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Intracellular studies on the role of calcium in regulating the activity and reactivity of locus coeruleus neurons in vivo
Brain Research, 273 (1983) 237-243 Elsevier
237
Intracellular Studies on the Role of Calcium in Regulating the Activity and Reactivity of Locus Coeruleus Neurons In Vivo G. K. AGHAJANIAN, C. P. VANDERMAELEN and R. ANDRADE Departments of Psychiatry and Pharmacology, Yale University School of Medicine, and the Connecticut Mental Health Center, 34 Park Street, New Haven, CT06508 (U.S.A.) (Accepted January llth, 1983) Key words: a2-adrenoceptors - - afterhyperpolarization - - calcium - - intracellular EGTA - locus coeruleus - - noradrenergic neuron - - potassium
EGTA, a specific calcium chelator, was injected intracellularly into presumed noradrenergic neurons of the rat locus coeruleus to evaluate the importance of calcium-dependent processes in regulating the activity and reactivity of these cells in vivo. The amplitude and duration of postactivation afterhyperpolarizations induced by intracellular depolarizing pulses were markedly reduced in EGTAtreated cells; this change was associated with: (1) an increase in spontaneous firing rate; (2) a reduction in postactivation inhibition of firing; and (3) an increased reactivity to sensory stimulation. In control cells the reversal potential of the afterhyperpolarization was at least 25 mV below 'resting' levels, indicating that an increase in potassium conductance was probably involved. Since EGTA virtually abolished the afterhyperpolarization, the data are consistent with the concept that the afterhyperpolarization is mediated by a calalum-activated potassium current. A calcium-dependent release of norepinephrine acting via a2-adrenoceptors might also contribute to the afterhyperpolarization. In conclusion, the influx of calcium into locus coeruleus neurons appears to serve a negative feedback function in the regulation of both spontaneous activity and reactivity to orthodromic stimulation.
INTRODUCTION Previous intracellular studies in vivo have shown that central noradrenergic neurons of the locus coeruleus undergo a prolonged afterhyperpolarization ( A H P ) when they are activated either by intracellular depolarizing pulses 2 or orthodromic stimulation 3. In spontaneously firing locus coeruleus neurons, the postactivation A H P is associated with a marked inhibition of tonic activity (i.e. postactivation inhibition). The late stages of the A H P can be attenuated by an a2-adrenoceptor antagonist, suggesting a role for a2-autoreceptors in mediating postactivation inhibitions 2. However, the early phase of the A H P is relatively resistant to a2-adrenoceptor blockade indicating that a mechanism in addition to a2-autoinhibition may be involved. A likely candidate would be a calcium-activated potassium current (IK(ca)) , triggered by the entry of Ca 2÷ into the cell through voltage-dependent Ca2+-channels during bursts of activity20,21. A H P s produced by a Ca2+-activated IK have been reported in a number of vertebrate 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
neurons including motoneurons6A5A6 hippocampal pyramidal cells4.13,22 cerebellar Purkinje cells 17 and inferior olivary neurons 18. In several instances, the calcium dependence of A H P s has been demonstrated by the intracellular injection of E G T A (ethyleneglycol-bis(fl-aminoethyl ether)- N,N'-tetra-acetic acid), which is highly specific in binding free calcium. This treatment virtually abolishes the Ca2+-dependent A H P and can lead to prolonged cell discharge12.14A6,21,23. In the present study, E G T A was injected into locus coeruleus neurons to evaluate the possible role of Ca 2+-dependent processes in mediating A H P s and postactivation inhibitions in these cells. METHODS Male albino rats (Charles River) weighing 275-325 g were anesthetized with chloral hydrate (400 mg/kg, i.p.) and m o u n t e d in a stereotaxic frame; body temperature was maintained at 35-37 *C. Micropipettes were pulled from 2 m m glass tubing (Pyrex). The tu-
238 bing had been preloaded with several filaments of fiberglass to promote filling by capillary action24. The micropipettes were filled with a solution of potassium acetate (1.0 M, pH 7.0) or 0.8 M potassium acetate containing 0.2 M potassium EGTA. Electrodes were then double bevelled by the thick slurry method 19. The second bevel was made at approximately a 45--60° turn from the initial bevel to give a final impedance of 18-34 MQ. The double bevelling helped prevent electrode blocking during passage through deep tissue, and facilitated passage of current. Because of highly unstable recording conditions in the locus coeruleus in vivo, 4 stabilizing pins (0 Clay Adams insect pins) were implanted 1.5-2 mm apart in a square pattern in the brain around the recording site (cf. ref. 2). The pins served to reduce the local transmission of pulsations and other movements. To obtain intracellular recordings from locus coeruleus neurons, a burr hole was made 1.2 mm posterior to lambda and 1.1 mm lateral to the midline. As described previously1, practical aids in finding the locus coeruleus included: (1) depth of 5.5-6.5 mm below brain surface; (2) a zone of relative electrical silence ventral to the cerebellum and dorsal to the locus coeruleus representing the fourth ventricle; (3) the presence just lateral to the locus coeruleus of cells of the mesencephalic nucleus of n. V, which are activated by displacement of the mandible; and (4) in the locus coeruleus itself, a closely packed population of slowly firing cells (1--4 spikes/s) all responding to noxious stimulation by a burst of firing followed by a long quiescent period (1-2 s). Two of the 7 control cells included for data analysis in this study were taken from a previous study in which cells meeting the above criteria had been definitively identified as noradrenergic locus coeruleus neurons by means of intracellular double-labeling techniques 3. Intracellular potentials were amplified by a W-P Instrument M707 amplifier, displayed on an oscilloscope, and stored on FM tape (frequency response of 0-1250 Hz). The animal was grounded through a NaCI salt bridge connected to a Ag-AgCI wire and the recording electrode was connected to the input stage of the amplifier by an Ag-AgCI wire. Current could be injected intracellularly through the recording electrode via a bridge circuit. Locus coeruleus neurons were impaled either by the passage of a DC pulse (e.g. 20 V, 2 ms) through the 'break-away box'
