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Serotonin receptor activation reduces calcium current in an acutely dissociated adult central neuron
Neuron,
Vol. 4, 751-758, May, 1990, Copyright
0 1990 by Cell Press
Serotonin Receptor Activation Reduces Calcium Current in an Acutelv Dissociated Adult Central Neuroh Nicholas J. Penington* and John S. Kelly Department of Pharmacology University of Edinburgh Medical Edinburgh, EH89JZ Scotland
School
Summary The release of serotonin (5-HT) from the terminals of serotonergic (raphe) neurons is under inhibitory feedback control. 5-HT, acting on raphe cell body autoreceptors, also mediates inhibitory postsynaptic potentials as a result of release from collaterals from neighboring raphe neurons. This may involve a ligand (5-HD-gated increase in the membrane potassium conductance, leading to a decrease in action potential frequency, which could indirectly reduce calcium influx into nerve terminals. In this report we demonstrate that 5-HT can also directly reduce calcium influx at potentials including and bracketing the peak of calcium current activation. Using acutely isolated, patch-clamped dorsal raphe neurons, we found that low concentrations of 5-HT and the 5-HTIA-selective agonist 8-OH-DPAT reversibly decrease whole-cell calcium current. This effect is antagonized by the putative 5-HTIA-selective antagonist NAN 190. Hence, the inhibition of calcium current may serve a physiological role in these cells and elsewhere in the brain. Introduction In this study we investigate whether calcium current (13 is modulated by the putative neurotransmitter substance serotonin (5-HT) in an acutely dissociated adult central neuron. If 5-HT reduces calcium current in these cells, it could be an important control mechanism in neurons responsible for the release of 5-HT in many of the brains higher centers. Raphe neurons have been shown to have large numbers of cell body autoreceptors for 5-HT (Akbari et al., 1989, Sot. Neuroscr., abstract), although their function is incompletely understood. One possible function of the autoreceptors is that 5-HT mediates an inhibitory postsynaptic potential resulting from serotonergic collaterals from neighboring raphe neurons (Wang and Aghajanian, 1977; Williams et al., 1988). Consequently, any action of 5-HT on Ica may have diverse effects on these cells, especially as low- and high-threshold calcium spikes are a prominent feature of raphe neuron electrophysiology (Penington et al., 1987, Neurosci. Lett. suppl. 29, abstract; Burlis and Aghajanian, 1987). *Present address: The University of Chicago, Pharmacological and Physiological Sciences, Street, Chicago, Illinois 60637.
Department of 947 East 58th
Several functions of neurons appear to be dependent on calcium ion influx through voltage-dependent calcium channels (for review see Augustine et al., 1987; Tsien et al., 1988). Furthermore, many investigators over the years have provided evidence that multiple calcium channels exist in various cell types (Hagiwara et al., 1975; Llinas and Yarom, 1981). These suggestions were confirmed when, using single-channel recording techniques, Nowycky et al. (1985) provided evidence for three distinct calcium channel conductance levels in cultured chick dorsal root ganglion (DRG) cells. By 1978, Dunlap and Fishbach had demonstrated that the Ica of chick DRG cells can be modulated by neurotransmitters acting at receptor sites distinct from the channel. Recently, significant improvements have been made in our understanding of the mechanisms at work during transmitter-mediated reduction of Ica. Modulation of ICa by transmitters usually involves a change in the activation kinetics (Marchetti et al., 1986; Tsunoo et al., 1986; Bean, 1989) and a clear voltage dependence; the effect of the transmitter is usually much smaller at very positive potentials (Tsunoo et al., 1986; Bean, 1989). G protein activation is a common feature of the transduction mechanism (Holz et al., 1986; Dolphin and Scott, 1987) that has been found to be tightly coupled to the calcium channel; in experiments in which norepinephrine was applied to the external membrane, little or no decrease in the lo, measured in cell-attached patch recordings was observed (Forscher et al., 1986). However, there are reports that protein kinase C activators can mimic the action of transmitters in decreasing IQ (Rane and Dunlap, 1986) and that inhibitors of protein kinase C can block the effect of norepinephrine (Rane et al., 1989; but see Hockberger et al., 1989).The question of which channel type is the main target for transmitter modulation has only recently been addressed. In some peripheral preparations, such as frog sympathetic neurons, in which one type of calcium channel seems to predominate, the N-channel has been shown in single-channel recordings to be modulated by a-adrenergic agents (Lipscombe et al., 1989). Elsewhere, the situation is less clear because of difficulties involved in separating the different components of Ica at the whole-cell level. Although Ica can be recorded from adult neurons in the slice preparation using a single microelectrode, it has been accompanied by technical problems associated with high access resistance and lack of space clamp. Recently, a small number of studies have characterized the I,, of hippocampal CAI, hypothalamic, and thalamic neurons acutely isolated from the adult rat brain (Kay and Wong, 1987; Akaike et al., 1989; Coulter et al., 1989). An added advantage of this approach is that these cells have not been exposed to tissue culture techniques, which, depending on the
growth conditions, can change the proportion and types of calcium channels incorporated in the cell membrane (Doerner et al., 1988). Thus, we exploited the advantages of this technique to determine the effects of 5-HT and 5-HTTA agonists and antagonists on autoreceptor control in raphe neurons.
A
B
Results
1nA 1
Multiple
Components
of Ica in Raphe Neurons
The action of 5-HT on Ica was studied in acutely isolated adult serotonergic neurons from the rat dorsal raphe nucleus. The experimental method allows the use of patch-clamp techniques and pipette and bath solutions designed to isolate currents carried through calcium channels by barium ions. This report is based on data obtained from 46 recordings from phasebright cells. In the whole-cell mode of recording (Figure I), a depolarizing step from -100 mV to -50 or -40 mV elicited a small (
The Effect of 5-HT on Ic. To examine the effect of 5-HT on all probable components of Ica (T-, .N-, and L-like), cells were held in voltage clamp at -100 mV and the current-voltage relation was determined. The cell was then repeatedly stepped at 0.05 Hz to the voltage at which the peak Ica occurred until a stable current (i.e., one showing
200ms
Figure 1. Representative Traces from a Cell Showing ponents of ICa
Three Com-
(A) A low-threshold transient current evoked at -40 mV from a negative holding potential (-100 mV). The current was smaller when elicited from more positive holding potentials and was nonexistent when elicited from a holding potential of -60 mV. (B) A sustained, slowly inactivating current was elicited at -20 mV (and at more positives potentials) from a holding potential of -50 mV, but if the holding potential was lowered to -100 mV, a larger, high-threshold transient current was evoked.
Figure 2. The Effect of 5-HT on I ca in an Acutely Raphe Neuron
Isolated
Dorsal
(A) The holding potential was -100 mV, and the cell was stepped to -10 mV, the potential at which the peak lea occurred. 5-HT (IO PM) reversibly decreased peak ICar in this case, to 25% of control. There appeared to be a reduction in the transient highthreshold component of Ic-. (B) A graph of peak Ica evoked every 20 s plotted against time. The bar indicates the period during which the pipette was in the bath close to the cell and leaking 5-HT. The action of 5-HT occurred with little delay; full recovery required 4 min.
5-HT Action
on Raphe Neuron
Calcium
Current
753
A.
B. 100 PAL
18 ms C.
