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Chapter 4 Classification of a calcium conductance in cold receptors
W . Hamann and A. lggo (Eds.) Progress in Brain Research, Val. 74
D 1988 Elsevier Science Publishers
29
B.V. (Biomedical Division)
CHAPTER 4
Classification of a calcium conductance in cold receptors Klaus Schaferl, Hans A. Braun2 and Ludgera Rempe1 'Instirut f i r Zoophysiologie, Universitat Hohenheim, 0-7000 Stuttgart 70, and 'Instirut fur Physiologie, Universitat Marburg, 0-3550 Marburg, FRG
Summary The small size of mammalian cold sensors, which have been shown to be free nerve endings, has so far prevented any attempt to study their transducer processes directly. However, recently developed techniques such as discharge pattern analysis and reliable isolated organ preparations have allowed to gain some insight into the conditions under which afferent activity in cold receptors is initiated. Afferent activity of mammalian cold sensors is generated by periodic receptor membrane processes. Frequency and amplitude of the impulse triggering cyclic membrane events are temperature dependent; the processes are supposed to be controlled by calcium currents and calcium-sensitive outward conductances. External menthol (10 - 50 pmol/l) induces changes of mean activity and discharge pattern qualitatively similar to those effected by reduced extracellular calcium concentrations. In molluscan neurons calcium currents are reversibly reduced by external menthol (100- 500 pmol/l). The mode of action appears to be a specific interaction of menthol with calcium channels, since the effect is stereochemically selective and cannot be evoked by intracellular application. Of the three classes of calcium conductances identified in dorsal root ganglion cells so far, menthol acts on the low-voltage-activated (LVA) type and high-voltage-activated (HVA) type only. These
two calcium conductances differ in their potential range of activation, their kinetics, and their sensitivity to calcium channel modulators. For instance, the LVA-type conductance has been shown to be rather inert against verapamil and 1,4dihydropyridine-type channel modulators. External verapamil (10- 100 pmol/l) either does not affect cold sensor discharges or inhibits afferent activity. These observations are at variance with the expected stimulating action of compounds interfering with calcium conductances (such as menthol). Analysis of the temporal pattern of cold sensor activity indicates that the cyclic sensory processes are independent of the release of action potentials. Interspike intervals of 'irregular' discharges are even-numbered multiples (up to 8) of the oscillation period. Likewise, the oscillation period of grouped discharges is constant in spite of a varying number of impulses per group. These various observations indicate that a verapamil-sensitive, action-potential-activated calcium conductance (such as the HVA type) is absent in cold sensors. The data suggest instead the existence of a calcium channel with properties of the LVA type.
Introduction Mammalian cold sensors are free nerve endings (Hensel et al., 1974), and their small size has so far prevented any direct study of their transducer pro-
30
cesses. However, various indirect experimental approaches have recently allowed to gain some insight into the conditions under which afferent activity of cold receptors is initiated (Braun et al., 1980, 1984; Schafer et al., 1982, 1984, 1986; Schafer, 1987). In particular the application of discharge pattern analysis and the development of a reliable isolated organ preparation have been proved to be effective techniques. Afferent activity of mammalian cold sensors is generated by periodic receptor membrane processes (Braun et al., 1980). Frequency and amplitude of the impulse triggering cyclic ekents are temperature dependent. The periodic processes are supposed to be controlled by calcium currents and calcium-stimulated outward conductances (Schafer et al., 1986). Changes of cold receptor activity induced by various levels of external calcium are in agreement with the hypothesis of calciumsensitive conductances being present in cold receptors (Schafer et al., 1982). In quantitative experiments by use of an isolated organ preparation these changes accord fairly well with the known properties of the corresponding neuronal channels, namely with the non-linear dependence of these conductances on the extracellular calcium concentration (Schafer, 1987). These various observations suggest that mammalian cold sensors might possess ionic conductances similar to those known to exist in other neurons. In avian and mammalian dorsal root ganglion cells three different classes of calcium currents have been recently identified (Carbone and Lux, 1984; Nowycky et al., 1985; Swandulla et al., 1987). They can be differentiated by their potential range of activation, by their kinetics of inactivation, and by their sensitivity to calcium channel modulators. The aim of the present study was to identify the type of calcium channel present in cold sensors by application of so-called calcium entry blockers along with an analysis of the induced changes of afferent activity. A successful classification of the type of calcium conductance involved in sensory transduction is considered to signify a substantial
progress in our understanding of sensory processes. Calcium control of afferent activity In various cold receptor populations beating activity and burst (grouped) discharges have been observed at maintained temperatures and during rapid cooling (Hensel, 1981). In all populations studied so far, analysis of the impulse activity revealed a cyclic pattern of impulse generation suggesting the existence of an underlying receptor potential oscillation, which initiates impulses in the afferent nerve when it exceeds a threshold value (Braun et al., 1984; Schafer et al., 1984, 1986). An example of afferent impulse activity together with the corresponding interval distribution of a single cold receptor at various constant temperatures is represented in Fig. 1. From the data shown in this figure the burst frequency (i.e., oscillation frequency) and number of impulses induced during each cycle can be calculated (Braun and Hensel, 1977; examples of such a calculation are given in Schafer et al., 1986). Consequently, the mean discharge rate is a function of the burst frequency, which increases with warming, and the number of impulses per burst, which increases with cooling. As a result, when the number of impulses per burst is multiplied by the burst frequency, the wellknown parabolic relation between mean activity and temperature is obtained (Braun et al., 1980). A calcium-stimulated outward current, acting as a negative feedback system, is generally viewed as an essential link in the cycle of events controlling periodic neuronal activity (Eckert and Lux, 1976; Gorman et al., 1982). Therefore, all experimental measures interfering with the availability of calcium to enter the receptive structure markedly affect the burst frequency, the number of impulses per burst, and, hence, the mean discharge rate (Schafer et al., 1982, 1984, 1986; Schafer, 1987). In our experiments, calcium supply was impaired by application of calcium chelators in whole animals, by perfusion of isolated organ preparations with solutions containing reduced calcium levels,
31
and by application of menthol, which has been shown to selectively interfere with neuronal calcium channels (Swandulla et al., 1986). Nevertheless, the induced changes of afferent activity are rather uniform, independently of the kind of treatment applied. As can be seen in Fig. 2, the effects of either reduced external calcium concentrations or of impairment of calcium entry into the receptor are qualitatively similar in experiments on whole animals and isolated organ preparations, which indicates the involvement of a common mechanism in all cases.
