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Long-duration spike afterhyperpolarizations in neurons from the guinea pig superior cervical ganglion
Neuroseienee Letters, 84 (1988) 191 196 Elsevier Scientific Publishers Ireland Ltd.
191
NSL 05066
Long-duration spike afterhyperpolarizations in neurons from the guinea pig superior cervical ganglion E d w a r d P. Christian a n d Daniel Weinreich Department (~/"Pharmacology and Experimental Therapeutics, University ~/ Mao, land School ~[ Medieine, Baltimore, MD 21201 (U.S.A.) (Received 9 July 1987: Revised version received 25 September 1987; Accepted 30 September 1987)
Key word~v Guinea pig; Superior cervical ganglion; lntracellular recording; Afterhyperpolarization: Neuromodulation; Histamine Intracellular recordings from guinea pig superior cervical ganglia maintained in vitro have revealed a protracted spike afterhyperpolarization in approximately 18% of the principal neurons that is substanlially longer ( > l s duration) than those previously reported. This long afterhyperpolarization is distinct from shorter duration aftcrpotentials in these cells because it can be selectively and reversibly blocked by cooling and by bath applied histamine. Cellular excitability is increased when the long-duration afterhyperpolarization is abolished and thus it deserves consideration as a site for modulation of synaptic transmission through the superior cervical ganglion.
Spike afterhyperpolarizations (AHPs) are a prominent feature of mammalian peripheral autonomic neurons [16]. Although these potentials have been shown almost exclusively to be mediated by K + conductances, several different AHPs have been distinguished with respect to their time courses, pharmacological properties, and conditions for activation, such as Ca 2~ influx or voltage dependency (see ref. 3 for review). Long-duration afterhyperpolarizations control the firing frequency in neurons [1,2, 7, 19], and thus can modulate impulse transmission through autonomic ganglia. Spike AHPs with a protracted time course (i.e. duration in the range of seconds) have been reported i,.z neurons in the guinea pig myenteric plexus [9, 17], in the rabbit nodose ganglion [6, 8], and in the guinea pig nodose ganglion [our unpublished observations]. In the mammalian superior cervical ganglion (SCG) by contrast, no AHPs lasting more than 500 ms have been documented under drug-free conditions [10. 1 I, 14, 16]. Here we show that approximately 18% of the principal neurons in the guinea pig SCG exhibit an AHP lasting several seconds. This long-duration AHP probably regulates excitability because its removal results in enhanced firing frequency. A preliminary report of this work has been presented [4]. Correspondence: E. Christian, Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 665 West Redwoord Street, Baltimore, MD 21201, U.S.A, 0304-3940/88/$ 03.50 @ 1988 Elsevier Scientific Publishers Ireland Ltd.
192
Adult guinea pigs (250 400 g) of either sex were sacrificed by a sharp blow to the head, exsanguinated, and the SCGs along with at least 2 cm of the cervical sympathetic trunk (CST) [18] were dissected free. Ganglia were trimmed of connective tissue, split longitudinally with a razor blade fragment and pinned flat to the Sylgard floor of a recording chamber mounted on a compound microscope. Tissue was perfused (3-5 ml/min) with warmed (35-3T'C) Locke solution (composition in mM: NaCI 136, KCI 5.6, NaHCO3 14.3, NaH2PO4 1.2, CaCIz 2.2, MgCI2 1.2, dextrose ll, choline-Cl 0.03) equilibrated with 95% O2~-5% CO2 (pH 7.2-7.4). Neurons were impaled with microelectrodes filled with a mixture of i M KAc and 2 M KCI (resistance 30-90 M~). Electrical activity was recorded with an Axoclamp II electrometer (Axon Ind.) in discontinuous current clamp mode (switching rate: 2.843.5 kHz). Synaptic inputs to neurons were stimulated with a glass suction electrode placed on the CST > 1 cm from the body of the ganglion. Drugs were applied by switching 3-way valves located close to the chamber to divert flow from the main reservoir to drug reservoirs. Intracellular signals were amplified, viewed on-line and stored on VCR tapes. Data were digitized from analog records with a PDP 11/23 computer (Digital Equipment) and reproduced for figures with an x--y plotter (Hewlett Packard).