200 PAL 18 ms Figure 3. 5-HT Has No Effect on the Low-Threshold, T-like Current and Slows the Rate of Activation of the High-Threshold, N-like Current in Dorsal Raphe Neurons (A) The cell was held at -100 mV and stepped to -50 mV. Control and 5-HT (IO PM) traces are superimposed; there was no effect on Fcurrent. All current sweeps in this figure have been leakagesubtracted. (6) Same as (A), but the step was to -40 mV, which activated a larger T-current and some sustained current. (C) The same cell showed only a moderately inactivating, highthreshold current when elicited from -100 mV; 5-HT not only reduced Ica, but slowed the rate of activation of the current; this was invariably seen if the holding potential was set at -60 mV, at which the current was always relatively noninactivating. Control and 5-HT (IO PM) traces are superimposed. The holding potential was -100 mV, with a step to -10 mV. The effect could be obtained repetitively and it was measured isochronally from the peak current in the control condition and after 5-HT.
negligible run-up or run-down) was observed. A pipette containing 5-HT creatine sulfate (IO nM-200 PM) dissolved in external bath solution was lowered into the bath, and the solution was leaked onto the cell. As shown in Figures 2 and 3, the peak Ica was depressed by, on average, 48.2% + 3.2% (mean f SEM; n = 21; 10 PM). The effect lasted for up to 4 min after removal of the pipette from the bath, but the inhibition recovered quicker when the bath was perfused at a faster rate. The extent and time course of the effect were shown to depend on the concentration of 5-HT applied, and 10 uM appeared to produce a maximal effect; 100 and 200 PM 5-HT caused an inhibition requiring a longer recovery. In another series of experiments, 5-HT was applied by switching the bath perfusate. The effect was identical and occurred only while 5-HT was present in the bath. In all cells that exhibited a high-threshold inactivating current (as in the top panel Figure 6), the characteristic effect of 5-HT was the elimination of this transient high-threshold
current component (that component not seen from a holding potential of -50 mV; Figure 1). However, there was also a smaller reduction in the sustained component. In cells showing less inactivating current (though a much larger current than could be elicited from a holding potential of -50 mV), 5-HT clearly slowed the rate of activation of lea in 20 out of 25 5-HT-responsive cells (Figure 3C). Both types of response could be repetitively elicited. To determine whether the two types of response represented a difference between the cells or the same effect of 5-HT on cells with differently shaped Icar cells were held at -50 mV to elicit only the nondecaying component of current and the effect of 5-HT was reexamined:Under these conditions, 5-HT always slowed the rate of activation of Ica in a fashion similar to that seen in Figure 3C and inhibited Ica by a similar relative amount (n = 9). This raises the possibility that the slowing of the activation rate may underlie the apparent abolition of the N-like inactivating component of current. When cells were held at -100 mV and the membrane potential was stepped to -50 mV, a protocol that maximized the T-like current, 5-HT had no reproducible effect on this component of current (Figures 3A and 3B) This confirms an observation made by Burlis and Aghajanian (1987), who observed no action of 5-HT on low-threshold calcium spikes in the dorsal raphe slice preparation. Voltage Dependence of the Effect of 5-HT The action of 5-HT was greatest at the peak of the current-voltage plot of peak IQ activation (Figure 4) and was not as large with small depolarizations or at very positive potentials This held true whether a cesium methanesulphonate or a Tris phosphate/TEA pipette solution was used. The smaller effect of 5-HT at positive voltages might result from an action of 5-HT on leakage conductance. Although 5-HT has no effect on leakage at -100 mV, if this proposed action were nonlinear with voltage, it might give a false impression of voltage dependence. The 5-HFinduced slowing of the current kinetics occurring at the peak of the current-voltage plot was abolished at positive potentials. Steps to +80 mV revealed no consistent increase in outward current in 5-HT-treated relative to control preparations, measured either at the instantaneous current or at the end of the step. Thus, we do not favor this interpretation. If a chloride current showing outward rectification was activated at positive potentials, it would shift the apparent reversal potential in 5-HT to a more negative membrane potential. Currents fitting this description did occur in a minority of cells, but the outward current at +80 mV was usually quite small (<500 PA). This current activated at potentials sufficiently positive SO that the experimentally determined reversal potential for ICa with and without 5-HT was not consistently altered. This was also true when using two
Neuron 754
A.
ably underlies any outward current. Thus, the peak should shift to the right instead of to the left, which is actually the case when using the Tris pipette solution. 5-HT was applied to 30 cells in this study, and 25 of them responded with a large inhibition of Ica. This represents slightly more than 80% of responsive cells, which agreeswell with the proportion of isolated cells of this size shown to contain 5-HT by immunohistochemistry (Lawrence et al., 1989, Neurosci. Lett., suppl. 36, abstract).
1000 I
-2 s
-1000
n
I PA
0
I PA (5HT)
The Action of a 5-HTTA -Selective Agonist Similar results were obtained with the selective 5-HTIA agonist &hydroxy-2-(di-n-propylamino)tetralin HBr (8OH-DPAT; n = 6). With this compound, significant inhibition was obtained with a concentration of 10 nM in 2 out of 4 cells to which it was applied. This concentration is only slightly higher than its Ko value of 4 nM (Gozlan et al., 1983) determined from binding studies. 8-OH-DPAT (100 nM) reversibly depressed peak Ica by 44%, and 1 PM 8-OH-DPAT suppressed peak I,-, by an average of 53.9% k 8.3% (mean f. SEM; n = 5; Figure 5B).
u4
-2000
L
r
1000
V ' o x
' (PA) 1 (PA) 5-H RECOVERY
Figure 4. Voltage Dependence and the Effect of 10 FM 5-HT
:
-2000
of the Peak (Leak-Subtracted)
Ica
(A) The pipette contained cesium methanesulphonate solution. 5-HT caused a maximal reduction in the amplitude of Ica near the peak of the current-voltage relation. Computer-generated curves that fit the data were selected by eye. The cell was held at -100 mV and stepped to the potentials indicated by the abscissae. (B) The action of IO PM 5-HTwas the same when the pipette soiution was Tris phosphate/TEA. Under these conditions, Ica reversed at the same potential (near +50 mV), but the peak current occurred at a potential that was IO-20 mV more negative than that seen when using cesium.
The Action of the Antagonist NAK 190 Inclusion of the potent 5-HTIA antagonist I-Q-methoxyphenyh-4-[4-(2-phthalimmido)butyl]piperazine HBr (NAN 190; Glennon et al., 1988a, 198813) at 1 PM or 100 nM in the application pipette with 5-HT (IO PM) preinduced by 5-HT, vented the decrease in I ca normally as illustrated in Figure 6 (average control inhibition to 5-HT 55.4% k 8.7%, mean F SEM; n = 5). In the presence of NAN 190 (applied to the same cell on which 5-HT was tested), the inhibition was 11.6% k 1.95% (mean + SEM). No response to 5-HT was ever obtained after the cell had been exposed to NAN 190. Recovery from antagonist therefore took longer than the process of Ica run-down, which usually was not very noticeable for the first 30 min of recording. Alone, NAN 190 at 1 PM had no effect on the size or shape of Ica (n = 4); IO PM 2-methyl 5-HT (n = 3) or phenyiephrine similarly had no effect (n = 3). This effectively rules out an action of 5-HT on a,-adrenoceptors, a receptor class for which NAN 190 has a high affinity in binding studies (Glennon et al., 1988a). Discussion
different pipette solutions that differed in their chloride concentrations. The peak Ica occurred at a potential IO-20 mV more negative when using Tris-filled pipettes. When using Tris phosphate, the peak ICa occurred around -10 mV; when using cesium methanesulphonate, it was close to 0 mV This is most likely to result from the large Tris molecule altering the charge of the inside of the cell membrane and thus the voltage dependence of calcium permeability. The shift in the peak current should not be due to a contaminating chloride current because the higher internal chloride concentration in the Tris pipette solution would reduce inward chloride ion movement, which presum-
The main findings of this study are that serotonergic dorsal raphe neurons possess at least two and possibly three components of current carried by either calcium or barium. The size of peak ICar when elicited from either -100 or -50 mV, is decreased by the application of 5-HT (100 nM to 10 PM). This effect appears to be mediated by a 5-HT receptor of the IA subtype, based on the action of the selective agonist 8-OHDPAT and the antagonism of the effect of 5-HT by the antagonist NAN 190. Interestingly, 5-HT,* receptor activation has previously been shown to decrease the action potential du-
5-HT Action on Raphe Neuron
Calcium
Current
755
5.HT
8-OH-DPAT
100 nM
1 uM
Figure 5. The Action of 5-HT Is Mimicked by the Selective 5-HT,, Agonist 8-OH-DPAT (A) The effects of 5-HT were dose-dependent, and 2 out of 4 cells tested responded to 100 nM 5-HT. (B) In another cell, the lowest threshold concentration for the response to 8-OHDPAT was 10 nM, but all of the responding ceils showed a large inhibition to 100 nM 8-OH-DPAT. This figure illustrates the action of 1 uM 8-OH-DPAT, which produced a S-HFlike slowing of the rate of Ica activation.
