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d-Lysergic acid diethylamide differentially affects the dual actions of 5-hydroxytryptamine on cortical neurons
Neuropharmacology Vol. 29, Printed in Great Britain. All
No. 8, pp. 705-712, rights reserved
1990 Copyright
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0028-3908/90 $3.00 + 0.00 1990 Pergamon Press plc
d-LYSERGIC ACID DIETHYLAMIDE DIFFERENTIALLY AFFECTS THE DUAL ACTIONS OF 5-HYDROXYTRYPTAMINE ON CORTICAL NEURONS PAMELAA. PIERCE and S. J. PEROUTKA* Department of Neurology, Stanford University Medical Center, Stanford, California 94305, U.S.A. (Accepted 5 February 1990)
Summary-S-Hydroxytryptamine (S-HT; 10m4M) produces an initial depolarization, followed by a long-lasting hyperpolarization, when focally applied to pyramidal neurons in the somatosensory cortex of the rat. Application of the selective S-HT,, agonist, 8-hydroxy-2-(di-N-propylamino)tetralin (g-OHDPAT; 10m6M) or d-lysergic acid diethylamide (d-LSD; 10m6M), produced only a hyperpolarizing response which was larger than the response to 5-HT. Application of I-OH-DPAT (10e6 M) and S-HT (10e4 M) together produced an initial depolarizing response, similar to the response with 5-HT alone, followed by a hyperpolarizing response which was 23 f 3% larger than with S-HT alone. By contrast, the application of d-LSD (10m6M) and 5-HT (10m4M) together produced either no depolarization (7 of 13 cells) or a significantly smaller depolarizing response (36 If:4% of the response to 5-HT alone), as well as a hyperpolarizing response which was 33 k 4% larger than with S-HT alone. Therefore, d-LSD displayed a unique pharmacological ability to both mimic and block the effect of 5-HT on single neurons in somatosensory cortex of the rat. Key words-d-LSD,
S-HT, S-HT,, receptor, S-HT, receptor, electrophysiology, cortex.
The neurophysiological effects of the neurotransmitter 5-hydroxytryptamine (5-HT) consist of both hyperpolarizing and depolarizing responses. The inhibitory effect of 5-HT on neurons in the CNS appears to be due to an increase in K+ conductance, mediated by 5-HT,, receptors (Segal and Gutnick, 1980; Aghajanian and Lakoski, 1984; Andrade, Malenka and Nicoll, 1986; Colino and Halliwell, 1987). The 5-HT-induced excitation, in contrast, is accompanied by a decrease in membrane K+ conductance (VanderMaelen and Aghajanian, 1980, 1982; Colino and Halliwell, 1987; Davies, Deisz, Prince and Peroutka, 1987) and has been attributed to activation of 5-HT, receptors (Roberts and Straughan, 1967; Penington and Reiffenstein, 1986; Davies et al., 1987). Both responses have been observed in individual neurons (Roberts and Straughan, 1967; Penington and Reiffenstein, 1986; Colino and Halliwell, 1987; Davies et al., 1987), indicating that both 5-HT,, and 5-HT, receptors can be present on single cells. The 5-HT, receptor has also been implicated in the CNS effects of hallucinogenic agents. The rank order potency of various hallucinogens at the 5-HT, receptor has been shown to correlate with dose levels of these agents in humans (Glennon, Titeler and McKenney, 1984). This correlation, together with findings from behavioral cue studies (Glennon, Young and Rosecrans, 1983; Colpaert, Meert, Niemegeers and Janssen, 1985; Nielsen, Ginn, Cunningham *To whom correspondence
should be addressed.