< 50
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msec -54 '-
< 500
mV
50 m s e c L_10 mV
msec -53
___....__._,,.~--'--~
200 I10mV
mV
msec
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> 1 sec -53
_10 mV
mV ]
Fig. 1. Different components of spike afterhyperpo]arization recorded in guinea pig SCG neurons. Voltage traces from 3 different cells each show a single action potential elicited by a 5-hA, 2-ms depolarizing current step and a resulting spike afterhyperpolarization (AHP). Temperature was maintained at 35 36°C. Positive-going phase of action potentials is truncated. M e m b r a n e potential here and in other figures is indicated by a labelled horizontal line. An A H P component lasting > 50 ms was seen in all neurons (upper trace). Some cells showed an additional component lasting < 500 ms (middle trace). Approximately 18% of neurons showed an A H P persisting > 1 s (AHP,jow; lower trace). Voltage deflection preceding action potential in lower trace was produced by a 0.1 nA, 300 ms hyperpolarizing current step to estimate input resistance.
193
One hundred and twenty-eight SCG neurons from 67 ganglia were recorded from intracellularly during the study. All of these neurons had a membrane potential more hyperpolarized than - 5 0 mV, input resistance > 30 Mf2 and spike overshoot > 10 mV. Fig. 1 demonstrates 3 AHPs with different time courses that were observed in these neurons. All neurons showed AHPs of short duration ( < 50 ms) following an action potential evoked by a 2 ms, 5 nA transmembrane depolarizing current step. In some neurons a longer AHP lasting up to 500 ms was also present, but its frequency of occurrence and characteristics were not assessed here. Twenty-three of the 128 neurons (18%) also showed a long-duration ( > 1 s) AHP component (AHP~Io,,). When 4 action potentials (100 ms interspike interval) were elicited in these cells (n = 23) at resting potential (range: - 5 0 to - 7 4 mV), the maximal amplitude of the AHP~tow ranged from 2 to 19 mV ( m e a n + S . D . = 9_+4,7 mV) and duration (from repolarization of the fourth spike to complete recovery of resting potential) ranged from 1.5 to 5.3 s (mean + S.D. = 2.6_+ 1.0 s). Cells could be clearly categorized as to whether they showed an AHP~low. In cells not exhibiting this component (n = 105), membrane potential always recovered fully within 500 ms of the fourth spike of a train. In strong contrast, the shortest duration AHPslow measured was 1500 ms (n = 23 neurons, see above). Neurons with or without an AHP~low could not be distinguished, however, by any other measured passive or active parameters (i.e. membrane potential, input resistance, spike amplitude, overshoot or duration). In 6 of the cells with an AHP~low we also tested for and confirmed the presence of a fast hexamethonium (300 /~M)-sensitive excitatory postsynaptic potential (EPSP) following stimulation of the CST. Our preliminary data suggest that the AHP~Iow is generated by a K + current because it is accompanied by a conductance increase (Fig. 2A) and is hulled by shifting membrane potential to approximately - 8 5 mV (Fig. 2B), near the predicted K + equilibrium potential [16]. The amplitude of the AHP~Iow summates with an increasing number of action potentials (Fig. 2C), suggesting that it is dependent on intracellular accumulation of some factor such as Ca 2+. Finally, the AHP~low could be reversibly abolished by lowering the perfusate temperature from 35 to 24"C (Fig. 2D). This is consistent with the AHP~lo,, being linked to metabolic processes. Peripheral neurons possessing a long AHP (seconds duration) also show powerful accommodation properties [19]. We are able to block the AHP~low in SCG neurons with histamine, and simultaneously examine effects on cell excitability (Fig. 3). Prior to histamine treament, the neuron showed a prominent AHP~tow and a long depolarizing current ramp elicited only a single spike (Fig. 3A). Exposure to histamine (10 llM) blocked the AHP~low, but not shorter duration AHP components ( < 500 ms; see above). The same depolarizing current ramp now produced a burst of 4 spikes (Fig. 3B); membrane potential, input resistance, action potential characteristics and threshold for the initial spike were not altered appreciably by histamine. Histamine blocked the AHP~tow in 3 of 3 additional experiments (mean AHP duration _+S.D. from fourth spike to recovery of resting potential: pre-histamine, 3.4_+ 1.3 s; posthistamine, 0.4 +0.1 s; n = 4) and increased the number of action potentials evoked by ramped depolarizing currents. These data suggest that the AHP,Io,, affects excitability in these SCG neurons.