.,‘I:: r--L- I--L
1nA 1COmV
-100 -lo
r--L-
F-L-
F-L-
IlF--d-tk
A””
r--t--rL-r--i1OOmV
Figure 6. The Effect of 10 PM 5-HT on Whole-Cell Ica in an Isolated Adult Neuron from the Dorsal Raphe Nucleus and Antagonism of the Effect by the 5-HT,* Antagonist NAN 190 The cell was held at -100 mV and stepped to -10 mV every 20 s. (a) Control. (b) Application of 10 uM 5-HT. (c) Recovery. Each current record is the average of four successive sweeps. 5.HT reversibly decreased Ic. by 55%. Record (b) was taken 20 s after the onset of the 5-HT application, and record (c) was taken 2.5 min after the 5-HT pipette was removed from the bath. Records (d)-(f) show the control (d), the record 20 s after the pipette containing 5-HT (IO uM) plus NAN 190 (1 uM) was inserted into the bath near the cell (e), and the recovery 2 min after the pipette was removed from the bath (f). Coadministration of the S-HT,* antagonist attenuated the effect of 5HT. During the application of 5-HT, there was no consistent shift in voltage dependence of the current-voltage relation, change in leakage conductance at -100 mV, or alteration in the holding current.
ration of bull frog DRG cells (Marszalec et al., 1988). 8-OH-DPAT did not, however, act as an agonist on this receptor, as it did in this study; it behaved only as an antagonist. The receptor coupled to the opening of potassium channels found on hippocampal CA1 cells is designated 5-HT,, and is also antagonized by 8-OHDPAT (Colino and Halliwell, 1987) or acts as a partial agonist (Andrade and Nicoll, 1987). A similar receptor occurring on rat dorsal raphe neurons is also coupled to the opening of potassium channels and is antagonized by the same 5-HT,A-selective antagonists that are effective in the hippocampal and bull frog DRG cells (Williams et al., 1988). However, intracellular recordings from raphe neurons show that 8-OH-DPAT is a pure potent agonist on the receptor coupled to the opening of potassium channels (Rainnie et al., 1987, Neurosci. Lett. suppl. 29, abstract; Williams et al., 1988). Sprouse and Aghajanian (1988) have suggested that CA1 and dorsal raphe neuron 5-HT receptors have different sensitivities to 5-HT agonists, 8-OHDPAT being a partial agonist on CA1 cells and a full agonist on dorsal raphe neurons. If 5-HT decreases Ica in other systems rather than just increasing potassium conductance, then it appears that the same receptor coupled to increases in potassium conductance is also coupled to a mechanism that decreases Ica. Both effects would work in the same direction to decrease calcium influx into the cell. However, an increase in potassium conductance alone would be less effective at the positive potentials reached by the strong depolarization of an action potential, since its action on potassium channels is reported to be inwardly rectifying (Williams et al., 1988). Recent work with acutely isolated DRG cells suggests that some transmitters shift the voltage depen-
NfXlKl”
756
dence of the maximum calcium channel opening probability to very positive potentials (Bean, 2989). As this effect is postulated to wear off or “relax”when the cell membrane is held at positive potentials, this suggestion is not inconsistent with our findings in central neurons. Single-channel recording is clearly required to determine unequivocally whether the gating properties of the channel opening are generally slowed, or whether a single class of “N” channels is selectively inhibited by 5-HT. Release of 5-HT in the vicinity of these cells would be expected to cause a decrease in the calcium influx, which occurs during a raphe neuron action potential. This in turn might decrease a component of the spike after-hyperpolarization thought to be a calcium-activated potassium conductance, termed lAHP, in hippocampal cells (Lancaster and Adams, 1986). Any change in lAHPsize would directly modify the firing frequency of the raphe neurons, which would be evident only after strong depolarizations that bring the cell to firing threshold (see Andrade and Nicoll, 1987). This may indirectly alter the amount of 5-HT released from the entire projection sites of the dorsal raphe system. In hippocampal CA1 cells recorded in the slice preparation, 5-HT decreases IAHPby a direct closure of potassium channels (Colino and Halliwell, 1987; Andrade and Nicoll, 1987), apparently independent of any reduction in Ica. Dorsal raphe cells, at least when isolated and patch-clamped, seem to differ from CA1 cells in that the size of their lamp may be regulated by the action of 5-HT on calcium influx. Under more physiological recording conditions (where Ica is difficult to assess accurately), 5-HT hyperpolarizes the cell body of raphe neurons by opening potassium channels (Lakoski and Aghajanian, 1984). This might indirectly alter Ica if it also happens in nerve terminals, but membrane potential and calcium ion influx are technically extremely difficult to study at such sites. In different preparations, diametrically opposed mechanisms have been proposed to account for the effect of membrane potential on transmitter release. For instance, depolarization of squid and vertebrate (Miledi and Slater, 1966; Ryall, 1978) and hyperpolarization of Aplysia and leech (Kretz et al., 1986; Nicholls and Wallace, 1978) neuron preparations decrease transmitter release. However, there may be some physiological significance for the action of 5-HT on lca, as the release of 5-HT from the terminals of raphe neurons is under autoinhibitory control (Langer and Moret, 1982). Thus the release of 5-HT may depend on the calcium influx into raphe terminals, which in turn may be modulated by 5-HT. The postulated autoinhibition of raphe neuron Ica mediated by a 5-HT receptor of the IA subtype may not be a good model of 5-HT-mediated inhibition of transmitter release at all sites in the brain. Binding and transmitter release studies, though not conclusive, suggest that in cortex a 5-HTTB site is responsible for inhibition of transmitter release (8-OH-DPAT is estimated to be 30,000 times less effective at displacing ligands from 5-HTTB com-