and Appel, 1985; Appel and Cunningham, 1986; Glennon, Titeler and Young, 1986), led to the hypothesis that hallucinogens act as direct agonists at the 5-HT, receptor. In the present study, intracellular recording techniques were used to determine the electrophysiological effects of 5-HT, the 5-HT,, agonist, 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT), and d-lysergic acid diethylamide (d-LSD) on pyramidal neurons in somatosensory cortex of the rat. By observing the neuronal membrane effects, mediated by the 5-HT,, and 5-HT, receptors, an attempt was made to determine agonist/antagonist actions of d-LSD on 5-HT1, and 5-HT, receptor sites in the cortex. No intracellular studies on the effects of d-LSD on the cortex have been reported to date. METHODS
Sprague-Dawley rats (100-l 50 g) were anesthetized with sodium pentobarbital (0.1 cm3/100 g, i.p.). The brains were then dissected. Slices from the somatosensory cortex were cut 400 p M thick using a Vibratome and transferred to an interface recording chamber. The slices were perfused with an artificial cerebrospinal fluid (CSF) saturated with 95% O,-5% CO2 and maintained between 33-34°C containing (in mM): NaCl 124, KC1 3, MgCl, 2, CaCl, 2, NaHCO, 26, NaH,PO, 1.2, glucose 10. Slices were allowed to equilibrate for one hour prior to recording. Microelectrodes were pulled, filled with 3 M potassium acetate and bevelled so that the resistance of 705
706
PAMELA A. PIERCE and
the electrode was 100-150 MO. Recordings were obtained from physiologically identified non-bursting cortical pyramidal neurons, located 60&900 p M below the surface of the pia, corresponding to lower layer 2/3 and upper layer 4. Input resistance was monitored by applying depolarizing or hyperpolarizing intracellular current pulses ( < 0.8 nA; 200 msec) through a bridge circuit and measuring the membrane voltage deflection. Drugs were dissolved in artificial CSF, injected into a microelectrode and focally applied to the slice by means of a timed N2 pulse, applied to the back of the pipette (15 psi). The duration of the pulse was adjusted for each electrode to produce a droplet approximately 100pm in diameter. Concentrations of drugs were chosen to be at least IOO-fold greater than the Ki value for the subtype of 5-HT receptor of interest (Peroutka, Lebovitz and Snyder, 1981; Peroutka, 1986). For antagonism experiments, application of both drugs was accomplished by mixing the solutions before injection into the puffer electrode. The following drugs and sources were used: 5-HT creatinine sulfate (Si~a), S-hydroxy-2-(di-~propylamino)tetralin hydrobromide (8-OH-DPAT) (Research Biochemicals, Inc.) and d-lysergic acid diethylamide tartrate (d-LSD) (National Institute for Drug Abuse). RESULTS
The electrophysiological effects of 5-HT, 8-OHDPAT and d-LSD were determined on 65 pyramidal neurons, located in lower layer 2/3 and upper layer 4 in slices of cortex using intracellular recording techniques. Cells displayed an average resting membrane potential of -73 & 1 mV (mean + SEM) and an average membrane resistance of 42 _t 2 Ma. In 6 out of 9 cells, pressure application of 5-HT (IO-” M) produced an initial depolarization, followed by a long-lasting hy~rpolarization which was present throughout the duration of the recording (up to 15 min; Figs 1A and 3). In the remaining 3 cells, 5-I-IT produced either a depolarizing response only (n = l), a hyperpolarizing response only (n = I), or no response (n = 1). Depolarizations induced by 5-HT averaged 4.5 f 0.4 mV and usually were accompanied by a decrease in membrane conductance averaging 24+ 9%, although a slight increase in conductance was observed in some cells (Fig. 1A). The 5-HT-induced depolarizations lasted an average of 1.4 + 0.3 min, with the longest depolarization lasting 2min (Fig. IB). A current-voltage (Z-I’) relationship graph before appIication of 5-HT and during the 5-HT-induced depolarization is shown in Figure l(C). The plots intersect at approximately - 90 mV, suggesting that a resting K+ conductance was decreased. The hyperpolarizing response to 5-HT averaged 3.9 + 0.5 mV and was accompanied by a 16 & 0.5% increase in membrane