194 A,
B,
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Fig. 2. Properties of the AHP~jo, in SCG neurons. Panels A - D each are from a different cell. Action potentials here and in Fig. 3 are truncated at variable levels due to low frequency (200 Hz) at which data were digitized. A: voltage (upper) and current (lower) records showing changes in membrane conductance during the AHPsiow. Transmembrane hyperpolarizing current steps (0.3 nA, 50 ms) were delivered at 100-ms intervals. The AHPsjo, was elicited by 4 action potentials produced by four 5-nA, 2-ms depolarizing current steps (100-ms interspike interval)• Note that voltage transients decreased in amplitude during the AHP~jow, signifying increased conductance. No appreciable change in conductance was noted when the membrane potential was hyperpolarized by current injection to the same level as reached during the AHP~]ow. B: voltage traces showing the effect of membrane potential on the AHP~ow. The AHP~o, in each trace was activated as described in A. Membrane potential was hyperpolarized from resting level ( - 6 0 mV; upper trace) in 2 lower traces by passing constant current through the electrode• The AHP~jow amplitude decreased with hyperpolarization and was nulled at - 85 mV. Little change in input resistance was noted over this range of membrane potentials. C: effect of varying number of action potentials used to activate the AHPs~ow. Action potentials were produced by passing a corresponding number of short depolarizing current steps as in A. Maximal amplitude and duration of the AHP~Io,~ is shown for each trace. The amplitude, but not duration of the AHP~o~ was directly dependent on the number of preceding spikes. D: effect of temperature on the AHPs~,,w. Five action potentials were produced by depolarizing current steps as in A to activate the AHPao, while the perfusate was maintained at 35 and 24°C. The AHP~ow was abolished at lower temperature, while the fast AHP after each spike showed a small decrease in amplitude and increase in duration. Input resistance was not affected by temperature changes.
Our findings thus demonstrate
a spike AHP in mammalian
S C G n e u r o n s t h a t is
c o n s i d e r a b l y l o n g e r in d u r a t i o n t h a n t h o s e p r e v i o u s l y r e p o r t e d . C o n s i d e r i n g t h a t t h e electrophysiological characteristics of SCG neurons in several mammalian h a v e b e e n e x t e n s i v e l y s t u d i e d , it is p e r p l e x i n g t h a t s u c h l o n g - d u r a t i o n
species
AHPs
have
not been documented. However, our experimental protocol may have provided parti-
195
B.
A. CONTROL
"LJ--~
-,o mv
HISTAMINE
-
-72
mV
0.5
sec
- i s-ec
Fig. 3. Effect of histamine on the AHP~o~, and on excitability of an SCG neuron. A: voltage record (upper trace) showing an AHPs~o~ activated by 4 action potentials as described in Fig. 2. Hyperpolarizing deflcction preceding the 4 spikes was produced by a 0.l-nA, 300-ms hyperpolarizing current step to evaluate input resistance. Middle trace shows voltage change produced during a ramped current injection (0 1.2 nA over 1 s; lower trace). B: data in same format as A, but recorded during a 3-min exposure to 10 itM histamine. Histamine selectively blocked the AHP~low without affecting shorter duration ( < 500 ms) components of the A H P and now a burst of 4 action potentials occurred during the same ramped current. Input resistance of the neuron was not affected appreciably by histamine. Calibration bars at right side refer to analogous traces from both A and B.
cularly favorable conditions for observing these events. First, we have shown that the AHP~low in the SCG is temperature sensitive; it is abolished by cooling from 35 to 24"C. Thus, studies performed at room temperature (e.g. see refs. 13, 14, 18) would have failed to detect the AHP~Iow. Second, the AHP~jow could be sensitive to the electrolyte used in the recording microelectrode; studies in paravertebral sympathetic ganglia from frogs revealed slow spontaneous hyperpolarizations that were sensitive to the anion used in the electrolyte [15]. Finally, there is the possibility that the AHP~Iow in SCG neurons is species-specific to the guinea pig and thus was not detected in other mammals where SCG neurons have been studied (e.g. rat, rabbit, cat). Considering that the AHP~low is present in only approximately 18% of SCG neurons, the questions arise whether these cells are unique morphologically or in terms of their afferent or efferent projections. We have established that cells exhibiting an AHP~low receive nicotinic cholinergic synaptic input, which is a well documented feature of principal postganglionic neurons in the SCG [5]. In addition, neurons with or without an AHP~lowcould not be differentiated by other electrophysiological characteristics, arguing that the AHP~Jow is not just an artifact of an exceptionally good or poor impalement. Finally, neurons with an AHP~low were randomly distributed anatomically in the ganglion. This suggests that these cells project in both rostral and caudal nerve branches from the ganglion [18] and therefore may not selectively inner-
