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A re-evaluation of the afterhyperpolarization mechanism in dorsal spinocerebellar tract neurons
543
SHORT COMMUNICATIONS
A re-evaluation of the afterhyperpolarization mechanism in dorsal spinocerebellar tract neurons In many types of neurons in the central nervous system, the somadendritic spike is followed by a long lasting afterhyperpolarization (AHP), which summates with repetitive activation. In a-motoneurons 1 and pyramidal tract neurons 9 this A H P is caused by a conductance increase of the membrane, presumably to potassium ions. An entirely different mechanism has recently been proposed by Kuno et al. 7 for the generation of the AHP in dorsal spinocerebellar tract (DSCT) neurons. They suggested that the A H P in these cells is caused by the activation of an electrogenic sodium pump and gave 4 main reasons: (1) no conductance increase could be detected during the A H P even after a short train of spikes; (2) during the A H P the membrane was polarized to a more negative level than the potassium equilibrium potential; (3) the AHP amplitude was largely independent of the membrane potential; (4) the half-decay time of the A H P had a Q10 of 2.4, which was believed to be more consistent with the temperature effect on active transport than on passive ionic conductance. The last argument by Kuno et al. 7 for an electrogenic pump seems to carry little weight since the rate o f change of the conductances underlying the action potential in squid axons have Q10 values o f about three4, 5. Furthermore, their arguments (1) and (3) contradict the findings in an earlier investigation of DSCT neurons in which the A H P following a single spike was found to be highly sensitive to hyperpolarizing current, and a small conductance increase was found after two spikes 3. We have therefore re-investigated the properties of the A H P in DSCT neurons. Experiments were performed on cats anesthetized with pentobarbital sodium (Nembutal, Abbott) 25-30 mg/kg. The DSCT neurons were identified by antidromic invasion from the ipsilateral anterior lobe of the cerebellum (dorsal column and the contralateral spinal half transected at low thoracic level), by their location in Clarke's column in L3 and rostral L4 segments and by their peripheral nerve input. Single micropipettes filled with 3 M KCI or 2 M potassium citrate were used both for recording and for current injection. Membrane conductance was measured with conventional pulse techniques. In 30 cells the membrane conductance was measured before and during the A H P which followed single or short trains of spikes. The spikes were in most cases elicited by just suprathreshold stimulation of the cerebellum, but in some cells by stimulation of the ipsilateral thoracic cord. In all cells a clear conductance increase could be detected, which, as illustrated in Fig. 1, was small after one spike, when the A H P was small, and rose in parallel with the increase in the A H P amplitude with an increased number of spikes. Both the A H P amplitude and conductance change reached a plateau after a small number of spikes. The conductance change was maximal Shortly after the spike and diminished progressively during the decay phase of the AHP. After a short train of spikes the conductance increase at the peak of the A H P was in the range of 1 0 - 6 5 ~ with a mean value of approximately 3 5 ~ . This should be compared with the conductance increases of 25 ~ (ref. 6) and 4 0 ~ (ref. 2) in spinal a-motoneurons after a single spike, which gives a considerable AHP. A Brain Research, 35 (1971) 543-546
544
SHORT COMMUNICATIONS
mV % conductanCechange / ~ 5' -50
A
mvl
o 4
50 msec
B
-40
/
x
El
4
6
3' -30 C
2- -20
D
x
E
-I0
I0 my]
I0 msec 0
0
;
;~
:~
8 no of spikes
Fig. 1. A H P amplitude and conductance change after different numbers of spikes. A, Antidromic spikes and A H P at threshold stimulation of the cerebellum taken with two different amplifications and sweep speeds. B, Resting membrane resistance. C-E, Membrane resistance during the A H P after 1, 4 and 8 antidromic spikes respectively. Test pulse 2 nA. In the graph the A H P amplitude (O) and conductance increase in per cent of the resting conductance (X) are plotted against the number of spikes. The open square (El) indicates the conductance increase'after 6 spikes elicited by a depolarizing current pulse through the recording electrode..
A ~yperpolInA ~
B ,.
C
.