of the M707 amplifier or by briefly inducing 'ringing' via the capacity compensation potentiometer. Input resistance was calculated from voltage deflections caused by constant current pulses passed through the recording electrode via the bridge circuit. The bridge was balanced intracellularly prior to resistance measurements. Storage oscilloscope traces were used for the figures, taken on line or reproduced from the FM tape. Statistical analysis was by means of Student's t-test. RESULTS A total of 14 locus coeruleus neurons met criteria for inclusion within the data sample. Acceptable cells had action potentials greater than 60 mV and retained normal spontaneous activity for extended periods (30 min to 2 h) following impalement. Seven of these cells were recorded with electrodes containing 1.0 M potassium acetate; in the remaining 7 cases, the electrodes contained 0.8 M potassium acetate plus 0.2 M potassium EGTA. Initially, most cells were activated due to irritation caused by the impalement; in such cases 1-2 nA of hyperpolarizing current was applied for 5-10 min to facilitate recovery. In general, when 1.0 M potassium acetate-containing electrodes were employed, approximately half of the impaled cells became deeply hyperpolarized (i.e. towards ---80 mV) and spontaneous activity was lost (such cells were not included in the present sample). A similar phenomenon, attributed to the development of high potassium conductance due to calcium loading, has been observed in rat sympathetic neurons 11. No cells impaled with potassium EGTAcontaining electrodes displayed a similar phenomenon. Of course, considerable amounts of EGTA, which is negatively charged, would have entered these cells by the application of hyperpolarizing current during the recovery period, as well as through leakage at later times 16. For these reasons, the obtaining of pre-EGTA data was not practical when EGTA-containing electrodes were used. Table I compares the characteristics of cells from potassium acetate-and EGTA-containing electrodes according to (1) spike amplitude, (2) spontaneous firing rate (determined after stabilization of membrane potential), (3) membrane potential (Vm, taken as the difference between extraceUular potential and the pla-
239
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r-q Fig. 1. Postactivation AHPs induced by intracellular depolarizing pulses: comparisons between AHPs when electrodes containing potassium acetate ('control electrode') versus potassium acetate-EGTA ('EGTA electrode') were used. In A, when a control electrode was used, there was a prolonged AHP following a burst of 6 spikes induced by a 1.2 nA depolarizing pulse; a return to 'resting' potential (ef. superimposed control sweep) was not complete within the period of time shown. In B when an EGTA electrode was used, the AHP, (also following a burst of 6 spikes) was greatly attenuated both in amplitude and duration (1.5 nA depolarizing pulse). The sweeps selected for display in A and B contained no spontaneous spikes to simplify the comparisons (however, of. Fig. 3). In C, in a cell recorded with a control electrode, superimposed traces were obtained following a series of hyperpolarizing pulses (1, 2, 3 and 4 nA) applied before and after a constant depolarizing pulse (2 hA); the resulting IV data for the preburst (open circles) and postburst (closod circles) voltage deflections was plotted in D, giving an apparent reversal potential for the AHP of between ----90and---95 mV (of. ref. 16). The linearity of the plots show a single slope resistance indicating ohmic properties of the membrane in this region. Current monitor traces are shown beneath intracellular traces.
teau region of the interspike interval); and (4) input resistance (Rinp~t). With respect to most of these measures, the two sets of cells were quite similar; however, spontaneous activity was significantly greater in the E G T A group. The most striking differences between the control and E G T A groups were in (1) the magnitude and duration of the A H P , (2) the duration of postactivation inhibition, and (3) the response to orthodromic stimulation. As illustrated in Fig. 1A, following a burst of spikes induced by an intracellular depolarizing pulse there was a prolonged A H P in a cell recorded with a potassium acetate-containing electrode; the A H P was almost totally absent in another cell in which an
E G T A electrode had been used (Fig. 1B). The mean peak amplitude of A H P was significantly greater in the 7 controls than in the 7 E G T A cells (8.5 vs 5.7 mV; P < 0.01) and the mean duration of A H P was much greater in the control than in the E G T A - t r e a ted cells (1520 + 780 S.D., vs 44 ms, + 17 S.D.; P < 0.001). Interestingly, in the E G T A treated cells, the A H P after a burst of spikes was not greater than after single spikes (cf. Fig. 3B). Because of its short duration the reversal potential for the A H P could not be determined after E G T A (cf. Fig. 3B). With 1.0 M potassium acetate-containing electrodes the reversal potential of the A H P was always at least 25 m V in the hyperpolarizing direction (Fig. 1C, D). A prolonged
240
A
B
Fig. 2. Attenuation of postactivation inhibition of firing by EGTA treatment. In A, a prolonged inhibition of firing (~2 s) occurred in a control cell following a burst of 6 spikes induced by a depolarizing intracellular pulse (1.0 hA) B shows the lack of substantial postactivation inhibition in an EGTA-treated cell following 2 bursts of 6 spikes (depolarizing pulse 1.5 nA); note that the intervals following the bursts were not longer than some of the intervals occurring between spontaneous spikes. This EGTA-treated cell had been impaled for about I h at the time of testing; the degree of attenuation of postactivation inhibition was progressive from early time points up to this 1 h sampling time.