196 v a t e o n l y c e r t a i n p o s t g a n g l i o n i c t a r g e t s [12]. N o n e t h e l e s s , issues r e g a r d i n g the specific c o n n e c t i v i t y o f n e u r o n s p o s s e s s i n g an AHP~low, a n d the i d e n t i t y o f p h a r m a c o l o g i c a l o r e n d o g e n o u s l y r e l e a s e d a g e n t s t h a t c a n m o d u l a t e n e u r o n a l e x c i t a b i l i t y via this long. A H P b e c o m e p r o v o c a t i v e t o p i c s for f u r t h e r i n v e s t i g a t i o n s . T h e a u t h o r s wish to t h a n k Drs. B r a d U n d e m , J o h n H o r n a n d Bill W o n d e r l i n for t h e i r critical r e a d i n g o f the m a n u s c r i p t . T h i s w o r k w a s s u p p o r t e d by N I H G r a n t N S 0 7 8 6 5 to E . P . C . a n d N S 2 5 5 9 8 to D . W . 1 Baldissera, F. and Gustafsson, B., Firing behavior of a neurone model based on the afterhyperpolarization conductance time course and algebraic summation. Adaption and steady state firing, Acta Physiol. Scand., 92 (1974) 27 47. 2 Barrett, E.F. and Barrett, J.N., Separation of two voltage-sensitive potassium currents, and demonstration of a tetrodotoxin-resistant calcium current in frog motoneurones, J. Physiol. (Lond.), 255 (1976) 737 744. 3 Brown, D.A., Adams, P.R. and Constanti, A., Voltage-sensitive K-currents in sympathetic neurons and their modulation by neurotransmitters, J. Auton., Nerv. Syst., 6 (1982) 23-35. 4 Christian, E.P. and Weinreich, D., Unusually long-lasting spike afterhyperpolarizations in guinea pig superior cervical ganglion, Fed. Proc. Fed. Am. Soc. Exp. Biol., 46 (1987) 505. 5 Eccles, R.M., Orthodromic activation of single ganglion cells, J. Physiol. (Lond.), 165 (1963) 387-391. 6 Fowler, J.C., Greene, R. and Weinreich, D., Two calcium-sensitive spike afterhyperpolarizations in visceral sensory neurones of the rabbit, J. Physiol. (Lond.), 365 (1985) 59-75. 7 Gustafsson, B., Lindstrom, S. and Zangger, P., Firing behavior of dorsal spinocerebeUar tract neurones, J. Physiol. (Lond.), 275 (1978) 321-343. 8 Higashi, H., Morita, K. and North, R.A., Calcium-dependent afterpotentials in visceral afferent neurones in the rabbit, J. Physiol. (Lond.), 355 (1984) 479-492. 9 Hirst, G.D.S., Holman, M.E. and Spence, I., Two types of neurones in the myenteric plexus of duodenum in the guinea-pig, J. Physiol. (Lond.), 236 (1974) 303-326. 10 Horn, J.P. and McAfee, D., Alpha-adrenergic inhibition of calcium-dependent potentials in rat sympathetic neurones, J. Physiol. (Lond.), 301 (1980) 191-204. [ l Kawai, T., Oka, J. and Watanabe, M., Hexamethonium increases the excitability of sympathetic neurons by the blockade of the Ca2+-activated K + channels, Life Sci., 36 (1985) 2339-2346. 12 Lichtman, J.W., Purves, D. and Yip, J.W., On the purpose of selective innervation of guinea-pig cervical ganglion cells, J. Physiol. (Lond.), 292 (1979) 69-84. 13 McAfee, D.A., Henon, B.K., Horn, J.P. and Yarowski, P., Calcium currents modulated by adrenergic receptors in sympathetic neurons, Fed. Proc. Fed. Am. Soc. Exp. Biol., 40 (1981) 2246-2249. 14 McAfee, D.A. and Yarowski, P.J., Calcium-dependent potentials in the mammalian sympathetic neurone, J. Physiol. (Lond.), 290 (1979) 507 523. 15 Morita, K. and Koketsu, K., Oscillation of lCa2~]~-linked K + conductance in bullfrog sympathetic ganglion cell is sensitive to intracellular anions, Nature (Lond.), 283 (1980) 204-205. 16 Nishi, S., Electrophysiological properties of sympathetic neurons. In A.G. Karczmar, K. Koketsu and S. Nishi (Eds.), Autonomic and Enteric Ganglia, Transmission and Its Pharmacology; Plenum, New York. 1986, pp. 79- 106. 17 Nishi, S. and North, R.A., lntracellular recording from the myenteric plexus of the guinea-pig ileum, J. Physiol. (Lond.}, 231 (1973) 471-491. 18 Purves, D., Functionaland structural changes in mammalian sympathetic neurones fotlowinginterruption of their axons, J. Physiol. (Lond.), 252 (1975) 429-463. 19 Weinreich, D. and Wonderlin, W.F., Inhibition of Ca + +-dependent spike afterhyperpolarization increases excitability of rabbit visceral sensory neurones, J. Physiol. (Lond.), in press.
