Research, 400 (1987) 139-144. 12 Ross, C.A., Ruggiero. D.A.. Park. D.H.. Hoh, T.H., Sved, A.F., Fernandez-Pardal. J.. Saavedra. J.M. and Reis, D.J., Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing CI adrenaline neurons on arterial pressure, heart rate and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 474-494. 13 Ryall, R.W., The effect of monoamines on sympathetic preganglionic neurons. Circ. Res.. Suppl. 3. 21 (1967) 83-87. 14 Yoshimura, M., Polosa, C. and Nishi, S., Electrophysiological properties of sympathetic pregangtionic neurons in the cat spinal cord in vitro, Pflagers Arch., 406 (1986) 91-98. 15 Yoshimura, M., Polosa, C. and Nishi, S., A transient outward rectification in the cat sympathetic preganglionic neuron, PflfigersArch., 408 (1987) 207-208. 16 Yoshimura, M., Polosa, C. and Nishi, S., Slow EPSP and the depolarizing action of noradrenaline on sympathetic neurons, Brain Research, in press.