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Fig. 3. Enhanced response to noxious stimulation in EGTA-treated cells. A~ shows a typical postactivation AHP in a control cell (0.5 nA hyperpolarizing pulses, 2.3 nA depolarizing pulse); A 2 shows the response of the same cell to steady pressure (arrow) applied to the contralateral hind paw by a smooth-ffled a!ii~ator spring clip; the response consisted of only a few initial spikes. B i shows an EGTA-treated cell in which the postactivation AHP was markedly attenuated (0.25 nA hyperpolarlzing pulses, 1.6 nA depolarizing pulse); B 2 shows a persistent train of spikes in this cell when a spring clip was applied to the contralateral hind paw. In A l and B 1superimposed control sweeps (i.e. no intracellular pulses) are shown.
241 TABLE I Electrode solution
Number of cells
Spike amplitude
Spontaneous firing rate*
Vm
R1
Potassium acetate 1.0 M Potassium acetate 0.8 + Potassium EGTA 0.2 M
7
72 mV (range, 66-80) 72 mV
0.95 spikes/s (range,0.1-2.5) 3.8 spikes/s
--57 mV (+2.4, S.D.) --55 V
25 M (+ 10, S.D.) 32 M
(range, 63-87)
(range, 0.4--10)
(+2.8, S.D.)
(+ 14, S.D.)
7
* P < 0.025. postactivation inhibition of firing occurred in association with AHPs when 1.0 M potassium acetate-containing electrodes were used (Fig. 2A). However, when EGTA-containing electrodes were used, postactivation inhibitions were almost totally abolished (Fig. 2B). In addition, the orthodromic activation of locus coeruleus neurons by noxious stimulation became greatly enhanced by E G T A treatment. For example, in all control cells, only a few initial spikes could be elicited by the steady application of pressure (spring clip) to the contralateral hind paw (Fig. 3A2); in contrast, the same steady pressure induced a sustained depolarization and a persistent train of spikes in all of the E G T A cells (Fig. 3B2). In two of the EGTA-treated cells the a2-adrenoceptor agonist clonidine was administered intraperitoneally (100 gg/kg); in one case an 8 mV and in the other a 10 mV hyperpolarization was produced. In both cells a total inhibition of spontaneous activity was associated with the clonidine-induced hyperpolarization. DISCUSSION The results of the present study provide evidence that calcium is important for regulating locus coeruleus neurons both with respect to spontaneous activity and reactivity to stimulation. Intracellular injection of the specific calcium chelator E G T A greatly attenuated AHPs normally found following bursts of locus coeruleus cell activity. This result corresponds to the effects of intracellular E G T A reported for various other invertebrate and vertebrate neurons (cf. Introduction). Thus, after E G T A treatment, postactivation AHPs were typically less than 50 ms in duration, compared to AHPs often lasting more than 1500 ms in control cells. The finding that E G T A caused a reduction in duration (as well as amplitude) of the AHP fits with the concept that intracellular free cal-
cium levels, which would be increased by the opening of voltage-dependent Ca 2+ channels during bursts of activity, are efficiently and rapidly reduced by the chelation process. The fact that the reversal potential of the AHP was strongly in the negative direction is consistent with the interpretation that the hyperpolarizing afterpotential per se is caused by an outward potassium current, as has been shown previously for various invertebrateT,21 and vertebrate neurons (of. ref. 10). This interpretation is further supported by the finding that the AHP of locus coeruleus neurons is not reversed by the use of chloride-containing intracellular electrodes either in vivo2 or in vitro in brain slices5. Taken together these data indicate that the AHP in locus coeruleus cells is mediated, at least in part, by a Ca2+-activated I K. The functional consequences of treating locus coeruleus cells with E G T A were threefold: (1) rate of spontaneous firing was increased; (2) postactivation inhibition of firing was reduced; and (3) reactivity to orthodromic activation was enhanced. It is possible to account for all of these effects in terms of an interference with the normal Ca2+-activated Ii~. In general, spontaneous repetitive firing (automaticity) can be explained by a decay in net IK during the latter part of the interspike interval, which together with a reactivation of inward current mechanisms leads to the succeeding spike9. In certain molluscan neurons with a very low rate of repetitive firing (e.g. 1 spike/s) the major influence on interspike interval has been shown to be a Ca2+-activated potassium current 22. The reason this mechanism may be particularly critical for regulating cells with slow rates of discharge is that Ca 2÷ diffusion and sequestration are relatively slow processes compared to changes in predominately voltage-dependent K ÷ channels. If such a mechanism holds for noradrenergic neurons of the locus coeruleus, then both their slow firing rate and auto-