pared with 5-HTJA receptors [Engel et. al., 19861). However, the suggestion that a ~-HT~B site is the only presynaptic 5-HT autoreceptor is not supported by lesion studies (Verge et al., 1986). A role for 5-HT1A receptors in the control of transmitter release is supported by the finding that 5-HT agonists inhibit synaptic potentials in rat locus ceruleus cells by an action on both 5-HTjA and 5-HT1* receptors (Bobker and W illiams, 1989). In guinea pig enteric cholinergic neurons, g-OHDPAT inhibits transmitter release by activating 5-HTIA receptors (Fozard and Kilbinger, 1988, Br. J. Pharmacol. 86, abstract). ica has been shown to be modulated by neurotransmitters in the bodies of various fetal cells including cultured peripheral sensory neurons (Dunlap and Fishbach, 1981; Dolphin and Scott, 1987; Gross et al., 1989; Ewald et al., 1988), sympathetic neurons (Galvan and Adams, 1982; lkeda and Schofield, 1989), cultured central neurons (Madison et al., 1987, Biophys. J., abstract), and cell lines (Tsunoo et al., 1986; Docherty and McFadzean, 1989). However, only rarely has transmitter modulation been investigated using conditions ideal for measuring Ica in adult central neurons (Williams and North, 1985). Interestingly, a good correlation exists between a neurotransmitterinduced decrease in lc, measured in the cell body of a presynaptic neuron and a decrease in transmitter release from its terminals (Kretz et al., 1986). As changes in membrane potential alone, without a change in calcium influx, are unlikely to alter transmitter release (Zucker and Haydon, 1988), the inverse may also be true, i.e., a change in internal calcium concentration without a change in membrane potential is not effective at evoking release (see results in crayfish muscle; Hochner et al., 1989), and the voltage dependence of transmitter release seems to be accounted for by the voltage dependence of ICa (Augustine et al., 1987). This mechanism could directly regulate terminal Ica by neurotransmitters and may be of importance in the vertebrate central nervous system. Experimental
Procedures
Isolation of Neurons Three coronal slices (500 pm) through the brainstem at the level of the dorsal raphe nucleus were prepared from adult rats (ZOO-250 g) in a conventional manner using a Vibroslice machine. The dorsal raphe nucleus was carefully dissected out of the slice with a razor blade. The pieces of tissue were then incubated in a PIPES-buffered saline containing 0.75 mg/ml trypsin (Sigma type XI) under pure oxygen for 90 min according to the method of Kay and W o n g (1987). The pieces of tissue were then triturated in Dulbecco’s modified Eagle’s medium, and the isolated cells were allowed to settle to the bottom of a Falcon tissue culture dish (35 mm), to which they adhered well enough to be perfused at l-2 ml per min. Recording was carried out at room temperature, l8”C-20°C. Steinbusch et al. (1981) have shown with immunohistochemistry that most of the cells in a thin raphe slice with a soma diameter greater than 20 p m contain S-HT. W e have confirmed and extended these observations (Lawrence et al., 1989, Neurosci. Lett., suppl. 36, abstract) by showing that the proportion of isolated cells larger than 20 p m which stain for 5-HT, using a method similar to that of Yamamoto et al. (1981), is greater than 85%.
5-HT Action 757
on Raphe Neuron
Calcium
Current
Patch-Clamp Recording Conditions Neurons with truncated dendrites and a cell soma diameter of at least 20 pm were voltage-clamped using a switching voltageclamp amplifier (Axoclamp 2A) and a single patch pipette in the whole-cell configuration (Hamill et al., 1981). Pipettes pulled from thick-walled borosilicate glass (resistance from 6-10 MB) allowedaswitchingfrequencyof8-13KHzwitha30% dutycycle. The seal resistance measured by the voltage response to a 50 pA step of current was often greater than 5 CD). For this reason, the current traces are not leak-subtracted, and the capacity transients are intact. The voltage-clamp data were filtered at 1 KHz and digitized for storage at 16 bits on a video recorder for later analysis on a PDPII computer. Later experiments were run on line by an IBM PC 286 clone (DCS) and a CED 1401 interface. The resultant data were written to disk for later analysis. Solutions The external saline was designed to isolate Ica and contained 147 m M TEA-Cl, 5 m M BaCIz, 20 m M HEPES, 10 m M D-glucose, 5 m M Caminopyridine, and 0.2 PM tetrodotoxin (pH 7.2 with TEA-OH). In some experiments, 2 m M CaClz was used in place of BaCI,. The patch pipette in 17 experiments contained 63 m M cesium methanesulphonate, 40 m M HEPES, 2 m M Mg-ATP, cesium-11 m M ECTA, and 300 pM GTP (pH 7.2 with CsOH). The remainder of pipettes contained 70 m M Trizma phosphate, 33 m M Trizma Base-11 m M EGTA, 2 m M MgATP, 300 KM GTP, and 40 m M TEA-Cl (pH 7.2 with Trizma Base). The osmolarity was adjusted with sucrose so that the pipette solution was 10 mOsm hypoosmotic to the bath solution. This latter solution was used in all figures unless otherwise specified. Drugs were either dissolved in the extracellular solution and added to the perfusate or applied by diffusion from a patch pipette (tip 15 pm) lowered close to the cell (50 pm) and then removed from the bath. As a control, the same procedure was carried out without addition of 5.HT to the application pipette; this did not affect Ica. Acknowledgments We thank Dr. Ian Dawson for making NAN 190, Dr. A. P. Fox for helpful discussions, and the Wellcome Trust for support. Received
December
26, 1989; revised
February
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Gross, R. A., Macdonald, R. L., and Ryan-Jastrow, T. (1989). 2Chloroadenosine reduces the N calcium current of cultured mouse sensory neurones in a pertussis toxin-sensitive manner. J. Physiol. 477, 585-595. Hagiwara, S., Ozawa, S., and Sand, 0. (1975). Voltage-clamp analysis of two inward current mechanisms in the egg cell membrane of a star fish. J. Gen. Physiol. 65, 617-644. Hamill, O., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 397, 85-100. Hochner, B., Parnas, H., and Parnas, I. (1989). Membrane depolarization evokes neurotransmitter release in the absence of calcium entry. Nature 342, 433-435. Hockberger, F’., Toselli, M., Swandulla, D., and Lux, H. D. (1989). Adiacylglycerol analogue reduces neuronal calcium currents independently of protein kinase C activation. Nature 338,340-342. Holz, G. G., Rane, S. G., and Dunlap, K. (1986). GTP-binding teins mediate transmitter inhibition of voltage-dependent cium channels. Nature 379, 670-672. Ikeda, S. R., and Schofield, G. G. (1989). Somatostatin cium current in rat sympathetic ganglion neurones. 409, 221-240.
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Kay, A. R., and Wong, R. K. S. (1987). Calcium current activation kinetics in isolated pyramidal neurones of the CA1 region of the mature guinea-pig hippocampus. J. Physiol. 392, 602-616. Kretz, R., Shapiro,
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Lakoski, J. M., and Aghajanian, G. K. (1984). Hyperpolarization of serotonergic neurons by serotonin and LSD: studies in brain slices showing increased K+ conductance. Brain Res. 305, 18% 185. Lancaster, B., and Adams, P R. (1986). Calcium dependent rent generating the after-hyperpolarization of hippocampal rons. J. Neurophysiol. 5.5, 1268-1282.