2nA
hyperpol I nA
2.5 nA
2nA
Iomvi 4.5 nA
50 msee 3hA ,]
I0 mse¢
,
rOmVJ
50 rnsec
Fig. 2. Effect ofhyperpolarization on the brief undershoot (A), an IPSP (B) and the A H P following a single (A) and a short train of spikes (C). The records in C are from a different cell than the records in A and B. The arrows in C indicate the take-off level of the last spike in the train.
Brain Research, 35 (1971) 543-546
SHORT COMMUNICATIONS
545
conductance increase within this range (approximately 15 ~) can also be deduced from the published records of Kuno et al. (see Kuno et al. 7, Fig. 4). These conductance changes were not due to descending or recurrent postsynaptic effects. At threshold stimulation the AHPs were of an 'all-or-none' nature ( e l Fig. 1A and the upper record in Fig. 2A). When the cells were depolarized or hyperpolarized so as to block somadendritic invasion there was no trace of synaptic potentials. The AHPs were also virtually unaffected by chloride ion injection which clearly reversed IPSPs. Furthermore, the same conductance changes were found after a similar train of spikes elicited by depolarizing current pulses injected through the recording electrode (open square in the graph of Fig. 1). The conductance changes found could be secondary to the hyperpolarization, if there was anomalous rectification in the cells8. This was tested by comparing the membrane conductance increase during the AHP with the conductance increase observed during a long pulse giving the same hyperpolarization of the membrane as the AHP. Anomalous rectification was indeed found in many cells although the conductance changes were usually not more than one-third of the changes during the AHPs. This excludes the possib!lity that the AHP conductance change is secondary to the hyperpolarization. In DSCT neurons a brief hyperpolarizing undershoot followed by a delayed depolarization is interposed between the spike and the AHP 3,7. The conclusion by Kuno et al. 7 that the AHP polarized the membrane to more negative levels than the potassium equilibrium potential was based on the assumption that the reversal potential of the brief undershoot approximates to the potassium equilibrium potential. However, as shown in Fig. 2A and B, the undershoot regularly reversed at a less negative potential than IPSPs. It is thus unlikely that the reversal potential of the undershoot approximates to the potassium equilibrium potential. Fig. 2 illustrates our invariable finding that the AHP, measured from the 'resting' potential level (baseline without train) to its peak amplitude, decreased markedly with hyperpolarization. This holds true both for the AHP following a single (A) as well as a short train of spikes (C). Such a decrease is also apparent in the records published by Kuno et al. 7, particularly in their Fig. 5. Their claim that the AHP is not potential dependent was based on measurements of the AHP amplitude, not from the 'resting' potential but from the level at which the last spike in the train took off. Two systematic errors were then introduced. Without polarizing current there is a gradual development of the AHP during the train, which if neglected will give erroneously low amplitudes for the AHP. During hyperpolarization, on the other hand, each spike is followed by a large delayed depolarization with considerable build-up during the train. Measurements from this depolarized level will give too high amplitudes for the AHP in the hyperpolarized cells ( e l arrows in Fig. 2C). Reversal of the AHP was not easily demonstrated since the somadendritic invasion of the spikes invariably blocked before the membrane was sufficiently hyperpolarized. By application of a hyperpolarizing current pulse immediately after the last spike in the train (eft Coombs et al. 1) the AHP could be reversed in a few cells. The exact reversal potential was difficult to determine because of the 'contamination' of Brain Research, 35 (1971) 543-546
546
SHORT COMMUNICATIONS
the A H P with the delayed d e p o l a r i z a t i o n . It seemed, however, to be at a m o r e negative level t h a n the reversal potential for IPSPs. E s t i m a t i o n o f the reversal p o t e n t i a l b y e x t r a p o l a t i o n is even m o r e difficult since it is c o m p l i c a t e d n o t only by the delayed d e p o l a r i z a t i o n b u t also by the a n o m a l o u s rectification. I n conclusion, all e x p e r i m e n t a l results clearly indicate t h a t a c o n d u c t a n c e process, p r e s u m a b l y an increase o f m e m b r a n e p e r m e a b i l i t y to p o t a s s i u m ions, is involved in the g e n e r a t i o n o f the A H P following single o r s h o r t trains o f spikes in D S C T neurons. T h e r e a p p e a r s to be no r e a s o n to p o s t u l a t e t h a t an electrogenic s o d i u m p u m p c o n t r i b u t e s to this A H P .