242 maticity5 could be explained in part by a slow decay in a Ca2+-activated I K. A m o r e rapid removal of intracellular free calcium by E G T A would then be expected to increase spontaneous firing rate, as was observed in the present experiments. This basic concept can be e x t e n d e d to account for the ability of E G T A to reduce postactivation inhibitions and to increase the o r t h o d r o m i c reactivity of locus coeruleus neurons. O u r observations on locus coeruleus neurons are consistent with the suggestion of Meech 21, based on studies in invertebrate neurons, that 'it seems likely that this calcium-mediated negative f e e d b a c k mechanism is a feature of neural processing in many animals'. This discussion has focussed primarily on the possibility that a Ca2+-activated IK may underly A H P s in the locus coeruleus. H o w e v e r , a n o t h e r CaZ+-dependent process which could be affected by E G T A is that of the release of transmitter involved in autoinhibition. N o r a d r e n e r g i c neurons possess receptors in their somatodendritic region towards their own transmitter s. These a u t o r e c e p t o r s , which are of the a2-type, could contribute to the A H P if there were a postactivation Ca2+-dependent release of norepinephrine from locus coeruleus neurons onto themselves 2. Preliminary e x p e r i m e n t s in the present
study indicate that the direct stimulation of autoreceptors by the exogenous a 2 - a d r e n o c e p t o r agonist clonidine hyperpolarizes locus coeruleus neurons despite E G T A treatment. The degree of hyperpolarization p r o d u c e d by clonidine in the E G T A - t r e a t e d cells was similar to that previously r e p o r t e d for the effect of clonidine on locus coeruleus neurons when nonE G T A - c o n t a i n i n g electrodes were usedL This suggests that hyperpolarizations p r o d u c e d by the direct stimulation of a2-adrenoceptors in the locus coeruleus are not Ca2+-dependent, On the o t h e r hand, since the release of e n d o g e n o u s agonist (e.g. from a locus coeruleus neuron onto itself) would be Ca 2÷d e p e n d e n t , a 2 - a d r e n o c e p t o r - m e d i a t e d A H P s in the locus coeruleus could be indirectly d e p e n d e n t on calcium. The relative importance of h y p e r p o l a r i z a t i o n s induced by the Ca2+-activated IK as c o m p a r e d to those m e d i a t e d by a2-adrenoceptors in the locus coeruleus remains to be d e t e r m i n e d . ACKNOWLEDGEMENTS W e thank L. Fields, N. M a r g i o t t a , and A . Z i m n e wicz for their valuable assistance in this work. Supp o r t e d by U S P H S G r a n t s MH-17871, MH-14276, GM-07324, and the State of Connecticut.
REFERENCES 1 Aghajanian, G. K., Cedarbaum, J. M. and Wang, R. Y., Evidence for norepinephrine mediated collateral inhibition of locus coeruleus neurons, Brain Research, 136 (1977) 570-577. 2 Aghajanian, G. K. and VanderMaelen, C. P., a2-Adrenoceptor-mediated hyperpolarization of locus coeruleus neurons: intracellular studies in vivo, Science, 215 (1982) 1394-1396. 3 Aghajanian, G. K. and VanderMaelen, C. P., Intraceilular identification of central noradrenergic and serotonergic neurons by a new double labeling procedure, J. Neurosci., 2 (1982) 1786-1792. 4 Alger, B. E. and Nicoll, R. A., Epileptiform burst afterhyperpolarization: calcium-dependent potassium potential in hippocampal CA1 pyramidal cells, Science, 210 (1980) 1122-1124. 5 Andrade, R., VanderMaelen, C. P. and Aghajanian, G. K., Locus coeruleus neurons: spontaneous activity in brain slices from naive and morphine-dependent rats, Soc. Neurosci. Abstr., 8 (1982) 922. 6 Barrett, E. F. and Barrett, J. N., Separation of two voltagesensitive potassium currents, and demonstration of a tetrodotoxin-resistant calcium current in frog motoneurons, J. Physiol. (Lond.), 255 (1976) 737-774. 7 Brodwick, M. S. and Jung, D., Post-stimulus hyperpolari-
8
9 10 11 12 13 14
15
zation and slow potassium conductance increase in Aplysia giant neurone, J. Physiol. (Lond.), 233 (1972) 549-570. Cedarbaum, J. M. and Aghajanian, G. K., Catecholamine receptors on locus coeruleus neurons: pharmacological characterization, Europ. J. Pharmacol. , 44 (1977) 375-385. Connor, J. A. and Stevens, C. F., Prediction of repetitive firing behavior from voltage clamp data on an isolated neurone soma, J. Physiol. (Lond.), 213 (1971) 31-53. Eccles, J. C., The Physiology of Synapses, Springer, New York, 1964. Galvan, M. and Adams, P. R., Control of calcium current in rat sympathetic neurons by norepinephrine, Brain Research, 244 (1982) 135-144. Hablitz, J. J., Altered burst responses in hippocampal CA3 neurons injected with EGTA, Exp. Brain Res., 42 (198t) 483--485. Hotson, J. F. and Prince, D. A., A calcium-activated hyperpolarization follows repetitive firing in hippocampal neurons, J. NeurophysioL, 43 (1980) 409--419. Kostyuk, P. G. and Krishtal, O. A., Effects of calcium and calcium-chelating agents on the inward and outward currents in the membrane of mollusc neurons, J. Physol. (Lond.), 270 (1977) 569--580. Krnjevi~ K. and Lisiewicz, A., Injections of calcium ions into spinal motoneurons, J. Physiol. (Lond.), 225 (1972) 363-390.