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Langer, 5. Z., and Moret, C. (1982). Citalopram antagonizes the stimulation by lysergic acid diethylamide of presynaptic inhibitory serotonin autoreceptors in rat hypothalamus. J. Pharmacol. Exp. Ther. 222, 220-226. Lipscombe, D., Kongsamut, S., and Tsien, R. W. (1989). a-Adrenergic inhibition of sympathetic neurotransmitter release mediated by modulation of N-type calcium-channel gating. Nature 340, 639-642. Llinas, R., and Yarom, Y. (1981). Properties and distribution of ionic conductances generating electroresponsiveness of mammalian inferior olivary neurones in vitro. 1. Physiol. 375,569-584. Marchetti, C., Carbone, E., and Lux, H. D. (1986). Effects of dopamineandnoradrenalineonCachannelsofcuIturedsensoryand sympathetic neurones of chick. Pfltigers Arch. 406, 104-111. Marszalec, W., Scroggs, R. S., and Anderson, E. C., (1988). Serotonininduced reduction of the calcium-dependent plateau in frog dorsal root ganglion cells is blocked by serotonergic agents acting at 5-hydroxytryptamineIA sites. J. Pharmacol. Exp. Ther. 247 399404. McCleskey, E. W., Fox, A. P., Feldman, D., Cruz, L. J., Olivera, B. M., Tsien, R. W., and Yoshikami, D. (1987). e+Conotoxin: direct and persistent blockade of specific types of calcium channels in neurones but not muscle. Proc. Natl. Acad. Sci. USA 84, 43274331. Miledi, R., and Slater, C. R. (1966). The action of calcium ronal synapses in the squid. J. Physiol. 784, 473-498. Nicholls, J., and Wallace, B. C. (1978). Modulation sion at an inhibitory synapse in thecentral nervous leech. J. Physiol. 287, 157-170.
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Verge, D., Daval, C., Marcinkiewicz, M., Patey, A., El Mestikawy, S., Gozlan, H., and Hamon, M. J. (1986). Quantitative autoradiography of multiple 5-HT, receptor subtypes in the brain of control or 5,7-dihydroxytryptamine-treated rats. Neuroscience 6, 3474-3482. Wang, R. Y., and Aghajanian, C. K. (1977). Antidromically identified serotonergic neurons in the rat midbrain raphe: evidence for collateral inhibition. Brain Res. 732, 186-193. Williams, J. T.,and North, R. A., (1985). Catecholamine inhibition of calcium action potentials in rat locus ceruleus neurones. Neuroscience 74, 102-109. Williams, J. T, Colmers, W. F., and Pan, Z. Z. (1988). Ligand and voltage activated inwardly rectifying currents in dorsal raphe neurons in vitro. J. Neurosci. 8, 3499-3506. Yamamoto, M., Steinbusch, H. W. M., and Jessel, T. M. (1981). Differential properties of identified serotonin neurones in dissociated cultures of embryonic iat brainstem. J. Cell Biol. 97, 142-152. Zucker, R. S., and Haydon, P. C. (1988). Membrane potential no direct role in evoking neurotransmitter release. Nature 360-362.
has 335,
Vol. 4, 751-758, May, 1990, Copyright
0 1990 by Cell Press
Serotonin Receptor Activation Reduces Calcium Current in an Acutelv Dissociated Adult Central Neuroh Nicholas J. Penington* and John S. Kelly Department of Pharmacology University of Edinburgh Medical Edinburgh, EH89JZ Scotland
School
Summary The release of serotonin (5-HT) from the terminals of serotonergic (raphe) neurons is under inhibitory feedback control. 5-HT, acting on raphe cell body autoreceptors, also mediates inhibitory postsynaptic potentials as a result of release from collaterals from neighboring raphe neurons. This may involve a ligand (5-HD-gated increase in the membrane potassium conductance, leading to a decrease in action potential frequency, which could indirectly reduce calcium influx into nerve terminals. In this report we demonstrate that 5-HT can also directly reduce calcium influx at potentials including and bracketing the peak of calcium current activation. Using acutely isolated, patch-clamped dorsal raphe neurons, we found that low concentrations of 5-HT and the 5-HTIA-selective agonist 8-OH-DPAT reversibly decrease whole-cell calcium current. This effect is antagonized by the putative 5-HTIA-selective antagonist NAN 190. Hence, the inhibition of calcium current may serve a physiological role in these cells and elsewhere in the brain. Introduction In this study we investigate whether calcium current (13 is modulated by the putative neurotransmitter substance serotonin (5-HT) in an acutely dissociated adult central neuron. If 5-HT reduces calcium current in these cells, it could be an important control mechanism in neurons responsible for the release of 5-HT in many of the brains higher centers. Raphe neurons have been shown to have large numbers of cell body autoreceptors for 5-HT (Akbari et al., 1989, Sot. Neuroscr., abstract), although their function is incompletely understood. One possible function of the autoreceptors is that 5-HT mediates an inhibitory postsynaptic potential resulting from serotonergic collaterals from neighboring raphe neurons (Wang and Aghajanian, 1977; Williams et al., 1988). Consequently, any action of 5-HT on Ica may have diverse effects on these cells, especially as low- and high-threshold calcium spikes are a prominent feature of raphe neuron electrophysiology (Penington et al., 1987, Neurosci. Lett. suppl. 29, abstract; Burlis and Aghajanian, 1987). *Present address: The University of Chicago, Pharmacological and Physiological Sciences, Street, Chicago, Illinois 60637.
Department of 947 East 58th
Several functions of neurons appear to be dependent on calcium ion influx through voltage-dependent calcium channels (for review see Augustine et al., 1987; Tsien et al., 1988). Furthermore, many investigators over the years have provided evidence that multiple calcium channels exist in various cell types (Hagiwara et al., 1975; Llinas and Yarom, 1981). These suggestions were confirmed when, using single-channel recording techniques, Nowycky et al. (1985) provided evidence for three distinct calcium channel conductance levels in cultured chick dorsal root ganglion (DRG) cells. By 1978, Dunlap and Fishbach had demonstrated that the Ica of chick DRG cells can be modulated by neurotransmitters acting at receptor sites distinct from the channel. Recently, significant improvements have been made in our understanding of the mechanisms at work during transmitter-mediated reduction of Ica. Modulation of ICa by transmitters usually involves a change in the activation kinetics (Marchetti et al., 1986; Tsunoo et al., 1986; Bean, 1989) and a clear voltage dependence; the effect of the transmitter is usually much smaller at very positive potentials (Tsunoo et al., 1986; Bean, 1989). G protein activation is a common feature of the transduction mechanism (Holz et al., 1986; Dolphin and Scott, 1987) that has been found to be tightly coupled to the calcium channel; in experiments in which norepinephrine was applied to the external membrane, little or no decrease in the lo, measured in cell-attached patch recordings was observed (Forscher et al., 1986). However, there are reports that protein kinase C activators can mimic the action of transmitters in decreasing IQ (Rane and Dunlap, 1986) and that inhibitors of protein kinase C can block the effect of norepinephrine (Rane et al., 1989; but see Hockberger et al., 1989).The question of which channel type is the main target for transmitter modulation has only recently been addressed. In some peripheral preparations, such as frog sympathetic neurons, in which one type of calcium channel seems to predominate, the N-channel has been shown in single-channel recordings to be modulated by a-adrenergic agents (Lipscombe et al., 1989). Elsewhere, the situation is less clear because of difficulties involved in separating the different components of Ica at the whole-cell level. Although Ica can be recorded from adult neurons in the slice preparation using a single microelectrode, it has been accompanied by technical problems associated with high access resistance and lack of space clamp. Recently, a small number of studies have characterized the I,, of hippocampal CAI, hypothalamic, and thalamic neurons acutely isolated from the adult rat brain (Kay and Wong, 1987; Akaike et al., 1989; Coulter et al., 1989). An added advantage of this approach is that these cells have not been exposed to tissue culture techniques, which, depending on the
growth conditions, can change the proportion and types of calcium channels incorporated in the cell membrane (Doerner et al., 1988). Thus, we exploited the advantages of this technique to determine the effects of 5-HT and 5-HTTA agonists and antagonists on autoreceptor control in raphe neurons.