191
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Long-duration spike afterhyperpolarizations in neurons from the guinea pig superior cervical ganglion E d w a r d P. Christian a n d Daniel Weinreich Department (~/"Pharmacology and Experimental Therapeutics, University ~/ Mao, land School ~[ Medieine, Baltimore, MD 21201 (U.S.A.) (Received 9 July 1987: Revised version received 25 September 1987; Accepted 30 September 1987)
Key word~v Guinea pig; Superior cervical ganglion; lntracellular recording; Afterhyperpolarization: Neuromodulation; Histamine Intracellular recordings from guinea pig superior cervical ganglia maintained in vitro have revealed a protracted spike afterhyperpolarization in approximately 18% of the principal neurons that is substanlially longer ( > l s duration) than those previously reported. This long afterhyperpolarization is distinct from shorter duration aftcrpotentials in these cells because it can be selectively and reversibly blocked by cooling and by bath applied histamine. Cellular excitability is increased when the long-duration afterhyperpolarization is abolished and thus it deserves consideration as a site for modulation of synaptic transmission through the superior cervical ganglion.
Spike afterhyperpolarizations (AHPs) are a prominent feature of mammalian peripheral autonomic neurons [16]. Although these potentials have been shown almost exclusively to be mediated by K + conductances, several different AHPs have been distinguished with respect to their time courses, pharmacological properties, and conditions for activation, such as Ca 2~ influx or voltage dependency (see ref. 3 for review). Long-duration afterhyperpolarizations control the firing frequency in neurons [1,2, 7, 19], and thus can modulate impulse transmission through autonomic ganglia. Spike AHPs with a protracted time course (i.e. duration in the range of seconds) have been reported i,.z neurons in the guinea pig myenteric plexus [9, 17], in the rabbit nodose ganglion [6, 8], and in the guinea pig nodose ganglion [our unpublished observations]. In the mammalian superior cervical ganglion (SCG) by contrast, no AHPs lasting more than 500 ms have been documented under drug-free conditions [10. 1 I, 14, 16]. Here we show that approximately 18% of the principal neurons in the guinea pig SCG exhibit an AHP lasting several seconds. This long-duration AHP probably regulates excitability because its removal results in enhanced firing frequency. A preliminary report of this work has been presented [4]. Correspondence: E. Christian, Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 665 West Redwoord Street, Baltimore, MD 21201, U.S.A, 0304-3940/88/$ 03.50 @ 1988 Elsevier Scientific Publishers Ireland Ltd.
192
Adult guinea pigs (250 400 g) of either sex were sacrificed by a sharp blow to the head, exsanguinated, and the SCGs along with at least 2 cm of the cervical sympathetic trunk (CST) [18] were dissected free. Ganglia were trimmed of connective tissue, split longitudinally with a razor blade fragment and pinned flat to the Sylgard floor of a recording chamber mounted on a compound microscope. Tissue was perfused (3-5 ml/min) with warmed (35-3T'C) Locke solution (composition in mM: NaCI 136, KCI 5.6, NaHCO3 14.3, NaH2PO4 1.2, CaCIz 2.2, MgCI2 1.2, dextrose ll, choline-Cl 0.03) equilibrated with 95% O2~-5% CO2 (pH 7.2-7.4). Neurons were impaled with microelectrodes filled with a mixture of i M KAc and 2 M KCI (resistance 30-90 M~). Electrical activity was recorded with an Axoclamp II electrometer (Axon Ind.) in discontinuous current clamp mode (switching rate: 2.843.5 kHz). Synaptic inputs to neurons were stimulated with a glass suction electrode placed on the CST > 1 cm from the body of the ganglion. Drugs were applied by switching 3-way valves located close to the chamber to divert flow from the main reservoir to drug reservoirs. Intracellular signals were amplified, viewed on-line and stored on VCR tapes. Data were digitized from analog records with a PDP 11/23 computer (Digital Equipment) and reproduced for figures with an x--y plotter (Hewlett Packard).
< 50
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msec -54 '-
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msec -53
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200 I10mV
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> 1 sec -53
_10 mV
mV ]
Fig. 1. Different components of spike afterhyperpo]arization recorded in guinea pig SCG neurons. Voltage traces from 3 different cells each show a single action potential elicited by a 5-hA, 2-ms depolarizing current step and a resulting spike afterhyperpolarization (AHP). Temperature was maintained at 35 36°C. Positive-going phase of action potentials is truncated. M e m b r a n e potential here and in other figures is indicated by a labelled horizontal line. An A H P component lasting > 50 ms was seen in all neurons (upper trace). Some cells showed an additional component lasting < 500 ms (middle trace). Approximately 18% of neurons showed an A H P persisting > 1 s (AHP,jow; lower trace). Voltage deflection preceding action potential in lower trace was produced by a 0.1 nA, 300 ms hyperpolarizing current step to estimate input resistance.