243 16 Krnjevi~, K., Puil, E. and Werman, R., EGTA and motoneuronal afterpotentials, J. Physiol. (Lond.), 275 (1978) 199-223. 17 Llin~is, R. and Sugimori, M., Eiectrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices, J. Physiol. (Lond.), 305 (1980) 171-195. 18 Llimis, R. and Yarom, Y., Electrophysiology of mammalian inferior olivary neurons in vitro: different types of voltage-dependent ionic conductances, J. Physiol. (Lond.), 315 (1981) 549-567. 19 Lederer, W. J., Spindler, A. J., and Eisner, D. A., Thick slurry bevelling: a new technique for bevelling extremely fine microelectrodes and micropipettes, Pfliigers Arch., 381 (1979) 287-288. 20 Meech, R. W., Intracellular calcium injection causes in-
21 22 23
24
creased potassium conductance in Aplysia nerve cells, Comp. Biochem. Physiol., 42A (1972) 493--499. Meech, R. W., Calcium influx induces a post-tetanic hyperpolarization in Aplysia neurons, Comp. Biochem. Physiol., 48A (1974) 387-395. Patridge, D. L., Current-voltage hysteresis and repetitive firing in molluscan neurons, Soc. Neurosci. Abstr., 7 (1981) 904. Schwartzkroin, P. A. and Stafstrom, C. E., Effects of EGTA on calcium-activated afterhyperpolarization in hippocampal CA3 pyramidal cells, Science, 210 (1980) 1125-1126. Tasaki, K., Tsukahara, Y., Ho, S., Wayner, M. J. and Yu, W. Y., A simple, direct and rapid method for filling microelectrodes, Physiol. Behav., 3 (1968) 1009-1010.
237
Intracellular Studies on the Role of Calcium in Regulating the Activity and Reactivity of Locus Coeruleus Neurons In Vivo G. K. AGHAJANIAN, C. P. VANDERMAELEN and R. ANDRADE Departments of Psychiatry and Pharmacology, Yale University School of Medicine, and the Connecticut Mental Health Center, 34 Park Street, New Haven, CT06508 (U.S.A.) (Accepted January llth, 1983) Key words: a2-adrenoceptors - - afterhyperpolarization - - calcium - - intracellular EGTA - locus coeruleus - - noradrenergic neuron - - potassium
EGTA, a specific calcium chelator, was injected intracellularly into presumed noradrenergic neurons of the rat locus coeruleus to evaluate the importance of calcium-dependent processes in regulating the activity and reactivity of these cells in vivo. The amplitude and duration of postactivation afterhyperpolarizations induced by intracellular depolarizing pulses were markedly reduced in EGTAtreated cells; this change was associated with: (1) an increase in spontaneous firing rate; (2) a reduction in postactivation inhibition of firing; and (3) an increased reactivity to sensory stimulation. In control cells the reversal potential of the afterhyperpolarization was at least 25 mV below 'resting' levels, indicating that an increase in potassium conductance was probably involved. Since EGTA virtually abolished the afterhyperpolarization, the data are consistent with the concept that the afterhyperpolarization is mediated by a calalum-activated potassium current. A calcium-dependent release of norepinephrine acting via a2-adrenoceptors might also contribute to the afterhyperpolarization. In conclusion, the influx of calcium into locus coeruleus neurons appears to serve a negative feedback function in the regulation of both spontaneous activity and reactivity to orthodromic stimulation.