S. J.
RROUTKA
conductance. An I-V graph before S-HT and during the hyperpolarizing response to S-HIT is shown in Figure l(D). The plots intersect at approximately -9OmV, suggesting that a resting K* conductance was increased. Pressure application of the 5-HTIA agonist 8-OHDPAT (10e6 M) produced only a hyperpolarizing response, along with an increase in conductance in 12 of 18 cells (Figs 2A and B). The duration of the hype~olarizing response to 8-OH-DPAT was shorter than for 5-HT, lasting less than 3 min. In 3 cells, only an increase in membrane conductance was observed, while 8-OH-DPAT produced no response in the remaining 3 cells. The 8-OH-DPAT-induced hyperpolarization averaged 5.9 _+ 1 mV with an average increase in membrane conductance of 14 + 3%. Lysergic acid diethyIamide (10e6 M) was pressureapplied onto a total of 9 cells. d-Lysergic acid diethylamide produced only a hyperpolarizing response, averaging 6.0 + 1.1 mV in 7 of the 9 cells. accompanied by an increase in conductance of 21 + 6% (Figs 2C and D). The duration of the hyperpolarizing response to d-LSD usually lasted the length of the recording (up to 15 min). however the cell shown in Figure 2(D) was the one exception. Two of the 9 cells showed no response to d-LSD. Application of 5-HT ( 10m4M) and 8-OH-DPAT ( lO-6 M) together produced an initial depoiarizdtion, followed by a hy~rpola~zation in 9 of 13 cells tested (Figs 3A and B). In the remaining 4 cells, either a hyperpolarizing response was produced (n = 1) or an increase in conductance was observed (n = l), while 2 cells showed no response. The combination of 5-HT and 8-OH-DPAT produced a membrane depolarization averaging 4.2 & 0.6 mV and lasting different from 1.I & 0.2 min. not signj~cantly the depolarizing response to 5-HT alone (P > 0.05; t-test). However, the hyperpolarizing response, due to the combination of 5-HT and 8-OH-DPAT averaged 4.8 + 0.7 mV, 23 + 3% larger than with 5-HT alone. In contrast, appIication of 5-HT (t0-4 M) and d-LSD (10m6 M) together produced either no depolarization (n = 7; Fig. 3C), or a significantly smaller depolarization than with 5-HT alone (1.6 k 0.2 mV; n = 6, P < 0.005). Hyperpolarizing responses to the combination of 5-HT and d-LSD were present in all of these cells (n = 13; Figs 3C and D). The 5-HT/d-LSD-induced hyperpolarization averaged 5.2 + 0.6 mV, 33 f 4% larger than with 5-HT alone. A total of 16 cells was tested; the remaining 3 cells showed no response. A summary of the membrane effects of 5-HT, 8-OH-DPAT and d-LSD is presented in Table I. The effects of 8-OH-DPAT and d-LSD on the depolarizing response to 5-HT are also displayed in Figure 4. These data demonstrate the ability of d-LSD to antagonize 5-HT-induced depolarizations. Six of the 13 cells responding to the combination of 5-HT and d-LSD displayed a significantly reduced membrane
707
Effects of d-LSD on cortical neurons 5-HT
(10-4M)
A
_______
C
D
fj-HT-lnduced
Depolarization
5-HT-hduced
Hyperpolarizetion
0
AfterJ-HT(lO-%i)
Fig. 1. The effects of 5-HT (low4 M) on pyramidal cells in rat somatosensory cortex of the rat. 5Hydroxytryptamine (5-HT) was applied at the time indicated by the black triangles. The values for the resting membrane potential are in mV and are marked by a dashed line (----). (A) Application of S-HT typically caused a depolarization, lasting approximately I min in duration, followed by a longer-lasting hy~rpola~zation. Depolarizing current pulses of 0.66 nA were used. (B) 5-Hydroxytryptamine produced a long-lasting depolarizing response (2 min), followed by a slight h~~la~zation in this pyramidal cell. The hy~rpolarizing current pulses (0.48 nA) allowed measurement of the 24% decrease in membrane conductance, during the depolarizing response. Due to the length of this response, the speed of the chart recorder was decreased 20-fold, twice during the trace (*). All action potentials present in these and the following figures were truncated by the chart recorder. (C) The current-voltage relationship, before S-HT (0) and during the initial depolarization to application of 5-HT (e). (D) Current-voltage relationship, before 5-HT (0) and during the hyperpolarizing response to application of S-HT (a). depolarization, compared to S-HT alone. Furthermore, 7 of the 13 cells displayed no depolarization to S-HT/d-LSD. No depolarization in the presence
of 5-HT occurred in only l/8 cells with 5-HT alone and l/10 cells with 5-HT in combination with 8-OH-DPAT.