193
One hundred and twenty-eight SCG neurons from 67 ganglia were recorded from intracellularly during the study. All of these neurons had a membrane potential more hyperpolarized than - 5 0 mV, input resistance > 30 Mf2 and spike overshoot > 10 mV. Fig. 1 demonstrates 3 AHPs with different time courses that were observed in these neurons. All neurons showed AHPs of short duration ( < 50 ms) following an action potential evoked by a 2 ms, 5 nA transmembrane depolarizing current step. In some neurons a longer AHP lasting up to 500 ms was also present, but its frequency of occurrence and characteristics were not assessed here. Twenty-three of the 128 neurons (18%) also showed a long-duration ( > 1 s) AHP component (AHP~Io,,). When 4 action potentials (100 ms interspike interval) were elicited in these cells (n = 23) at resting potential (range: - 5 0 to - 7 4 mV), the maximal amplitude of the AHP~tow ranged from 2 to 19 mV ( m e a n + S . D . = 9_+4,7 mV) and duration (from repolarization of the fourth spike to complete recovery of resting potential) ranged from 1.5 to 5.3 s (mean + S.D. = 2.6_+ 1.0 s). Cells could be clearly categorized as to whether they showed an AHP~low. In cells not exhibiting this component (n = 105), membrane potential always recovered fully within 500 ms of the fourth spike of a train. In strong contrast, the shortest duration AHPslow measured was 1500 ms (n = 23 neurons, see above). Neurons with or without an AHP~low could not be distinguished, however, by any other measured passive or active parameters (i.e. membrane potential, input resistance, spike amplitude, overshoot or duration). In 6 of the cells with an AHP~low we also tested for and confirmed the presence of a fast hexamethonium (300 /~M)-sensitive excitatory postsynaptic potential (EPSP) following stimulation of the CST. Our preliminary data suggest that the AHP~Iow is generated by a K + current because it is accompanied by a conductance increase (Fig. 2A) and is hulled by shifting membrane potential to approximately - 8 5 mV (Fig. 2B), near the predicted K + equilibrium potential [16]. The amplitude of the AHP~Iow summates with an increasing number of action potentials (Fig. 2C), suggesting that it is dependent on intracellular accumulation of some factor such as Ca 2+. Finally, the AHP~low could be reversibly abolished by lowering the perfusate temperature from 35 to 24"C (Fig. 2D). This is consistent with the AHP~lo,, being linked to metabolic processes. Peripheral neurons possessing a long AHP (seconds duration) also show powerful accommodation properties [19]. We are able to block the AHP~low in SCG neurons with histamine, and simultaneously examine effects on cell excitability (Fig. 3). Prior to histamine treament, the neuron showed a prominent AHP~tow and a long depolarizing current ramp elicited only a single spike (Fig. 3A). Exposure to histamine (10 llM) blocked the AHP~low, but not shorter duration AHP components ( < 500 ms; see above). The same depolarizing current ramp now produced a burst of 4 spikes (Fig. 3B); membrane potential, input resistance, action potential characteristics and threshold for the initial spike were not altered appreciably by histamine. Histamine blocked the AHP~tow in 3 of 3 additional experiments (mean AHP duration _+S.D. from fourth spike to recovery of resting potential: pre-histamine, 3.4_+ 1.3 s; posthistamine, 0.4 +0.1 s; n = 4) and increased the number of action potentials evoked by ramped depolarizing currents. These data suggest that the AHP,Io,, affects excitability in these SCG neurons.
194 A,
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I/VVVVVVVVVV .
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.