INTRODUCTION Previous intracellular studies in vivo have shown that central noradrenergic neurons of the locus coeruleus undergo a prolonged afterhyperpolarization ( A H P ) when they are activated either by intracellular depolarizing pulses 2 or orthodromic stimulation 3. In spontaneously firing locus coeruleus neurons, the postactivation A H P is associated with a marked inhibition of tonic activity (i.e. postactivation inhibition). The late stages of the A H P can be attenuated by an a2-adrenoceptor antagonist, suggesting a role for a2-autoreceptors in mediating postactivation inhibitions 2. However, the early phase of the A H P is relatively resistant to a2-adrenoceptor blockade indicating that a mechanism in addition to a2-autoinhibition may be involved. A likely candidate would be a calcium-activated potassium current (IK(ca)) , triggered by the entry of Ca 2÷ into the cell through voltage-dependent Ca2+-channels during bursts of activity20,21. A H P s produced by a Ca2+-activated IK have been reported in a number of vertebrate 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
neurons including motoneurons6A5A6 hippocampal pyramidal cells4.13,22 cerebellar Purkinje cells 17 and inferior olivary neurons 18. In several instances, the calcium dependence of A H P s has been demonstrated by the intracellular injection of E G T A (ethyleneglycol-bis(fl-aminoethyl ether)- N,N'-tetra-acetic acid), which is highly specific in binding free calcium. This treatment virtually abolishes the Ca2+-dependent A H P and can lead to prolonged cell discharge12.14A6,21,23. In the present study, E G T A was injected into locus coeruleus neurons to evaluate the possible role of Ca 2+-dependent processes in mediating A H P s and postactivation inhibitions in these cells. METHODS Male albino rats (Charles River) weighing 275-325 g were anesthetized with chloral hydrate (400 mg/kg, i.p.) and m o u n t e d in a stereotaxic frame; body temperature was maintained at 35-37 *C. Micropipettes were pulled from 2 m m glass tubing (Pyrex). The tu-
238 bing had been preloaded with several filaments of fiberglass to promote filling by capillary action24. The micropipettes were filled with a solution of potassium acetate (1.0 M, pH 7.0) or 0.8 M potassium acetate containing 0.2 M potassium EGTA. Electrodes were then double bevelled by the thick slurry method 19. The second bevel was made at approximately a 45--60° turn from the initial bevel to give a final impedance of 18-34 MQ. The double bevelling helped prevent electrode blocking during passage through deep tissue, and facilitated passage of current. Because of highly unstable recording conditions in the locus coeruleus in vivo, 4 stabilizing pins (0 Clay Adams insect pins) were implanted 1.5-2 mm apart in a square pattern in the brain around the recording site (cf. ref. 2). The pins served to reduce the local transmission of pulsations and other movements. To obtain intracellular recordings from locus coeruleus neurons, a burr hole was made 1.2 mm posterior to lambda and 1.1 mm lateral to the midline. As described previously1, practical aids in finding the locus coeruleus included: (1) depth of 5.5-6.5 mm below brain surface; (2) a zone of relative electrical silence ventral to the cerebellum and dorsal to the locus coeruleus representing the fourth ventricle; (3) the presence just lateral to the locus coeruleus of cells of the mesencephalic nucleus of n. V, which are activated by displacement of the mandible; and (4) in the locus coeruleus itself, a closely packed population of slowly firing cells (1--4 spikes/s) all responding to noxious stimulation by a burst of firing followed by a long quiescent period (1-2 s). Two of the 7 control cells included for data analysis in this study were taken from a previous study in which cells meeting the above criteria had been definitively identified as noradrenergic locus coeruleus neurons by means of intracellular double-labeling techniques 3. Intracellular potentials were amplified by a W-P Instrument M707 amplifier, displayed on an oscilloscope, and stored on FM tape (frequency response of 0-1250 Hz). The animal was grounded through a NaCI salt bridge connected to a Ag-AgCI wire and the recording electrode was connected to the input stage of the amplifier by an Ag-AgCI wire. Current could be injected intracellularly through the recording electrode via a bridge circuit. Locus coeruleus neurons were impaled either by the passage of a DC pulse (e.g. 20 V, 2 ms) through the 'break-away box'
of the M707 amplifier or by briefly inducing 'ringing' via the capacity compensation potentiometer. Input resistance was calculated from voltage deflections caused by constant current pulses passed through the recording electrode via the bridge circuit. The bridge was balanced intracellularly prior to resistance measurements. Storage oscilloscope traces were used for the figures, taken on line or reproduced from the FM tape. Statistical analysis was by means of Student's t-test. RESULTS A total of 14 locus coeruleus neurons met criteria for inclusion within the data sample. Acceptable cells had action potentials greater than 60 mV and retained normal spontaneous activity for extended periods (30 min to 2 h) following impalement. Seven of these cells were recorded with electrodes containing 1.0 M potassium acetate; in the remaining 7 cases, the electrodes contained 0.8 M potassium acetate plus 0.2 M potassium EGTA. Initially, most cells were activated due to irritation caused by the impalement; in such cases 1-2 nA of hyperpolarizing current was applied for 5-10 min to facilitate recovery. In general, when 1.0 M potassium acetate-containing electrodes were employed, approximately half of the impaled cells became deeply hyperpolarized (i.e. towards ---80 mV) and spontaneous activity was lost (such cells were not included in the present sample). A similar phenomenon, attributed to the development of high potassium conductance due to calcium loading, has been observed in rat sympathetic neurons 11. No cells impaled with potassium EGTAcontaining electrodes displayed a similar phenomenon. Of course, considerable amounts of EGTA, which is negatively charged, would have entered these cells by the application of hyperpolarizing current during the recovery period, as well as through leakage at later times 16. For these reasons, the obtaining of pre-EGTA data was not practical when EGTA-containing electrodes were used. Table I compares the characteristics of cells from potassium acetate-and EGTA-containing electrodes according to (1) spike amplitude, (2) spontaneous firing rate (determined after stabilization of membrane potential), (3) membrane potential (Vm, taken as the difference between extraceUular potential and the pla-
239
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v
r-q Fig. 1. Postactivation AHPs induced by intracellular depolarizing pulses: comparisons between AHPs when electrodes containing potassium acetate ('control electrode') versus potassium acetate-EGTA ('EGTA electrode') were used. In A, when a control electrode was used, there was a prolonged AHP following a burst of 6 spikes induced by a 1.2 nA depolarizing pulse; a return to 'resting' potential (ef. superimposed control sweep) was not complete within the period of time shown. In B when an EGTA electrode was used, the AHP, (also following a burst of 6 spikes) was greatly attenuated both in amplitude and duration (1.5 nA depolarizing pulse). The sweeps selected for display in A and B contained no spontaneous spikes to simplify the comparisons (however, of. Fig. 3). In C, in a cell recorded with a control electrode, superimposed traces were obtained following a series of hyperpolarizing pulses (1, 2, 3 and 4 nA) applied before and after a constant depolarizing pulse (2 hA); the resulting IV data for the preburst (open circles) and postburst (closod circles) voltage deflections was plotted in D, giving an apparent reversal potential for the AHP of between ----90and---95 mV (of. ref. 16). The linearity of the plots show a single slope resistance indicating ohmic properties of the membrane in this region. Current monitor traces are shown beneath intracellular traces.