A
B
Results
1nA 1
Multiple
Components
of Ica in Raphe Neurons
The action of 5-HT on Ica was studied in acutely isolated adult serotonergic neurons from the rat dorsal raphe nucleus. The experimental method allows the use of patch-clamp techniques and pipette and bath solutions designed to isolate currents carried through calcium channels by barium ions. This report is based on data obtained from 46 recordings from phasebright cells. In the whole-cell mode of recording (Figure I), a depolarizing step from -100 mV to -50 or -40 mV elicited a small (
The Effect of 5-HT on Ic. To examine the effect of 5-HT on all probable components of Ica (T-, .N-, and L-like), cells were held in voltage clamp at -100 mV and the current-voltage relation was determined. The cell was then repeatedly stepped at 0.05 Hz to the voltage at which the peak Ica occurred until a stable current (i.e., one showing
200ms
Figure 1. Representative Traces from a Cell Showing ponents of ICa
Three Com-
(A) A low-threshold transient current evoked at -40 mV from a negative holding potential (-100 mV). The current was smaller when elicited from more positive holding potentials and was nonexistent when elicited from a holding potential of -60 mV. (B) A sustained, slowly inactivating current was elicited at -20 mV (and at more positives potentials) from a holding potential of -50 mV, but if the holding potential was lowered to -100 mV, a larger, high-threshold transient current was evoked.
Figure 2. The Effect of 5-HT on I ca in an Acutely Raphe Neuron
Isolated
Dorsal
(A) The holding potential was -100 mV, and the cell was stepped to -10 mV, the potential at which the peak lea occurred. 5-HT (IO PM) reversibly decreased peak ICar in this case, to 25% of control. There appeared to be a reduction in the transient highthreshold component of Ic-. (B) A graph of peak Ica evoked every 20 s plotted against time. The bar indicates the period during which the pipette was in the bath close to the cell and leaking 5-HT. The action of 5-HT occurred with little delay; full recovery required 4 min.
5-HT Action
on Raphe Neuron
Calcium
Current
753
A.
B. 100 PAL
18 ms C.
200 PAL 18 ms Figure 3. 5-HT Has No Effect on the Low-Threshold, T-like Current and Slows the Rate of Activation of the High-Threshold, N-like Current in Dorsal Raphe Neurons (A) The cell was held at -100 mV and stepped to -50 mV. Control and 5-HT (IO PM) traces are superimposed; there was no effect on Fcurrent. All current sweeps in this figure have been leakagesubtracted. (6) Same as (A), but the step was to -40 mV, which activated a larger T-current and some sustained current. (C) The same cell showed only a moderately inactivating, highthreshold current when elicited from -100 mV; 5-HT not only reduced Ica, but slowed the rate of activation of the current; this was invariably seen if the holding potential was set at -60 mV, at which the current was always relatively noninactivating. Control and 5-HT (IO PM) traces are superimposed. The holding potential was -100 mV, with a step to -10 mV. The effect could be obtained repetitively and it was measured isochronally from the peak current in the control condition and after 5-HT.
negligible run-up or run-down) was observed. A pipette containing 5-HT creatine sulfate (IO nM-200 PM) dissolved in external bath solution was lowered into the bath, and the solution was leaked onto the cell. As shown in Figures 2 and 3, the peak Ica was depressed by, on average, 48.2% + 3.2% (mean f SEM; n = 21; 10 PM). The effect lasted for up to 4 min after removal of the pipette from the bath, but the inhibition recovered quicker when the bath was perfused at a faster rate. The extent and time course of the effect were shown to depend on the concentration of 5-HT applied, and 10 uM appeared to produce a maximal effect; 100 and 200 PM 5-HT caused an inhibition requiring a longer recovery. In another series of experiments, 5-HT was applied by switching the bath perfusate. The effect was identical and occurred only while 5-HT was present in the bath. In all cells that exhibited a high-threshold inactivating current (as in the top panel Figure 6), the characteristic effect of 5-HT was the elimination of this transient high-threshold
current component (that component not seen from a holding potential of -50 mV; Figure 1). However, there was also a smaller reduction in the sustained component. In cells showing less inactivating current (though a much larger current than could be elicited from a holding potential of -50 mV), 5-HT clearly slowed the rate of activation of lea in 20 out of 25 5-HT-responsive cells (Figure 3C). Both types of response could be repetitively elicited. To determine whether the two types of response represented a difference between the cells or the same effect of 5-HT on cells with differently shaped Icar cells were held at -50 mV to elicit only the nondecaying component of current and the effect of 5-HT was reexamined:Under these conditions, 5-HT always slowed the rate of activation of Ica in a fashion similar to that seen in Figure 3C and inhibited Ica by a similar relative amount (n = 9). This raises the possibility that the slowing of the activation rate may underlie the apparent abolition of the N-like inactivating component of current. When cells were held at -100 mV and the membrane potential was stepped to -50 mV, a protocol that maximized the T-like current, 5-HT had no reproducible effect on this component of current (Figures 3A and 3B) This confirms an observation made by Burlis and Aghajanian (1987), who observed no action of 5-HT on low-threshold calcium spikes in the dorsal raphe slice preparation. Voltage Dependence of the Effect of 5-HT The action of 5-HT was greatest at the peak of the current-voltage plot of peak IQ activation (Figure 4) and was not as large with small depolarizations or at very positive potentials This held true whether a cesium methanesulphonate or a Tris phosphate/TEA pipette solution was used. The smaller effect of 5-HT at positive voltages might result from an action of 5-HT on leakage conductance. Although 5-HT has no effect on leakage at -100 mV, if this proposed action were nonlinear with voltage, it might give a false impression of voltage dependence. The 5-HFinduced slowing of the current kinetics occurring at the peak of the current-voltage plot was abolished at positive potentials. Steps to +80 mV revealed no consistent increase in outward current in 5-HT-treated relative to control preparations, measured either at the instantaneous current or at the end of the step. Thus, we do not favor this interpretation. If a chloride current showing outward rectification was activated at positive potentials, it would shift the apparent reversal potential in 5-HT to a more negative membrane potential. Currents fitting this description did occur in a minority of cells, but the outward current at +80 mV was usually quite small (<500 PA). This current activated at potentials sufficiently positive SO that the experimentally determined reversal potential for ICa with and without 5-HT was not consistently altered. This was also true when using two
Neuron 754
A.
ably underlies any outward current. Thus, the peak should shift to the right instead of to the left, which is actually the case when using the Tris pipette solution. 5-HT was applied to 30 cells in this study, and 25 of them responded with a large inhibition of Ica. This represents slightly more than 80% of responsive cells, which agreeswell with the proportion of isolated cells of this size shown to contain 5-HT by immunohistochemistry (Lawrence et al., 1989, Neurosci. Lett., suppl. 36, abstract).