Calcium conductances of sensory neurons Three different classes of calcium channels have been recently characterized in vertebrate sensory neurons (Carbone and Lux, 1984; Nowycky et al., 1985). One of them, activated at high voltages (HVA type), resembles the classical calcium channel of other excitable cells. A second channel, activated at low voltages (LVA type), differs both kinetically and pharmacologically from the classical one. Its time-dependent inactivation is fast and complete, and it is rather inert against large doses
Fig 1. Discharge pattern of a feline lingual cold receptor at various constant temperatures. Left diagrams: interval distribution; right diagrams: impulse activity, The mean discharge rates (in s-I) are: 5.4 (40°C); 7.2 (35°C); 9.2 (30°C); 10.4 (25°C); 12.0 (20°C); 11 .O (15°C). Intervals shorter than 100 ms are intraburst intervals. Dimensions of graphs and stimulus temperatures as indicated. (Methods are given in Braun et al., 1980; Schafer et al., 1986).
32
channels present in sensory neurons by different mechanisms. The high-voltage-activated, long lasting conductance (HVA type) is impaired by an accelerated inactivation, whereas the lowvoltage-activated, transient current (LVA type) is reduced without its inactivation time course being affected. Menthol has practically no effect on a third type of calcium current recently identified by Nowycky et al. (1985) in the same preparation. This fast inactivating channel activates at high voltages and deactivates at low voltages (N type).
of organic calcium channel modulators such as verapamil and 1,4-dihydropyridine derivatives (Boll and Lux, 1985). In mammalian sensory neurons, LVA channels occasionally contribute more than HVA channels to the total calcium current of the cell membrane (Swandulla et al., 1987). Observations in molluscan neurons and in avian and mammalian dorsal root ganglion cells suggest a specific impeding action of menthol (2-isopropyl5-methyl-cyclohexanol) on calcium currents (Swandulla et al., 1986, 1987). The effects of menthol develop in seconds, they are fully reversible and stereochemically selective, and they cannot be evoked by intracellular application. Related compounds, such as cyclohexanol, fail to produce any comparable effect, even in considerably higher concentrations, which suggests a specific interaction of menthol with calcium channels. Menthol reduces currents through two types of calcium
Calcium conductance of cold sensors Reduced extracellular calcium levels induce depolarizing shifts of membrane potential in invertebrate and vertebrate bursting neurons, whereas elevated calcium levels result in hyperpolarization; the induced changes of potential are
10.0 -J
5.0
-
DYNAMIC RESPONSE AT = -5OC
2.0 1.0 -
0.2 J
10
20
15
25
30
TEMPERATURE ["C]
35
40
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-
0.2
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15
20
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30
35
40
INITIAL TEMPERATURE ["C]
Fig. 2. Relative change of cold receptor activity induced by menthol and by reduced external calcium. Control values are represented by '1 .OO'. Left diagram: static firing rate; right diagram: dynamic peak frequency following 5°C cooling from adapting temperature. Circles, lingual cold receptors, isolated organ preparation, 0.5 mmol/l calcium (n = 11). data from Schafer (1987); diamonds, lingual cold receptors, isolated organ preparation, 50 pmol/l menthol (n = 3), data from Schafer et al. (1986); squares, nasal cold receptors, whole animal, 0.57 k 0.11 mg/kg/min menthol i.v. (n = 8). data from Schafer et al. (1986); triangles, nasal cold receptors, whole animal, 0.77 0.29 mg/kg/min EDTA i.v. (n = 9), data from Schafer et al. (1982). EDTA chelates calcium ions. Methods are given in the cited references.