PAMELA A.PIERCE and S. J. PEROUTKA
708
6-OH-DPATt10-6M)
d-LSD (10%)
-J
C
IOmV
IOSW
Y_____
-74__-
0
_-I
5llW
4sec s____--
-68 ___
Fig. 2. The effects of 8-OH-DPAT (IO-’ M) and d-LSD (10e6 M) on pyramidal cells of the somatosensory cortex. (A) Application of I-OH-DPAT caused only a hyperpolarizing response and an 18% increase in membrane conductance in this cell (I = 0.49 nA pulses). (B) 8-OH-DPAT caused a short hyperpolarization and a 27% increase in membrane conductance (I = 0.50 nA pulses). (C) Application of d-LSD produced only a hyperpolarizing response. with an increase in membrane conductance of 17% in this cell (I = 0.62 nA pulses). (Dj d-LSD caused a short hyperpolarization and a 10% increase in membrane conductance in this cell (I = 0.41 nA pulses). DISCUSSION
The major finding of the present study was that d-LSD both blocked and mimicked the effects of 5-HT at the single cell level in the somatosensory
cortex. In a large percentage of cortical pyramidal neurons, S-HT causes both a depolarizing response and a hyperpolarizing response. The selective 5-HT,, agonist. 8-OH-DPAT caused only a hyperpolarizing response and did not affect the depolarizing effects of 5-HT. d-Lysergic acid diethylamide was unique in that it inhibited 5-HT-induced membrane depolarization apparently by its antagonism of putative 5-HTz receptors (Roberts and Straughan, 1967; Penington
and Reiffenstein, 1986; Davies et al., 1987) and simultaneously mimicked the hyperpolarizing effects of 5-HT, possibly by activation of putative 5-HT,, receptors (Segal and Cutnick, 1980; Aghajanian and Lakoski, 1984; Andrade er al., 1986; Colino and Halliwell, 1987). Limited intracellular electrophysiological studies on the cortical effects of S-HT have been reported (Nedergaard, Engberg and Flatmen, 1986; Davies et al., 1987: Reynolds, Baskys and Carien, 1988). Relevant to the present study, Davies and colleagues recorded intracellularly from layer 4 of the cortex of the guinea pig and found that 5-HT produced both
Effects of d-LSD on cortical neurons
109
5-HT/8-OH-DPAT
S-W/d-LSD
c b
--_-
4sec
_-I
IOmV
10 set
Fig. 3. Effects of applications of 5-HT (1W4 M) together with either 8-OH-DPAT (1W6 M) or d-LSD (IOe6M) on pyramidal cells of the somatosensory cortex. (A and B) Application of S-HT and II-OH-DPAT together, produced a membrane depolarization, followed by a hyperpola~zation similar to the response with S-HT alone. Depolarizing current pulses of 0.53 and I .OnA were used, respectively. (C and D) Application of 5-HT and d-LSD together produced either a hyperpolarization only, shown in (C), or a significantly reduced depolarization, followed by a hyperpolarization. An example of this response is shown in (D). Depolarizing current pulses of 0.45 and 0.7 nA were used, respectively. Table I. ElTects of 5-HT. 8.OH-DPAT and d-LSD on the resting membrane potential of pyramidal cells in the somatosensory cortex Drug
N
5-HT ( W4 M) 5-HT/&OH-DPAT 5-HT/d-LSD S-OH-DPAT (IO-*Mf d-LSD (IO-” M)
9 I3 I6 18 9
Neuronal Membrane Response (mV) Depolarization Hyperpolarization No response 4.5 + 4.2 + 1.6 2 -
0.4 (7) 0.6 (9) 0.2 (6) (0) (0)
3.9 It:0.5 (7) 4.8 f 0.7 (10) 5.2 + 0.6 (13) 5.9 f l(l2) 6.0 f 1 (7)
(1) (3) (3) (6) (2)
Int~~llular recordings of pyramidal cells in somato~nsory cortex of the rat were performed as described in the text. N is the total number number of neurons tested in each drugapplication group. Changes in membrane potential are listed as the mean -i: standard error in mV for the number of neurons shown in parentheses.