1000 I
-2 s
-1000
n
I PA
0
I PA (5HT)
The Action of a 5-HTTA -Selective Agonist Similar results were obtained with the selective 5-HTIA agonist &hydroxy-2-(di-n-propylamino)tetralin HBr (8OH-DPAT; n = 6). With this compound, significant inhibition was obtained with a concentration of 10 nM in 2 out of 4 cells to which it was applied. This concentration is only slightly higher than its Ko value of 4 nM (Gozlan et al., 1983) determined from binding studies. 8-OH-DPAT (100 nM) reversibly depressed peak Ica by 44%, and 1 PM 8-OH-DPAT suppressed peak I,-, by an average of 53.9% k 8.3% (mean f. SEM; n = 5; Figure 5B).
u4
-2000
L
r
1000
V ' o x
' (PA) 1 (PA) 5-H RECOVERY
Figure 4. Voltage Dependence and the Effect of 10 FM 5-HT
:
-2000
of the Peak (Leak-Subtracted)
Ica
(A) The pipette contained cesium methanesulphonate solution. 5-HT caused a maximal reduction in the amplitude of Ica near the peak of the current-voltage relation. Computer-generated curves that fit the data were selected by eye. The cell was held at -100 mV and stepped to the potentials indicated by the abscissae. (B) The action of IO PM 5-HTwas the same when the pipette soiution was Tris phosphate/TEA. Under these conditions, Ica reversed at the same potential (near +50 mV), but the peak current occurred at a potential that was IO-20 mV more negative than that seen when using cesium.
The Action of the Antagonist NAK 190 Inclusion of the potent 5-HTIA antagonist I-Q-methoxyphenyh-4-[4-(2-phthalimmido)butyl]piperazine HBr (NAN 190; Glennon et al., 1988a, 198813) at 1 PM or 100 nM in the application pipette with 5-HT (IO PM) preinduced by 5-HT, vented the decrease in I ca normally as illustrated in Figure 6 (average control inhibition to 5-HT 55.4% k 8.7%, mean F SEM; n = 5). In the presence of NAN 190 (applied to the same cell on which 5-HT was tested), the inhibition was 11.6% k 1.95% (mean + SEM). No response to 5-HT was ever obtained after the cell had been exposed to NAN 190. Recovery from antagonist therefore took longer than the process of Ica run-down, which usually was not very noticeable for the first 30 min of recording. Alone, NAN 190 at 1 PM had no effect on the size or shape of Ica (n = 4); IO PM 2-methyl 5-HT (n = 3) or phenyiephrine similarly had no effect (n = 3). This effectively rules out an action of 5-HT on a,-adrenoceptors, a receptor class for which NAN 190 has a high affinity in binding studies (Glennon et al., 1988a). Discussion
different pipette solutions that differed in their chloride concentrations. The peak Ica occurred at a potential IO-20 mV more negative when using Tris-filled pipettes. When using Tris phosphate, the peak ICa occurred around -10 mV; when using cesium methanesulphonate, it was close to 0 mV This is most likely to result from the large Tris molecule altering the charge of the inside of the cell membrane and thus the voltage dependence of calcium permeability. The shift in the peak current should not be due to a contaminating chloride current because the higher internal chloride concentration in the Tris pipette solution would reduce inward chloride ion movement, which presum-
The main findings of this study are that serotonergic dorsal raphe neurons possess at least two and possibly three components of current carried by either calcium or barium. The size of peak ICar when elicited from either -100 or -50 mV, is decreased by the application of 5-HT (100 nM to 10 PM). This effect appears to be mediated by a 5-HT receptor of the IA subtype, based on the action of the selective agonist 8-OHDPAT and the antagonism of the effect of 5-HT by the antagonist NAN 190. Interestingly, 5-HT,* receptor activation has previously been shown to decrease the action potential du-
5-HT Action on Raphe Neuron
Calcium
Current
755
5.HT
8-OH-DPAT
100 nM
1 uM
Figure 5. The Action of 5-HT Is Mimicked by the Selective 5-HT,, Agonist 8-OH-DPAT (A) The effects of 5-HT were dose-dependent, and 2 out of 4 cells tested responded to 100 nM 5-HT. (B) In another cell, the lowest threshold concentration for the response to 8-OHDPAT was 10 nM, but all of the responding ceils showed a large inhibition to 100 nM 8-OH-DPAT. This figure illustrates the action of 1 uM 8-OH-DPAT, which produced a S-HFlike slowing of the rate of Ica activation.
.,‘I:: r--L- I--L
1nA 1COmV
-100 -lo
r--L-
F-L-
F-L-
IlF--d-tk
A””
r--t--rL-r--i1OOmV
Figure 6. The Effect of 10 PM 5-HT on Whole-Cell Ica in an Isolated Adult Neuron from the Dorsal Raphe Nucleus and Antagonism of the Effect by the 5-HT,* Antagonist NAN 190 The cell was held at -100 mV and stepped to -10 mV every 20 s. (a) Control. (b) Application of 10 uM 5-HT. (c) Recovery. Each current record is the average of four successive sweeps. 5.HT reversibly decreased Ic. by 55%. Record (b) was taken 20 s after the onset of the 5-HT application, and record (c) was taken 2.5 min after the 5-HT pipette was removed from the bath. Records (d)-(f) show the control (d), the record 20 s after the pipette containing 5-HT (IO uM) plus NAN 190 (1 uM) was inserted into the bath near the cell (e), and the recovery 2 min after the pipette was removed from the bath (f). Coadministration of the S-HT,* antagonist attenuated the effect of 5HT. During the application of 5-HT, there was no consistent shift in voltage dependence of the current-voltage relation, change in leakage conductance at -100 mV, or alteration in the holding current.
ration of bull frog DRG cells (Marszalec et al., 1988). 8-OH-DPAT did not, however, act as an agonist on this receptor, as it did in this study; it behaved only as an antagonist. The receptor coupled to the opening of potassium channels found on hippocampal CA1 cells is designated 5-HT,, and is also antagonized by 8-OHDPAT (Colino and Halliwell, 1987) or acts as a partial agonist (Andrade and Nicoll, 1987). A similar receptor occurring on rat dorsal raphe neurons is also coupled to the opening of potassium channels and is antagonized by the same 5-HT,A-selective antagonists that are effective in the hippocampal and bull frog DRG cells (Williams et al., 1988). However, intracellular recordings from raphe neurons show that 8-OH-DPAT is a pure potent agonist on the receptor coupled to the opening of potassium channels (Rainnie et al., 1987, Neurosci. Lett. suppl. 29, abstract; Williams et al., 1988). Sprouse and Aghajanian (1988) have suggested that CA1 and dorsal raphe neuron 5-HT receptors have different sensitivities to 5-HT agonists, 8-OHDPAT being a partial agonist on CA1 cells and a full agonist on dorsal raphe neurons. If 5-HT decreases Ica in other systems rather than just increasing potassium conductance, then it appears that the same receptor coupled to increases in potassium conductance is also coupled to a mechanism that decreases Ica. Both effects would work in the same direction to decrease calcium influx into the cell. However, an increase in potassium conductance alone would be less effective at the positive potentials reached by the strong depolarization of an action potential, since its action on potassium channels is reported to be inwardly rectifying (Williams et al., 1988). Recent work with acutely isolated DRG cells suggests that some transmitters shift the voltage depen-
NfXlKl”
756