Department of Physiology, University of G6teborg, G6teborg (Sweden)
B. GUSTAFSSON S. LINDSTROM M. TAKATA
1 COOMBS,J. S., ECCLES, J. C., AND FATT, P., The electrical properties of the motoneurone mem-
brane, J. Physiol. (Lond.), 130 (1955) 291-325. 2 ECCLES,J. C., The Physiology of Nerve Cells, The Johns Hopkins Press, Baltimore, 1957, 270 pp. 3 EIDE, E., FEDINA,L., JANSEN,J., LUNDBERG,A., ANDVYKLICK~, L., Properties of Clarke's column neurones, Acta physiol, scand., 77 (1969) 125-144. 5 HODGKIN,A. L., HUXLEY, A. F., AND KATZ, B., Measurement of current-voltage relations in the membrane of the giant axon of Loligo, J. Physiol. (Lond.), 116 (1952) 424-448. 4 HODGKtN,A. L., AND HUXLEY, A. F., The components of the membrane conductance in the giant axon of Loligo, J. Physiol. (Lond.), 116 (1952) 473-496. 6 ITO, M., AND OSHIMA, T., Temporal summation of after-hyperpolarization following a motoneurone spike, Nature (Lond.), 195 (1962) 910-911. 7 KUNO, M., MIYAHARA,J. T., AND WEAKLY,J. N., Post-tetanic hyperpolarization produced by an electrogenic pump in dorsal spinocerebellar tract neurones of the cat, J. Physiol. (Lond.), 210 (1970) 839-855. 8 NELSON, P. G., AND FRANK, K., Anomalous rectification in cat spinal motoneurons and effect of polarizing currents on excitatory postsynaptic potential, J. Neurophysiol., 30 (1967) 1097-1113. 9 TAKAHASHI,K., Slow and fast groups of pyramidal tract cells and their respective membrane properties, J. Neurophysiol., 28 (1965) 908-924. (Accepted September 24th, 1971)
Brain Research, 35 (1971) 543-546
SHORT COMMUNICATIONS
A re-evaluation of the afterhyperpolarization mechanism in dorsal spinocerebellar tract neurons In many types of neurons in the central nervous system, the somadendritic spike is followed by a long lasting afterhyperpolarization (AHP), which summates with repetitive activation. In a-motoneurons 1 and pyramidal tract neurons 9 this A H P is caused by a conductance increase of the membrane, presumably to potassium ions. An entirely different mechanism has recently been proposed by Kuno et al. 7 for the generation of the AHP in dorsal spinocerebellar tract (DSCT) neurons. They suggested that the A H P in these cells is caused by the activation of an electrogenic sodium pump and gave 4 main reasons: (1) no conductance increase could be detected during the A H P even after a short train of spikes; (2) during the A H P the membrane was polarized to a more negative level than the potassium equilibrium potential; (3) the AHP amplitude was largely independent of the membrane potential; (4) the half-decay time of the A H P had a Q10 of 2.4, which was believed to be more consistent with the temperature effect on active transport than on passive ionic conductance. The last argument by Kuno et al. 7 for an electrogenic pump seems to carry little weight since the rate o f change of the conductances underlying the action potential in squid axons have Q10 values o f about three4, 5. Furthermore, their arguments (1) and (3) contradict the findings in an earlier investigation of DSCT neurons in which the A H P following a single spike was found to be highly sensitive to hyperpolarizing current, and a small conductance increase was found after two spikes 3. We have therefore re-investigated the properties of the A H P in DSCT neurons. Experiments were performed on cats anesthetized with pentobarbital sodium (Nembutal, Abbott) 25-30 mg/kg. The DSCT neurons were identified by antidromic invasion from the ipsilateral anterior lobe of the cerebellum (dorsal column and the contralateral spinal half transected at low thoracic level), by their location in Clarke's column in L3 and rostral L4 segments and by their peripheral nerve input. Single micropipettes filled with 3 M KCI or 2 M potassium citrate were used both for recording and for current injection. Membrane conductance was measured with conventional pulse techniques. In 30 cells the membrane conductance was measured before and during the A H P which followed single or short trains of spikes. The spikes were in most cases elicited by just