*
33
associated with concomitant modifications of discharge rate (Gola and Selveston, 1981; Legendre et al., 1985). Activity of these neurons is controlled by calcium currents and by calcium-sensitive conductances. Cold receptor activity is comparably suppressed by excess external calcium and enhanced by reduced calcium (Schafer et al., 1982; Schafer, 1987). The considerable conformity of the calcium-induced changes of either the neuronal membrane potential or the sensory afferent discharge supports the view that the activity of both cell types is controlled by identical mechanisms, namely by calcium-stimulated outward conductances. Calcium seems not to be inA
15
volved in affecting the membrane potential as a current carrier, since elevation of external calcium neither induces depolarization nor increases afferent activity. The data indicate that a calciumactivated outward current is involved, which masks the direct effect of the calcium current by its considerably higher conductance (Akaike et al., 1978; Lux and Hofmeier, 1982). Figs. 2 and 3 show that the menthol-induced changes of cold receptor discharge are fairly identical to those induced by reduced external calcium. Both menthol and reduced calcium exert their effects on the mean firing rate by comparable modifications of cyclic receptor activity (Schafer et al.,
--
0.5 mmol Calcium
= -
.
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5
mmol Calcium
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35
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.P
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51
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. 1 min
15. 10 pmol Verapamil
.
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100 pmol Verapamii
'
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' T * 30%
--
1 rnin
Fig. 3. Effect of reduced external calcium and of calcium entry blockers on mean discharge rate of cold receptors. Data from six lingual cold receptors out of a total of 43 which were studied. Isolated organ preparation. T, thermode temperature; discharge rate was averaged over periods of 10 s. The isolated organ preparation was perfused for the periods indicated by bars with solutions containing either reduced or elevated calcium levels (A, B), various menthol concentrations (C, D), or various verapamil concentrations (E, F). Under control conditions, the isolated organ preparation was perfused with a modified Krebs solution (for details, see Schafer, 1987).
34
1986; Schafer and Braun, unpublished). Since menthol has been proved to selectively impair the calcium entry, we compared menthol with the classical calcium entry blocker, verapamil. When tested in six specific cold receptors, either external verapamil in doses of 10 - 500 pmol/l was ineffective or cold receptor discharge was inhibited. In every case the observed effects were at variance with the expected stimulating action of compounds interfering with the calcium entry such as menthol (Fig. 3). Since verapamil is mainly effective in reducing current through the ubiquitous calcium channel (HVA type) (Boll and Lux, 1985), these data do not support the view of an action-potential-activated calcium conductance being present at the cold receptor membrane. Cyclic receptor activity, however, is strongly controlled by calcium. If calcium entry through the HVA-type conductance is negligible, the periodic receptor activity should be independent of the release of afferent impulses by the sensor. In fact, various analyses of the temporal pattern of cold receptor discharges have indicated the existence of an endogenously
instantaneous frequency
BP: k50 t 49 mscc
c
I5 O C
SBs6
BP.653 2 5 9 mscc
ISOC SB.7
BP.652 t 99msec
10% SB. 5 BP.799:
70mscc
10 o c SB. 8 BP.780 :79 mscc
Fig. 5 . Instantaneous frequency diagram of the temporal pattern of grouped (burst) discharges. Ordinate, instantaneous frequency in s - (reciprocal value of the duration of successive intervals); abscissa, time in ms. Data of a 2 min period at every constant temperature. BP, burst period (i.e., period of the underlying cyclic receptor events); SB, impulses per burst. Method of calculation is given in Braun et al. (1980).
’
Fig. 4.Temporal pattern of cold receptor discharge. Lingual cold receptor, isolated organ preparation. Perfusion with a solution containing 0.5 mmol/l calcium (for details, see Schafer, 1987). Thermode temperature, 40°C. (A) Impulse activity. (B) Duration of successive intervals. (C) Interval distribution. Arrows indicate the basic period of the cyclic receptor events and the even-numbered multiples. Values were calculated from the data presented in C (the method of calculation and examples are given in Braun et al., 1980; and in Schafer et al., 1986; respectively).