dence of the maximum calcium channel opening probability to very positive potentials (Bean, 2989). As this effect is postulated to wear off or “relax”when the cell membrane is held at positive potentials, this suggestion is not inconsistent with our findings in central neurons. Single-channel recording is clearly required to determine unequivocally whether the gating properties of the channel opening are generally slowed, or whether a single class of “N” channels is selectively inhibited by 5-HT. Release of 5-HT in the vicinity of these cells would be expected to cause a decrease in the calcium influx, which occurs during a raphe neuron action potential. This in turn might decrease a component of the spike after-hyperpolarization thought to be a calcium-activated potassium conductance, termed lAHP, in hippocampal cells (Lancaster and Adams, 1986). Any change in lAHPsize would directly modify the firing frequency of the raphe neurons, which would be evident only after strong depolarizations that bring the cell to firing threshold (see Andrade and Nicoll, 1987). This may indirectly alter the amount of 5-HT released from the entire projection sites of the dorsal raphe system. In hippocampal CA1 cells recorded in the slice preparation, 5-HT decreases IAHPby a direct closure of potassium channels (Colino and Halliwell, 1987; Andrade and Nicoll, 1987), apparently independent of any reduction in Ica. Dorsal raphe cells, at least when isolated and patch-clamped, seem to differ from CA1 cells in that the size of their lamp may be regulated by the action of 5-HT on calcium influx. Under more physiological recording conditions (where Ica is difficult to assess accurately), 5-HT hyperpolarizes the cell body of raphe neurons by opening potassium channels (Lakoski and Aghajanian, 1984). This might indirectly alter Ica if it also happens in nerve terminals, but membrane potential and calcium ion influx are technically extremely difficult to study at such sites. In different preparations, diametrically opposed mechanisms have been proposed to account for the effect of membrane potential on transmitter release. For instance, depolarization of squid and vertebrate (Miledi and Slater, 1966; Ryall, 1978) and hyperpolarization of Aplysia and leech (Kretz et al., 1986; Nicholls and Wallace, 1978) neuron preparations decrease transmitter release. However, there may be some physiological significance for the action of 5-HT on lca, as the release of 5-HT from the terminals of raphe neurons is under autoinhibitory control (Langer and Moret, 1982). Thus the release of 5-HT may depend on the calcium influx into raphe terminals, which in turn may be modulated by 5-HT. The postulated autoinhibition of raphe neuron Ica mediated by a 5-HT receptor of the IA subtype may not be a good model of 5-HT-mediated inhibition of transmitter release at all sites in the brain. Binding and transmitter release studies, though not conclusive, suggest that in cortex a 5-HTTB site is responsible for inhibition of transmitter release (8-OH-DPAT is estimated to be 30,000 times less effective at displacing ligands from 5-HTTB com-
pared with 5-HTJA receptors [Engel et. al., 19861). However, the suggestion that a ~-HT~B site is the only presynaptic 5-HT autoreceptor is not supported by lesion studies (Verge et al., 1986). A role for 5-HT1A receptors in the control of transmitter release is supported by the finding that 5-HT agonists inhibit synaptic potentials in rat locus ceruleus cells by an action on both 5-HTjA and 5-HT1* receptors (Bobker and W illiams, 1989). In guinea pig enteric cholinergic neurons, g-OHDPAT inhibits transmitter release by activating 5-HTIA receptors (Fozard and Kilbinger, 1988, Br. J. Pharmacol. 86, abstract). ica has been shown to be modulated by neurotransmitters in the bodies of various fetal cells including cultured peripheral sensory neurons (Dunlap and Fishbach, 1981; Dolphin and Scott, 1987; Gross et al., 1989; Ewald et al., 1988), sympathetic neurons (Galvan and Adams, 1982; lkeda and Schofield, 1989), cultured central neurons (Madison et al., 1987, Biophys. J., abstract), and cell lines (Tsunoo et al., 1986; Docherty and McFadzean, 1989). However, only rarely has transmitter modulation been investigated using conditions ideal for measuring Ica in adult central neurons (Williams and North, 1985). Interestingly, a good correlation exists between a neurotransmitterinduced decrease in lc, measured in the cell body of a presynaptic neuron and a decrease in transmitter release from its terminals (Kretz et al., 1986). As changes in membrane potential alone, without a change in calcium influx, are unlikely to alter transmitter release (Zucker and Haydon, 1988), the inverse may also be true, i.e., a change in internal calcium concentration without a change in membrane potential is not effective at evoking release (see results in crayfish muscle; Hochner et al., 1989), and the voltage dependence of transmitter release seems to be accounted for by the voltage dependence of ICa (Augustine et al., 1987). This mechanism could directly regulate terminal Ica by neurotransmitters and may be of importance in the vertebrate central nervous system. Experimental
Procedures
Isolation of Neurons Three coronal slices (500 pm) through the brainstem at the level of the dorsal raphe nucleus were prepared from adult rats (ZOO-250 g) in a conventional manner using a Vibroslice machine. The dorsal raphe nucleus was carefully dissected out of the slice with a razor blade. The pieces of tissue were then incubated in a PIPES-buffered saline containing 0.75 mg/ml trypsin (Sigma type XI) under pure oxygen for 90 min according to the method of Kay and W o n g (1987). The pieces of tissue were then triturated in Dulbecco’s modified Eagle’s medium, and the isolated cells were allowed to settle to the bottom of a Falcon tissue culture dish (35 mm), to which they adhered well enough to be perfused at l-2 ml per min. Recording was carried out at room temperature, l8”C-20°C. Steinbusch et al. (1981) have shown with immunohistochemistry that most of the cells in a thin raphe slice with a soma diameter greater than 20 p m contain S-HT. W e have confirmed and extended these observations (Lawrence et al., 1989, Neurosci. Lett., suppl. 36, abstract) by showing that the proportion of isolated cells larger than 20 p m which stain for 5-HT, using a method similar to that of Yamamoto et al. (1981), is greater than 85%.
5-HT Action 757
on Raphe Neuron
Calcium
Current
Patch-Clamp Recording Conditions Neurons with truncated dendrites and a cell soma diameter of at least 20 pm were voltage-clamped using a switching voltageclamp amplifier (Axoclamp 2A) and a single patch pipette in the whole-cell configuration (Hamill et al., 1981). Pipettes pulled from thick-walled borosilicate glass (resistance from 6-10 MB) allowedaswitchingfrequencyof8-13KHzwitha30% dutycycle. The seal resistance measured by the voltage response to a 50 pA step of current was often greater than 5 CD). For this reason, the current traces are not leak-subtracted, and the capacity transients are intact. The voltage-clamp data were filtered at 1 KHz and digitized for storage at 16 bits on a video recorder for later analysis on a PDPII computer. Later experiments were run on line by an IBM PC 286 clone (DCS) and a CED 1401 interface. The resultant data were written to disk for later analysis. Solutions The external saline was designed to isolate Ica and contained 147 m M TEA-Cl, 5 m M BaCIz, 20 m M HEPES, 10 m M D-glucose, 5 m M Caminopyridine, and 0.2 PM tetrodotoxin (pH 7.2 with TEA-OH). In some experiments, 2 m M CaClz was used in place of BaCI,. The patch pipette in 17 experiments contained 63 m M cesium methanesulphonate, 40 m M HEPES, 2 m M Mg-ATP, cesium-11 m M ECTA, and 300 pM GTP (pH 7.2 with CsOH). The remainder of pipettes contained 70 m M Trizma phosphate, 33 m M Trizma Base-11 m M EGTA, 2 m M MgATP, 300 KM GTP, and 40 m M TEA-Cl (pH 7.2 with Trizma Base). The osmolarity was adjusted with sucrose so that the pipette solution was 10 mOsm hypoosmotic to the bath solution. This latter solution was used in all figures unless otherwise specified. Drugs were either dissolved in the extracellular solution and added to the perfusate or applied by diffusion from a patch pipette (tip 15 pm) lowered close to the cell (50 pm) and then removed from the bath. As a control, the same procedure was carried out without addition of 5.HT to the application pipette; this did not affect Ica. Acknowledgments We thank Dr. Ian Dawson for making NAN 190, Dr. A. P. Fox for helpful discussions, and the Wellcome Trust for support. Received
December
26, 1989; revised
February
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