suprathreshold stimulation of the cerebellum, but in some cells by stimulation of the ipsilateral thoracic cord. In all cells a clear conductance increase could be detected, which, as illustrated in Fig. 1, was small after one spike, when the A H P was small, and rose in parallel with the increase in the A H P amplitude with an increased number of spikes. Both the A H P amplitude and conductance change reached a plateau after a small number of spikes. The conductance change was maximal Shortly after the spike and diminished progressively during the decay phase of the AHP. After a short train of spikes the conductance increase at the peak of the A H P was in the range of 1 0 - 6 5 ~ with a mean value of approximately 3 5 ~ . This should be compared with the conductance increases of 25 ~ (ref. 6) and 4 0 ~ (ref. 2) in spinal a-motoneurons after a single spike, which gives a considerable AHP. A Brain Research, 35 (1971) 543-546
544
SHORT COMMUNICATIONS
mV % conductanCechange / ~ 5' -50
A
mvl
o 4
50 msec
B
-40
/
x
El
4
6
3' -30 C
2- -20
D
x
E
-I0
I0 my]
I0 msec 0
0
;
;~
:~
8 no of spikes
Fig. 1. A H P amplitude and conductance change after different numbers of spikes. A, Antidromic spikes and A H P at threshold stimulation of the cerebellum taken with two different amplifications and sweep speeds. B, Resting membrane resistance. C-E, Membrane resistance during the A H P after 1, 4 and 8 antidromic spikes respectively. Test pulse 2 nA. In the graph the A H P amplitude (O) and conductance increase in per cent of the resting conductance (X) are plotted against the number of spikes. The open square (El) indicates the conductance increase'after 6 spikes elicited by a depolarizing current pulse through the recording electrode..
A ~yperpolInA ~
B ,.
C
.
2nA
hyperpol I nA
2.5 nA
2nA
Iomvi 4.5 nA
50 msee 3hA ,]
I0 mse¢
,
rOmVJ
50 rnsec
Fig. 2. Effect ofhyperpolarization on the brief undershoot (A), an IPSP (B) and the A H P following a single (A) and a short train of spikes (C). The records in C are from a different cell than the records in A and B. The arrows in C indicate the take-off level of the last spike in the train.
Brain Research, 35 (1971) 543-546
SHORT COMMUNICATIONS
545
conductance increase within this range (approximately 15 ~) can also be deduced from the published records of Kuno et al. (see Kuno et al. 7, Fig. 4). These conductance changes were not due to descending or recurrent postsynaptic effects. At threshold stimulation the AHPs were of an 'all-or-none' nature ( e l Fig. 1A and the upper record in Fig. 2A). When the cells were depolarized or hyperpolarized so as to block somadendritic invasion there was no trace of synaptic potentials. The AHPs were also virtually unaffected by chloride ion injection which clearly reversed IPSPs. Furthermore, the same conductance changes were found after a similar train of spikes elicited by depolarizing current pulses injected through the recording electrode (open square in the graph of Fig. 1). The conductance changes found could be secondary to the hyperpolarization, if there was anomalous rectification in the cells8. This was tested by comparing the membrane conductance increase during the AHP with the conductance increase observed during a long pulse giving the same hyperpolarization of the membrane as the AHP. Anomalous rectification was indeed found in many cells although the conductance changes were usually not more than one-third of the changes during the AHPs. This excludes the possib!lity that the AHP conductance change is secondary to the hyperpolarization. In DSCT neurons a brief hyperpolarizing undershoot followed by a delayed depolarization is interposed between the spike and the AHP 3,7. The conclusion by Kuno et al. 7 that the AHP polarized the membrane to more negative levels than the potassium equilibrium potential was based on the assumption that the reversal potential of the brief undershoot approximates to the potassium equilibrium potential. However, as shown in Fig. 2A and B, the undershoot regularly reversed at a less negative potential than IPSPs. It is thus