oscillating receptor process (Braun et al., 1980, 1984; Schafer et al., 1986). At higher adapting temperatures, these cyclic processes occasionally fail to initiate the appropriate impulse, which results in ‘irregular’ discharges. In that case, all interspike intervals are even-numbered multiples of the oscillation period, and sporadically intervals of a duration up to 8 times the oscillation period are seen (Fig. 4). At lower adapting temperatures, the periodic receptor processes mainly initiate groups of impulses during each cycle (Braun et al., 1980; Schafer et al., 1986). At a given temperature, burst period (i.e., oscillation period) and burst duration are fairly constant in spite of a varying number of
35
impulses induced per group (Fig. 5 ) . This additionally accords with the assumption that action potentials do not contribute to the initiation and maintenance of cyclic sensory processes. Moreover, it indicates that cyclic activity might be differently controlled in cold receptors and in molluscan bursting pacemaker neurons. In these neurons, which have been repeatedly proposed as models for cold receptor function (Braun et al., 1980, 1984; Schafer et al., 1982, 1986), the maintenance of cyclic activity evidently depends on the initiation of action potentials (Kramer and Zucker, 1985). Taken together, these various observations indicate that calcium channels of cold receptors are menthol-sensitive, but verapamil-insensitive, and that action potentials contribute only insignificantly, if at all, to the calcium entry into the sensor. The data strongly suggest that the cold receptor membrane possesses calcium channels with properties of the recently characterized LVA channel. Since the action of menthol is rather specific, it can be concluded that the N-type calcium conductance is absent in cold receptor membranes. In this connection it might be of interest that the LVA type of calcium channel is assumed to be involved in the maintenance of rhythmic neuronal activity (Llinas and Yarom, 1981). Therefore, for the first time, by application of indirect methods of investigation, it was possible to identify a type of ionic conductance involved in sensory transduction of thermoreceptors. Acknowledgement
This work was supported by the Deutsche Forschungsgemeinschaft . References Akaike, N., Lee, K.S. and Brown, A.M. (1978) The calcium current of Helix neuron. J. Gen. Physiol., 71: 509-531. Boll, W. and Lux, H.D. (1985) Action of organic antagonists on neuronal calcium currents. Neurosci. Lett., 56: 335 - 339. Braun, H.A. and Hensel, H. (1977) A computer program for identification and analysis of neuronal burst discharges. Pflugers Arch., 368: R48.
Braun, H.A., Bade, H. and Hensel, H. (1980) Static and dynamic discharge patterns of bursting cold fibres related to hypothetical receptor mechanisms. Pflugers Arch., 386: 1-9. Braun, H.A., Schafer, K., Wissig, H. and Hensel, H. (1984) Periodic transduction processes in thermosensitive receptors. In: W. Hamann and A. Iggo (Eds.), Sensory Receptor Mechanisms. World Sci. Publ. Corp., Singapore, pp. 147 - 156. Carbone, E. and Lux, H.D. (1984) A low voltage-activated calcium conductance in embryonic chick sensory neurons. Biophys. J . , .46: 413 -418. Eckert, R. and Lux, H.D. (1976) A voltage-sensitive persistent calcium conductance in neuronal somata of Helix. J . Physiol. (London), 254: 129- 152. Gola, M. and Selverston, A. (1981) Ionic requirements for bursting activity in lobster stomatogastric neurons. J . Comp. Physiol., 145: 191 -207. Gorman, A.L.F., Hermann, A. and Thomas, M.V. (1982) Ionic requirements for membrane oscillations and their dependence on the calcium concentration in a molluscan pacemaker neurone. J. Physiol. (London), 327: 185 -218. Hensel, H. (1981) Thermoreception and Temperature Regulafion. Academic Press, London. Hensel, H., Andres, K.H. and v. During, M. (1974) Structure and function of cold receptors. Pflugers Arch., 352: 1 - 10. Kramer, R.H. and Zucker, R.S. (1985) Calcium-induced inactivation of calcium current causes the interburst hyperpolarization of Aplysia bursting neurones. J . Physiol. (London), 362: 131 - 160. Legendre, P . , McKenzie, J.S., Dupouy, B. and Vincent, J.D. (1985) Evidence for bursting pacemaker neurones in cultured spinal cord cells. Neuroscience, 16: 753 - 767. Llinas, R. and Yarom, Y. (1981) Properties and distribution of ionic conductances generating electroresponsiveness of mammalian inferior olivary neurones in vitro. J . Physiol. (London), 315: 569-584. Lux, H.D. and Hofmeier, G. (1982) Properties of a calciumand voltage-activated potassium current in Helix pomatia neurons. Pflugers Arch., 394: 61 -69. Nowycky, M.C., Fox, A.P. and Tsien, R.W. (1985) Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature (London), 316: 440 - 443. Schafer, K. (1987) A quantitative study of the dependence of feline cold receptor activity o n the calcium concentration. Pflugers Arch., 409: 208-213. Schafer, K., Braun, H.A. and Hensel, H. (1982) Static and dynamic activity of cold receptors at various calcium levels. J. Neurophysiol., 47: 1017- 1028. Schafer, K., Braun, H.A. and Hensel, H. (1984) Temperature transduction in the skin. In: J.R.S. Hales (Ed.), Thermal Physiology, Raven Press, New York, pp. 1 - 11. Schafer, K., Braun, H.A. and Isenberg, C. (1986) Effect of menthol o n cold receptor activity. Analysis of receptor processes. J . Gen. Physiol., 88: 757 - 776.
36
Schafer, K., Braun, H.A. and Kiirten, L. (1987) Analysis of discharge pattern of cold- and warm-receptor activity of vampire bats and mice. Verh. Dtsch. Zoo/. Ges., 80: 279. Swandulla, D., Schafer, K . and Lux, H.D. (1986) Calcium channel current inactivation is selectively modulated by men-
thol. Neurosci. Lett.. 68: 23 - 28. Swandulla, D., Carbone, E., Schafer, K. and Lux, H.D.(1987) Effect of menthol on two types of calcium currents in cultured sensory neurons of vertebrates. Pf/ugers Arch., 409: 52 - 59.