teau region of the interspike interval); and (4) input resistance (Rinp~t). With respect to most of these measures, the two sets of cells were quite similar; however, spontaneous activity was significantly greater in the E G T A group. The most striking differences between the control and E G T A groups were in (1) the magnitude and duration of the A H P , (2) the duration of postactivation inhibition, and (3) the response to orthodromic stimulation. As illustrated in Fig. 1A, following a burst of spikes induced by an intracellular depolarizing pulse there was a prolonged A H P in a cell recorded with a potassium acetate-containing electrode; the A H P was almost totally absent in another cell in which an
E G T A electrode had been used (Fig. 1B). The mean peak amplitude of A H P was significantly greater in the 7 controls than in the 7 E G T A cells (8.5 vs 5.7 mV; P < 0.01) and the mean duration of A H P was much greater in the control than in the E G T A - t r e a ted cells (1520 + 780 S.D., vs 44 ms, + 17 S.D.; P < 0.001). Interestingly, in the E G T A treated cells, the A H P after a burst of spikes was not greater than after single spikes (cf. Fig. 3B). Because of its short duration the reversal potential for the A H P could not be determined after E G T A (cf. Fig. 3B). With 1.0 M potassium acetate-containing electrodes the reversal potential of the A H P was always at least 25 m V in the hyperpolarizing direction (Fig. 1C, D). A prolonged
240
A
B
Fig. 2. Attenuation of postactivation inhibition of firing by EGTA treatment. In A, a prolonged inhibition of firing (~2 s) occurred in a control cell following a burst of 6 spikes induced by a depolarizing intracellular pulse (1.0 hA) B shows the lack of substantial postactivation inhibition in an EGTA-treated cell following 2 bursts of 6 spikes (depolarizing pulse 1.5 nA); note that the intervals following the bursts were not longer than some of the intervals occurring between spontaneous spikes. This EGTA-treated cell had been impaled for about I h at the time of testing; the degree of attenuation of postactivation inhibition was progressive from early time points up to this 1 h sampling time.
B1
A1
l~ms
_ Ji . ~ .~ E Ir
loom, f" .
.
.
~
~.~J
llllll
.
i
l.i,,i.l
L----..--,I
A2
IIIllW" ~
B2
v
o,s t
i
Fig. 3. Enhanced response to noxious stimulation in EGTA-treated cells. A~ shows a typical postactivation AHP in a control cell (0.5 nA hyperpolarizing pulses, 2.3 nA depolarizing pulse); A 2 shows the response of the same cell to steady pressure (arrow) applied to the contralateral hind paw by a smooth-ffled a!ii~ator spring clip; the response consisted of only a few initial spikes. B i shows an EGTA-treated cell in which the postactivation AHP was markedly attenuated (0.25 nA hyperpolarlzing pulses, 1.6 nA depolarizing pulse); B 2 shows a persistent train of spikes in this cell when a spring clip was applied to the contralateral hind paw. In A l and B 1superimposed control sweeps (i.e. no intracellular pulses) are shown.
241 TABLE I Electrode solution
Number of cells
Spike amplitude
Spontaneous firing rate*
Vm
R1
Potassium acetate 1.0 M Potassium acetate 0.8 + Potassium EGTA 0.2 M
7
72 mV (range, 66-80) 72 mV
0.95 spikes/s (range,0.1-2.5) 3.8 spikes/s
--57 mV (+2.4, S.D.) --55 V
25 M (+ 10, S.D.) 32 M
(range, 63-87)
(range, 0.4--10)
(+2.8, S.D.)
(+ 14, S.D.)