unlikely that the reversal potential of the undershoot approximates to the potassium equilibrium potential. Fig. 2 illustrates our invariable finding that the AHP, measured from the 'resting' potential level (baseline without train) to its peak amplitude, decreased markedly with hyperpolarization. This holds true both for the AHP following a single (A) as well as a short train of spikes (C). Such a decrease is also apparent in the records published by Kuno et al. 7, particularly in their Fig. 5. Their claim that the AHP is not potential dependent was based on measurements of the AHP amplitude, not from the 'resting' potential but from the level at which the last spike in the train took off. Two systematic errors were then introduced. Without polarizing current there is a gradual development of the AHP during the train, which if neglected will give erroneously low amplitudes for the AHP. During hyperpolarization, on the other hand, each spike is followed by a large delayed depolarization with considerable build-up during the train. Measurements from this depolarized level will give too high amplitudes for the AHP in the hyperpolarized cells ( e l arrows in Fig. 2C). Reversal of the AHP was not easily demonstrated since the somadendritic invasion of the spikes invariably blocked before the membrane was sufficiently hyperpolarized. By application of a hyperpolarizing current pulse immediately after the last spike in the train (eft Coombs et al. 1) the AHP could be reversed in a few cells. The exact reversal potential was difficult to determine because of the 'contamination' of Brain Research, 35 (1971) 543-546
546
SHORT COMMUNICATIONS
the A H P with the delayed d e p o l a r i z a t i o n . It seemed, however, to be at a m o r e negative level t h a n the reversal potential for IPSPs. E s t i m a t i o n o f the reversal p o t e n t i a l b y e x t r a p o l a t i o n is even m o r e difficult since it is c o m p l i c a t e d n o t only by the delayed d e p o l a r i z a t i o n b u t also by the a n o m a l o u s rectification. I n conclusion, all e x p e r i m e n t a l results clearly indicate t h a t a c o n d u c t a n c e process, p r e s u m a b l y an increase o f m e m b r a n e p e r m e a b i l i t y to p o t a s s i u m ions, is involved in the g e n e r a t i o n o f the A H P following single o r s h o r t trains o f spikes in D S C T neurons. T h e r e a p p e a r s to be no r e a s o n to p o s t u l a t e t h a t an electrogenic s o d i u m p u m p c o n t r i b u t e s to this A H P .
Department of Physiology, University of G6teborg, G6teborg (Sweden)
B. GUSTAFSSON S. LINDSTROM M. TAKATA
1 COOMBS,J. S., ECCLES, J. C., AND FATT, P., The electrical properties of the motoneurone mem-
brane, J. Physiol. (Lond.), 130 (1955) 291-325. 2 ECCLES,J. C., The Physiology of Nerve Cells, The Johns Hopkins Press, Baltimore, 1957, 270 pp. 3 EIDE, E., FEDINA,L., JANSEN,J., LUNDBERG,A., ANDVYKLICK~, L., Properties of Clarke's column neurones, Acta physiol, scand., 77 (1969) 125-144. 5 HODGKIN,A. L., HUXLEY, A. F., AND KATZ, B., Measurement of current-voltage relations in the membrane of the giant axon of Loligo, J. Physiol. (Lond.), 116 (1952) 424-448. 4 HODGKtN,A. L., AND HUXLEY, A. F., The components of the membrane conductance in the giant axon of Loligo, J. Physiol. (Lond.), 116 (1952) 473-496. 6 ITO, M., AND OSHIMA, T., Temporal summation of after-hyperpolarization following a motoneurone spike, Nature (Lond.), 195 (1962) 910-911. 7 KUNO, M., MIYAHARA,J. T., AND WEAKLY,J. N., Post-tetanic hyperpolarization produced by an electrogenic pump in dorsal spinocerebellar tract neurones of the cat, J. Physiol. (Lond.), 210 (1970) 839-855. 8 NELSON, P. G., AND FRANK, K., Anomalous rectification in cat spinal motoneurons and effect of polarizing currents on excitatory postsynaptic potential, J. Neurophysiol., 30 (1967) 1097-1113. 9 TAKAHASHI,K., Slow and fast groups of pyramidal tract cells and their respective membrane properties, J. Neurophysiol., 28 (1965) 908-924. (Accepted September 24th, 1971)
Brain Research, 35 (1971) 543-546