D 1988 Elsevier Science Publishers
29
B.V. (Biomedical Division)
CHAPTER 4
Classification of a calcium conductance in cold receptors Klaus Schaferl, Hans A. Braun2 and Ludgera Rempe1 'Instirut f i r Zoophysiologie, Universitat Hohenheim, 0-7000 Stuttgart 70, and 'Instirut fur Physiologie, Universitat Marburg, 0-3550 Marburg, FRG
Summary The small size of mammalian cold sensors, which have been shown to be free nerve endings, has so far prevented any attempt to study their transducer processes directly. However, recently developed techniques such as discharge pattern analysis and reliable isolated organ preparations have allowed to gain some insight into the conditions under which afferent activity in cold receptors is initiated. Afferent activity of mammalian cold sensors is generated by periodic receptor membrane processes. Frequency and amplitude of the impulse triggering cyclic membrane events are temperature dependent; the processes are supposed to be controlled by calcium currents and calcium-sensitive outward conductances. External menthol (10 - 50 pmol/l) induces changes of mean activity and discharge pattern qualitatively similar to those effected by reduced extracellular calcium concentrations. In molluscan neurons calcium currents are reversibly reduced by external menthol (100- 500 pmol/l). The mode of action appears to be a specific interaction of menthol with calcium channels, since the effect is stereochemically selective and cannot be evoked by intracellular application. Of the three classes of calcium conductances identified in dorsal root ganglion cells so far, menthol acts on the low-voltage-activated (LVA) type and high-voltage-activated (HVA) type only. These
two calcium conductances differ in their potential range of activation, their kinetics, and their sensitivity to calcium channel modulators. For instance, the LVA-type conductance has been shown to be rather inert against verapamil and 1,4dihydropyridine-type channel modulators. External verapamil (10- 100 pmol/l) either does not affect cold sensor discharges or inhibits afferent activity. These observations are at variance with the expected stimulating action of compounds interfering with calcium conductances (such as menthol). Analysis of the temporal pattern of cold sensor activity indicates that the cyclic sensory processes are independent of the release of action potentials. Interspike intervals of 'irregular' discharges are even-numbered multiples (up to 8) of the oscillation period. Likewise, the oscillation period of grouped discharges is constant in spite of a varying number of impulses per group. These various observations indicate that a verapamil-sensitive, action-potential-activated calcium conductance (such as the HVA type) is absent in cold sensors. The data suggest instead the existence of a calcium channel with properties of the LVA type.
Introduction Mammalian cold sensors are free nerve endings (Hensel et al., 1974), and their small size has so far prevented any direct study of their transducer pro-
30
cesses. However, various indirect experimental approaches have recently allowed to gain some insight into the conditions under which afferent activity of cold receptors is initiated (Braun et al., 1980, 1984; Schafer et al., 1982, 1984, 1986; Schafer, 1987). In particular the application of discharge pattern analysis and the development of a reliable isolated organ preparation have been proved to be effective techniques. Afferent activity of mammalian cold sensors is generated by periodic receptor membrane processes (Braun et al., 1980). Frequency and amplitude of the impulse triggering cyclic ekents are temperature dependent. The periodic processes are supposed to be controlled by calcium currents and calcium-stimulated outward conductances (Schafer et al., 1986). Changes of cold receptor activity induced by various levels of external calcium are in agreement with the hypothesis of calciumsensitive conductances being present in cold receptors (Schafer et al., 1982). In quantitative experiments by use of an isolated organ preparation these changes accord fairly well with the known properties of the corresponding neuronal channels, namely with the non-linear dependence of these conductances on the extracellular calcium concentration (Schafer, 1987). These various observations suggest that mammalian cold sensors might possess ionic conductances similar to those known to exist in other neurons. In avian and mammalian dorsal root ganglion cells three different classes of calcium currents have been recently identified (Carbone and Lux, 1984; Nowycky et al., 1985; Swandulla et al., 1987). They can be differentiated by their potential range of activation, by their kinetics of inactivation, and by their sensitivity to calcium channel modulators. The aim of the present study was to identify the type of calcium channel present in cold sensors by application of so-called calcium entry blockers along with an analysis of the induced changes of afferent activity. A successful classification of the type of calcium conductance involved in sensory transduction is considered to signify a substantial
progress in our understanding of sensory processes. Calcium control of afferent activity In various cold receptor populations beating activity and burst (grouped) discharges have been observed at maintained temperatures and during rapid cooling (Hensel, 1981). In all populations studied so far, analysis of the impulse activity revealed a cyclic pattern of impulse generation suggesting the existence of an underlying receptor potential oscillation, which initiates impulses in the afferent nerve when it exceeds a threshold value (Braun et al., 1984; Schafer et al., 1984, 1986). An example of afferent impulse activity together with the corresponding interval distribution of a single cold receptor at various constant temperatures is represented in Fig. 1. From the data shown in this figure the burst frequency (i.e., oscillation frequency) and number of impulses induced during each cycle can be calculated (Braun and Hensel, 1977; examples of such a calculation are given in Schafer et al., 1986). Consequently, the mean discharge rate is a function of the burst frequency, which increases with warming, and the number of impulses per burst, which increases with cooling. As a result, when the number of impulses per burst is multiplied by the burst frequency, the wellknown parabolic relation between mean activity and temperature is obtained (Braun et al., 1980). A calcium-stimulated outward current, acting as a negative feedback system, is generally viewed as an essential link in the cycle of events controlling periodic neuronal activity (Eckert and Lux, 1976; Gorman et al., 1982). Therefore, all experimental measures interfering with the availability of calcium to enter the receptive structure markedly affect the burst frequency, the number of impulses per burst, and, hence, the mean discharge rate (Schafer et al., 1982, 1984, 1986; Schafer, 1987). In our experiments, calcium supply was impaired by application of calcium chelators in whole animals, by perfusion of isolated organ preparations with solutions containing reduced calcium levels,
31
and by application of menthol, which has been shown to selectively interfere with neuronal calcium channels (Swandulla et al., 1986). Nevertheless, the induced changes of afferent activity are rather uniform, independently of the kind of treatment applied. As can be seen in Fig. 2, the effects of either reduced external calcium concentrations or of impairment of calcium entry into the receptor are qualitatively similar in experiments on whole animals and isolated organ preparations, which indicates the involvement of a common mechanism in all cases.