7
* P < 0.025. postactivation inhibition of firing occurred in association with AHPs when 1.0 M potassium acetate-containing electrodes were used (Fig. 2A). However, when EGTA-containing electrodes were used, postactivation inhibitions were almost totally abolished (Fig. 2B). In addition, the orthodromic activation of locus coeruleus neurons by noxious stimulation became greatly enhanced by E G T A treatment. For example, in all control cells, only a few initial spikes could be elicited by the steady application of pressure (spring clip) to the contralateral hind paw (Fig. 3A2); in contrast, the same steady pressure induced a sustained depolarization and a persistent train of spikes in all of the E G T A cells (Fig. 3B2). In two of the EGTA-treated cells the a2-adrenoceptor agonist clonidine was administered intraperitoneally (100 gg/kg); in one case an 8 mV and in the other a 10 mV hyperpolarization was produced. In both cells a total inhibition of spontaneous activity was associated with the clonidine-induced hyperpolarization. DISCUSSION The results of the present study provide evidence that calcium is important for regulating locus coeruleus neurons both with respect to spontaneous activity and reactivity to stimulation. Intracellular injection of the specific calcium chelator E G T A greatly attenuated AHPs normally found following bursts of locus coeruleus cell activity. This result corresponds to the effects of intracellular E G T A reported for various other invertebrate and vertebrate neurons (cf. Introduction). Thus, after E G T A treatment, postactivation AHPs were typically less than 50 ms in duration, compared to AHPs often lasting more than 1500 ms in control cells. The finding that E G T A caused a reduction in duration (as well as amplitude) of the AHP fits with the concept that intracellular free cal-
cium levels, which would be increased by the opening of voltage-dependent Ca 2+ channels during bursts of activity, are efficiently and rapidly reduced by the chelation process. The fact that the reversal potential of the AHP was strongly in the negative direction is consistent with the interpretation that the hyperpolarizing afterpotential per se is caused by an outward potassium current, as has been shown previously for various invertebrateT,21 and vertebrate neurons (of. ref. 10). This interpretation is further supported by the finding that the AHP of locus coeruleus neurons is not reversed by the use of chloride-containing intracellular electrodes either in vivo2 or in vitro in brain slices5. Taken together these data indicate that the AHP in locus coeruleus cells is mediated, at least in part, by a Ca2+-activated I K. The functional consequences of treating locus coeruleus cells with E G T A were threefold: (1) rate of spontaneous firing was increased; (2) postactivation inhibition of firing was reduced; and (3) reactivity to orthodromic activation was enhanced. It is possible to account for all of these effects in terms of an interference with the normal Ca2+-activated Ii~. In general, spontaneous repetitive firing (automaticity) can be explained by a decay in net IK during the latter part of the interspike interval, which together with a reactivation of inward current mechanisms leads to the succeeding spike9. In certain molluscan neurons with a very low rate of repetitive firing (e.g. 1 spike/s) the major influence on interspike interval has been shown to be a Ca2+-activated potassium current 22. The reason this mechanism may be particularly critical for regulating cells with slow rates of discharge is that Ca 2÷ diffusion and sequestration are relatively slow processes compared to changes in predominately voltage-dependent K ÷ channels. If such a mechanism holds for noradrenergic neurons of the locus coeruleus, then both their slow firing rate and auto-
242 maticity5 could be explained in part by a slow decay in a Ca2+-activated I K. A m o r e rapid removal of intracellular free calcium by E G T A would then be expected to increase spontaneous firing rate, as was observed in the present experiments. This basic concept can be e x t e n d e d to account for the ability of E G T A to reduce postactivation inhibitions and to increase the o r t h o d r o m i c reactivity of locus coeruleus neurons. O u r observations on locus coeruleus neurons are consistent with the suggestion of Meech 21, based on studies in invertebrate neurons, that 'it seems likely that this calcium-mediated negative f e e d b a c k mechanism is a feature of neural processing in many animals'. This discussion has focussed primarily on the possibility that a Ca2+-activated IK may underly A H P s in the locus coeruleus. H o w e v e r , a n o t h e r CaZ+-dependent process which could be affected by E G T A is that of the release of transmitter involved in autoinhibition. N o r a d r e n e r g i c neurons possess receptors in their somatodendritic region towards their own transmitter s. These a u t o r e c e p t o r s , which are of the a2-type, could contribute to the A H P if there were a postactivation Ca2+-dependent release of norepinephrine from locus coeruleus neurons onto themselves 2. Preliminary e x p e r i m e n t s in the present
study indicate that the direct stimulation of autoreceptors by the exogenous a 2 - a d r e n o c e p t o r agonist clonidine hyperpolarizes locus coeruleus neurons despite E G T A treatment. The degree of hyperpolarization p r o d u c e d by clonidine in the E G T A - t r e a t e d cells was similar to that previously r e p o r t e d for the effect of clonidine on locus coeruleus neurons when nonE G T A - c o n t a i n i n g electrodes were usedL This suggests that hyperpolarizations p r o d u c e d by the direct stimulation of a2-adrenoceptors in the locus coeruleus are not Ca2+-dependent, On the o t h e r hand, since the release of e n d o g e n o u s agonist (e.g. from a locus coeruleus neuron onto itself) would be Ca 2÷d e p e n d e n t , a 2 - a d r e n o c e p t o r - m e d i a t e d A H P s in the locus coeruleus could be indirectly d e p e n d e n t on calcium. The relative importance of h y p e r p o l a r i z a t i o n s induced by the Ca2+-activated IK as c o m p a r e d to those m e d i a t e d by a2-adrenoceptors in the locus coeruleus remains to be d e t e r m i n e d . ACKNOWLEDGEMENTS W e thank L. Fields, N. M a r g i o t t a , and A . Z i m n e wicz for their valuable assistance in this work. Supp o r t e d by U S P H S G r a n t s MH-17871, MH-14276, GM-07324, and the State of Connecticut.
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