1 sec
Do •
35 °
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II II
-6o rnv
IIIII
~
1 sec
.0 mV
,.,
]
-62 mV
-56 mV 1 sec l~rnV
I
Fig. 2. Properties of the AHP~jo, in SCG neurons. Panels A - D each are from a different cell. Action potentials here and in Fig. 3 are truncated at variable levels due to low frequency (200 Hz) at which data were digitized. A: voltage (upper) and current (lower) records showing changes in membrane conductance during the AHPsiow. Transmembrane hyperpolarizing current steps (0.3 nA, 50 ms) were delivered at 100-ms intervals. The AHPsjo, was elicited by 4 action potentials produced by four 5-nA, 2-ms depolarizing current steps (100-ms interspike interval)• Note that voltage transients decreased in amplitude during the AHP~jow, signifying increased conductance. No appreciable change in conductance was noted when the membrane potential was hyperpolarized by current injection to the same level as reached during the AHP~]ow. B: voltage traces showing the effect of membrane potential on the AHP~ow. The AHP~o, in each trace was activated as described in A. Membrane potential was hyperpolarized from resting level ( - 6 0 mV; upper trace) in 2 lower traces by passing constant current through the electrode• The AHP~jow amplitude decreased with hyperpolarization and was nulled at - 85 mV. Little change in input resistance was noted over this range of membrane potentials. C: effect of varying number of action potentials used to activate the AHPs~ow. Action potentials were produced by passing a corresponding number of short depolarizing current steps as in A. Maximal amplitude and duration of the AHP~Io,~ is shown for each trace. The amplitude, but not duration of the AHP~o~ was directly dependent on the number of preceding spikes. D: effect of temperature on the AHPs~,,w. Five action potentials were produced by depolarizing current steps as in A to activate the AHPao, while the perfusate was maintained at 35 and 24°C. The AHP~ow was abolished at lower temperature, while the fast AHP after each spike showed a small decrease in amplitude and increase in duration. Input resistance was not affected by temperature changes.
Our findings thus demonstrate
a spike AHP in mammalian
S C G n e u r o n s t h a t is
c o n s i d e r a b l y l o n g e r in d u r a t i o n t h a n t h o s e p r e v i o u s l y r e p o r t e d . C o n s i d e r i n g t h a t t h e electrophysiological characteristics of SCG neurons in several mammalian h a v e b e e n e x t e n s i v e l y s t u d i e d , it is p e r p l e x i n g t h a t s u c h l o n g - d u r a t i o n
species
AHPs
have
not been documented. However, our experimental protocol may have provided parti-
195
B.
A. CONTROL
"LJ--~
-,o mv
HISTAMINE
-
-72
mV
0.5
sec
- i s-ec
Fig. 3. Effect of histamine on the AHP~o~, and on excitability of an SCG neuron. A: voltage record (upper trace) showing an AHPs~o~ activated by 4 action potentials as described in Fig. 2. Hyperpolarizing deflcction preceding the 4 spikes was produced by a 0.l-nA, 300-ms hyperpolarizing current step to evaluate input resistance. Middle trace shows voltage change produced during a ramped current injection (0 1.2 nA over 1 s; lower trace). B: data in same format as A, but recorded during a 3-min exposure to 10 itM histamine. Histamine selectively blocked the AHP~low without affecting shorter duration ( < 500 ms) components of the A H P and now a burst of 4 action potentials occurred during the same ramped current. Input resistance of the neuron was not affected appreciably by histamine. Calibration bars at right side refer to analogous traces from both A and B.
cularly favorable conditions for observing these events. First, we have shown that the AHP~low in the SCG is temperature sensitive; it is abolished by cooling from 35 to 24"C. Thus, studies performed at room temperature (e.g. see refs. 13, 14, 18) would have failed to detect the AHP~Iow. Second, the AHP~jow could be sensitive to the electrolyte used in the recording microelectrode; studies in paravertebral sympathetic ganglia from frogs revealed slow spontaneous hyperpolarizations that were sensitive to the anion used in the electrolyte [15]. Finally, there is the possibility that the AHP~Iow in SCG neurons is species-specific to the guinea pig and thus was not detected in other mammals where SCG neurons have been studied (e.g. rat, rabbit, cat). Considering that the AHP~low is present in only approximately 18% of SCG neurons, the questions arise whether these cells are unique morphologically or in terms of their afferent or efferent projections. We have established that cells exhibiting an AHP~low receive nicotinic cholinergic synaptic input, which is a well documented feature of principal postganglionic neurons in the SCG [5]. In addition, neurons with or without an AHP~lowcould not be differentiated by other electrophysiological characteristics, arguing that the AHP~Jow is not just an artifact of an exceptionally good or poor impalement. Finally, neurons with an AHP~low were randomly distributed anatomically in the ganglion. This suggests that these cells project in both rostral and caudal nerve branches from the ganglion [18] and therefore may not selectively inner-