Calcium conductances of sensory neurons Three different classes of calcium channels have been recently characterized in vertebrate sensory neurons (Carbone and Lux, 1984; Nowycky et al., 1985). One of them, activated at high voltages (HVA type), resembles the classical calcium channel of other excitable cells. A second channel, activated at low voltages (LVA type), differs both kinetically and pharmacologically from the classical one. Its time-dependent inactivation is fast and complete, and it is rather inert against large doses
Fig 1. Discharge pattern of a feline lingual cold receptor at various constant temperatures. Left diagrams: interval distribution; right diagrams: impulse activity, The mean discharge rates (in s-I) are: 5.4 (40°C); 7.2 (35°C); 9.2 (30°C); 10.4 (25°C); 12.0 (20°C); 11 .O (15°C). Intervals shorter than 100 ms are intraburst intervals. Dimensions of graphs and stimulus temperatures as indicated. (Methods are given in Braun et al., 1980; Schafer et al., 1986).
32
channels present in sensory neurons by different mechanisms. The high-voltage-activated, long lasting conductance (HVA type) is impaired by an accelerated inactivation, whereas the lowvoltage-activated, transient current (LVA type) is reduced without its inactivation time course being affected. Menthol has practically no effect on a third type of calcium current recently identified by Nowycky et al. (1985) in the same preparation. This fast inactivating channel activates at high voltages and deactivates at low voltages (N type).
of organic calcium channel modulators such as verapamil and 1,4-dihydropyridine derivatives (Boll and Lux, 1985). In mammalian sensory neurons, LVA channels occasionally contribute more than HVA channels to the total calcium current of the cell membrane (Swandulla et al., 1987). Observations in molluscan neurons and in avian and mammalian dorsal root ganglion cells suggest a specific impeding action of menthol (2-isopropyl5-methyl-cyclohexanol) on calcium currents (Swandulla et al., 1986, 1987). The effects of menthol develop in seconds, they are fully reversible and stereochemically selective, and they cannot be evoked by intracellular application. Related compounds, such as cyclohexanol, fail to produce any comparable effect, even in considerably higher concentrations, which suggests a specific interaction of menthol with calcium channels. Menthol reduces currents through two types of calcium
Calcium conductance of cold sensors Reduced extracellular calcium levels induce depolarizing shifts of membrane potential in invertebrate and vertebrate bursting neurons, whereas elevated calcium levels result in hyperpolarization; the induced changes of potential are
10.0 -J
5.0
-
DYNAMIC RESPONSE AT = -5OC
2.0 1.0 -
0.2 J
10
20
15
25
30
TEMPERATURE ["C]
35
40
0.5
-
0.2
J
15
20
25
30
35
40
INITIAL TEMPERATURE ["C]
Fig. 2. Relative change of cold receptor activity induced by menthol and by reduced external calcium. Control values are represented by '1 .OO'. Left diagram: static firing rate; right diagram: dynamic peak frequency following 5°C cooling from adapting temperature. Circles, lingual cold receptors, isolated organ preparation, 0.5 mmol/l calcium (n = 11). data from Schafer (1987); diamonds, lingual cold receptors, isolated organ preparation, 50 pmol/l menthol (n = 3), data from Schafer et al. (1986); squares, nasal cold receptors, whole animal, 0.57 k 0.11 mg/kg/min menthol i.v. (n = 8). data from Schafer et al. (1986); triangles, nasal cold receptors, whole animal, 0.77 0.29 mg/kg/min EDTA i.v. (n = 9), data from Schafer et al. (1982). EDTA chelates calcium ions. Methods are given in the cited references.