196 v a t e o n l y c e r t a i n p o s t g a n g l i o n i c t a r g e t s [12]. N o n e t h e l e s s , issues r e g a r d i n g the specific c o n n e c t i v i t y o f n e u r o n s p o s s e s s i n g an AHP~low, a n d the i d e n t i t y o f p h a r m a c o l o g i c a l o r e n d o g e n o u s l y r e l e a s e d a g e n t s t h a t c a n m o d u l a t e n e u r o n a l e x c i t a b i l i t y via this long. A H P b e c o m e p r o v o c a t i v e t o p i c s for f u r t h e r i n v e s t i g a t i o n s . T h e a u t h o r s wish to t h a n k Drs. B r a d U n d e m , J o h n H o r n a n d Bill W o n d e r l i n for t h e i r critical r e a d i n g o f the m a n u s c r i p t . T h i s w o r k w a s s u p p o r t e d by N I H G r a n t N S 0 7 8 6 5 to E . P . C . a n d N S 2 5 5 9 8 to D . W . 1 Baldissera, F. and Gustafsson, B., Firing behavior of a neurone model based on the afterhyperpolarization conductance time course and algebraic summation. Adaption and steady state firing, Acta Physiol. Scand., 92 (1974) 27 47. 2 Barrett, E.F. and Barrett, J.N., Separation of two voltage-sensitive potassium currents, and demonstration of a tetrodotoxin-resistant calcium current in frog motoneurones, J. Physiol. (Lond.), 255 (1976) 737 744. 3 Brown, D.A., Adams, P.R. and Constanti, A., Voltage-sensitive K-currents in sympathetic neurons and their modulation by neurotransmitters, J. Auton., Nerv. Syst., 6 (1982) 23-35. 4 Christian, E.P. and Weinreich, D., Unusually long-lasting spike afterhyperpolarizations in guinea pig superior cervical ganglion, Fed. Proc. Fed. Am. Soc. Exp. Biol., 46 (1987) 505. 5 Eccles, R.M., Orthodromic activation of single ganglion cells, J. Physiol. (Lond.), 165 (1963) 387-391. 6 Fowler, J.C., Greene, R. and Weinreich, D., Two calcium-sensitive spike afterhyperpolarizations in visceral sensory neurones of the rabbit, J. Physiol. (Lond.), 365 (1985) 59-75. 7 Gustafsson, B., Lindstrom, S. and Zangger, P., Firing behavior of dorsal spinocerebeUar tract neurones, J. Physiol. (Lond.), 275 (1978) 321-343. 8 Higashi, H., Morita, K. and North, R.A., Calcium-dependent afterpotentials in visceral afferent neurones in the rabbit, J. Physiol. (Lond.), 355 (1984) 479-492. 9 Hirst, G.D.S., Holman, M.E. and Spence, I., Two types of neurones in the myenteric plexus of duodenum in the guinea-pig, J. Physiol. (Lond.), 236 (1974) 303-326. 10 Horn, J.P. and McAfee, D., Alpha-adrenergic inhibition of calcium-dependent potentials in rat sympathetic neurones, J. Physiol. (Lond.), 301 (1980) 191-204. [ l Kawai, T., Oka, J. and Watanabe, M., Hexamethonium increases the excitability of sympathetic neurons by the blockade of the Ca2+-activated K + channels, Life Sci., 36 (1985) 2339-2346. 12 Lichtman, J.W., Purves, D. and Yip, J.W., On the purpose of selective innervation of guinea-pig cervical ganglion cells, J. Physiol. (Lond.), 292 (1979) 69-84. 13 McAfee, D.A., Henon, B.K., Horn, J.P. and Yarowski, P., Calcium currents modulated by adrenergic receptors in sympathetic neurons, Fed. Proc. Fed. Am. Soc. Exp. Biol., 40 (1981) 2246-2249. 14 McAfee, D.A. and Yarowski, P.J., Calcium-dependent potentials in the mammalian sympathetic neurone, J. Physiol. (Lond.), 290 (1979) 507 523. 15 Morita, K. and Koketsu, K., Oscillation of lCa2~]~-linked K + conductance in bullfrog sympathetic ganglion cell is sensitive to intracellular anions, Nature (Lond.), 283 (1980) 204-205. 16 Nishi, S., Electrophysiological properties of sympathetic neurons. In A.G. Karczmar, K. Koketsu and S. Nishi (Eds.), Autonomic and Enteric Ganglia, Transmission and Its Pharmacology; Plenum, New York. 1986, pp. 79- 106. 17 Nishi, S. and North, R.A., lntracellular recording from the myenteric plexus of the guinea-pig ileum, J. Physiol. (Lond.}, 231 (1973) 471-491. 18 Purves, D., Functionaland structural changes in mammalian sympathetic neurones fotlowinginterruption of their axons, J. Physiol. (Lond.), 252 (1975) 429-463. 19 Weinreich, D. and Wonderlin, W.F., Inhibition of Ca + +-dependent spike afterhyperpolarization increases excitability of rabbit visceral sensory neurones, J. Physiol. (Lond.), in press.