*
33
associated with concomitant modifications of discharge rate (Gola and Selveston, 1981; Legendre et al., 1985). Activity of these neurons is controlled by calcium currents and by calcium-sensitive conductances. Cold receptor activity is comparably suppressed by excess external calcium and enhanced by reduced calcium (Schafer et al., 1982; Schafer, 1987). The considerable conformity of the calcium-induced changes of either the neuronal membrane potential or the sensory afferent discharge supports the view that the activity of both cell types is controlled by identical mechanisms, namely by calcium-stimulated outward conductances. Calcium seems not to be inA
15
volved in affecting the membrane potential as a current carrier, since elevation of external calcium neither induces depolarization nor increases afferent activity. The data indicate that a calciumactivated outward current is involved, which masks the direct effect of the calcium current by its considerably higher conductance (Akaike et al., 1978; Lux and Hofmeier, 1982). Figs. 2 and 3 show that the menthol-induced changes of cold receptor discharge are fairly identical to those induced by reduced external calcium. Both menthol and reduced calcium exert their effects on the mean firing rate by comparable modifications of cyclic receptor activity (Schafer et al.,
--
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.
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--
1 rnin
Fig. 3. Effect of reduced external calcium and of calcium entry blockers on mean discharge rate of cold receptors. Data from six lingual cold receptors out of a total of 43 which were studied. Isolated organ preparation. T, thermode temperature; discharge rate was averaged over periods of 10 s. The isolated organ preparation was perfused for the periods indicated by bars with solutions containing either reduced or elevated calcium levels (A, B), various menthol concentrations (C, D), or various verapamil concentrations (E, F). Under control conditions, the isolated organ preparation was perfused with a modified Krebs solution (for details, see Schafer, 1987).
34
1986; Schafer and Braun, unpublished). Since menthol has been proved to selectively impair the calcium entry, we compared menthol with the classical calcium entry blocker, verapamil. When tested in six specific cold receptors, either external verapamil in doses of 10 - 500 pmol/l was ineffective or cold receptor discharge was inhibited. In every case the observed effects were at variance with the expected stimulating action of compounds interfering with the calcium entry such as menthol (Fig. 3). Since verapamil is mainly effective in reducing current through the ubiquitous calcium channel (HVA type) (Boll and Lux, 1985), these data do not support the view of an action-potential-activated calcium conductance being present at the cold receptor membrane. Cyclic receptor activity, however, is strongly controlled by calcium. If calcium entry through the HVA-type conductance is negligible, the periodic receptor activity should be independent of the release of afferent impulses by the sensor. In fact, various analyses of the temporal pattern of cold receptor discharges have indicated the existence of an endogenously
instantaneous frequency
BP: k50 t 49 mscc
c
I5 O C
SBs6
BP.653 2 5 9 mscc
ISOC SB.7
BP.652 t 99msec
10% SB. 5 BP.799:
70mscc
10 o c SB. 8 BP.780 :79 mscc
Fig. 5 . Instantaneous frequency diagram of the temporal pattern of grouped (burst) discharges. Ordinate, instantaneous frequency in s - (reciprocal value of the duration of successive intervals); abscissa, time in ms. Data of a 2 min period at every constant temperature. BP, burst period (i.e., period of the underlying cyclic receptor events); SB, impulses per burst. Method of calculation is given in Braun et al. (1980).
’
Fig. 4.Temporal pattern of cold receptor discharge. Lingual cold receptor, isolated organ preparation. Perfusion with a solution containing 0.5 mmol/l calcium (for details, see Schafer, 1987). Thermode temperature, 40°C. (A) Impulse activity. (B) Duration of successive intervals. (C) Interval distribution. Arrows indicate the basic period of the cyclic receptor events and the even-numbered multiples. Values were calculated from the data presented in C (the method of calculation and examples are given in Braun et al., 1980; and in Schafer et al., 1986; respectively).
oscillating receptor process (Braun et al., 1980, 1984; Schafer et al., 1986). At higher adapting temperatures, these cyclic processes occasionally fail to initiate the appropriate impulse, which results in ‘irregular’ discharges. In that case, all interspike intervals are even-numbered multiples of the oscillation period, and sporadically intervals of a duration up to 8 times the oscillation period are seen (Fig. 4). At lower adapting temperatures, the periodic receptor processes mainly initiate groups of impulses during each cycle (Braun et al., 1980; Schafer et al., 1986). At a given temperature, burst period (i.e., oscillation period) and burst duration are fairly constant in spite of a varying number of
35
impulses induced per group (Fig. 5 ) . This additionally accords with the assumption that action potentials do not contribute to the initiation and maintenance of cyclic sensory processes. Moreover, it indicates that cyclic activity might be differently controlled in cold receptors and in molluscan bursting pacemaker neurons. In these neurons, which have been repeatedly proposed as models for cold receptor function (Braun et al., 1980, 1984; Schafer et al., 1982, 1986), the maintenance of cyclic activity evidently depends on the initiation of action potentials (Kramer and Zucker, 1985). Taken together, these various observations indicate that calcium channels of cold receptors are menthol-sensitive, but verapamil-insensitive, and that action potentials contribute only insignificantly, if at all, to the calcium entry into the sensor. The data strongly suggest that the cold receptor membrane possesses calcium channels with properties of the recently characterized LVA channel. Since the action of menthol is rather specific, it can be concluded that the N-type calcium conductance is absent in cold receptor membranes. In this connection it might be of interest that the LVA type of calcium channel is assumed to be involved in the maintenance of rhythmic neuronal activity (Llinas and Yarom, 1981). Therefore, for the first time, by application of indirect methods of investigation, it was possible to identify a type of ionic conductance involved in sensory transduction of thermoreceptors. Acknowledgement
This work was supported by the Deutsche Forschungsgemeinschaft . References Akaike, N., Lee, K.S. and Brown, A.M. (1978) The calcium current of Helix neuron. J. Gen. Physiol., 71: 509-531. Boll, W. and Lux, H.D. (1985) Action of organic antagonists on neuronal calcium currents. Neurosci. Lett., 56: 335 - 339. Braun, H.A. and Hensel, H. (1977) A computer program for identification and analysis of neuronal burst discharges. Pflugers Arch., 368: R48.
Braun, H.A., Bade, H. and Hensel, H. (1980) Static and dynamic discharge patterns of bursting cold fibres related to hypothetical receptor mechanisms. Pflugers Arch., 386: 1-9. Braun, H.A., Schafer, K., Wissig, H. and Hensel, H. (1984) Periodic transduction processes in thermosensitive receptors. In: W. Hamann and A. Iggo (Eds.), Sensory Receptor Mechanisms. World Sci. Publ. Corp., Singapore, pp. 147 - 156. Carbone, E. and Lux, H.D. (1984) A low voltage-activated calcium conductance in embryonic chick sensory neurons. Biophys. J . , .46: 413 -418. Eckert, R. and Lux, H.D. (1976) A voltage-sensitive persistent calcium conductance in neuronal somata of Helix. J . Physiol. (London), 254: 129- 152. Gola, M. and Selverston, A. (1981) Ionic requirements for bursting activity in lobster stomatogastric neurons. J . Comp. Physiol., 145: 191 -207. Gorman, A.L.F., Hermann, A. and Thomas, M.V. (1982) Ionic requirements for membrane oscillations and their dependence on the calcium concentration in a molluscan pacemaker neurone. J. Physiol. (London), 327: 185 -218. Hensel, H. (1981) Thermoreception and Temperature Regulafion. Academic Press, London. Hensel, H., Andres, K.H. and v. During, M. (1974) Structure and function of cold receptors. Pflugers Arch., 352: 1 - 10. Kramer, R.H. and Zucker, R.S. (1985) Calcium-induced inactivation of calcium current causes the interburst hyperpolarization of Aplysia bursting neurones. J . Physiol. (London), 362: 131 - 160. Legendre, P . , McKenzie, J.S., Dupouy, B. and Vincent, J.D. (1985) Evidence for bursting pacemaker neurones in cultured spinal cord cells. Neuroscience, 16: 753 - 767. Llinas, R. and Yarom, Y. (1981) Properties and distribution of ionic conductances generating electroresponsiveness of mammalian inferior olivary neurones in vitro. J . Physiol. (London), 315: 569-584. Lux, H.D. and Hofmeier, G. (1982) Properties of a calciumand voltage-activated potassium current in Helix pomatia neurons. Pflugers Arch., 394: 61 -69. Nowycky, M.C., Fox, A.P. and Tsien, R.W. (1985) Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature (London), 316: 440 - 443. Schafer, K. (1987) A quantitative study of the dependence of feline cold receptor activity o n the calcium concentration. Pflugers Arch., 409: 208-213. Schafer, K., Braun, H.A. and Hensel, H. (1982) Static and dynamic activity of cold receptors at various calcium levels. J. Neurophysiol., 47: 1017- 1028. Schafer, K., Braun, H.A. and Hensel, H. (1984) Temperature transduction in the skin. In: J.R.S. Hales (Ed.), Thermal Physiology, Raven Press, New York, pp. 1 - 11. Schafer, K., Braun, H.A. and Isenberg, C. (1986) Effect of menthol o n cold receptor activity. Analysis of receptor processes. J . Gen. Physiol., 88: 757 - 776.
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Schafer, K., Braun, H.A. and Kiirten, L. (1987) Analysis of discharge pattern of cold- and warm-receptor activity of vampire bats and mice. Verh. Dtsch. Zoo/. Ges., 80: 279. Swandulla, D., Schafer, K . and Lux, H.D. (1986) Calcium channel current inactivation is selectively modulated by men-
thol. Neurosci. Lett.. 68: 23 - 28. Swandulla, D., Carbone, E., Schafer, K. and Lux, H.D.(1987) Effect of menthol on two types of calcium currents in cultured sensory neurons of vertebrates. Pf/ugers Arch., 409: 52 - 59.