PAMELA
5--KF
S-i-IT/ WH-OPAT
A. PIERCE and S. J. PEROIJTKA
5-H&f d-LSD
Fig. 4. Effects of 8-OH-DPAT and d-LSD on the depolar-
izing response to 5-HT. Only those cells responding to anolication of drugs are induded. (Al 5-HT-induced depokizations were unaffected by S-Ofi-DPAT but were reduced in the presence of d-LSD. Numbers in parenthesis are the fraction of ce& displaying a depolarizing response to 5-HT. fB) The percentage ofcetis which did not depolarize in the presence of 5-HT. Only cells showing a response to S-HT were included in the ~lculations.
a depolarizing elect, which could be blocked by ritanserin and cinanserin and a hy~~ola~~ng effect which was mimicked by &OH-DPAT. Membrane depolarization and increased neuronal excitability induced by 5-HT have been shown to be blocked by 5-HT, receptor antagonists in both cortex and hippocampus (Roberts and Straughan, 1967; Penington and Reiffenstein, 1986: Davies ef al., 1987). Specifically, this response has been blocked by the S-HT, antagonists ritanserin, cinanserin, methy sergide, metergohne, c~roheptadine and ketanserin. In the present study, d-LSD, but not I-OH-DPAT, prevented the depolarizing response to S-FIT in cortical pyramidal neurons, suggesting that d-LSD acts as an antagonist at S-HT2 receptors in the cortex. Moreover, the finding that 8-OH-DPAT and d-LSD did not produce depolarizing responses indicates that, at the applied concentrations, these drugs do not display agonist activity at the 5-HT, receptor in cortex.
The hy~rpola~zin~ effect of 5HT has been extensively studied in the hippocampus (Segal and Gutnick, 1980; Andrade et al., 1986; Colino and HalliwelI, 1987). S-Hydrox~ryp~mine, apparently acting through the S-HT,, receptor, causes an increase in KC conductance, resulting in a hyperpolarization of the cell and a decrease in firing rate. This response has been blocked using the 5-HT,, antagonists spiperone and propranolol and mimicked using the 5-H-I,, agonists &OH-DPAT and S-methoxytryptamine. The present finding that 5-HT, 8-OH-DPAT and d-LSD produced a hyperpolarization of cortical pyramidal cells suggest that these agents act as agonists at the putative 5-HT,, receptor in somatosensory cortex. In addition to the results of the present study, numerous physiological, behavioral and biochenlical data also suggest that d-LSD acts as an antagonist at 5-HT, receptors. Recently, nanomolar concentrations of d-LSD were shown to inhibit S-HTz-mediated hydrolysis of phosphatidyhnositol in the cortex of the rat (Pierce and Peroutka, 1988). In addition, d-LSD antagonizes S-HT-induced contraction of the guinea pig trachea (Heller and Baraban, 1987) and the rat uterus (Gaddum, Khan. Hathway and Stephens, 1955; Cerletti and Doepfner, 1958; Hashimoto, Hayashi, Nakahara, Niwagu~hi and Ishii, 1977), both shown to be 5-HT,-mediated effects (Van Nueten, Leysen, Vanhoutte and Ianssen, 1982; Millar, Facoory and Laverty, 1982; Cohen, Schenck, Colbert and Wittenauer, 1985). Moreover, d-LSD antagonizes 5-HT-stimulated edema of the paw of the rat (Doepfner and Cerletti, 1958) tryptamine induced seizures (Tedeschi, Tedeschi and Fellows. 1959) and head-twitch (Corne, Pickering and Warner, 1963; Gerber, Barbaz, Martin, Neale, Williams and Liebman, 1985). These effects have also been shown to be mediated by 5-HTz receptors (Leysen, Niemegeers, Tollenaere and Laduron, 1978; Peroutka et al., 1981; Ortmann, Bischoff, Radeke, Beuch and Dehni-St&a, 1982). While these data suggest that d-LSD inhibits 5-HTinduced effects through 5-HTz receptors, the possibility of mixed agonist-antagonist or weak partial agonist activity of d-LSD cannot be ruled out. For instance, head-twitch studies indicate that doseresponse curves for d-LSD are bell-shaped with weak partial agonist activity present at small doses and antagonist activity occurring at larger doses (Bedard and Pycock, 1977; Vetulani, Bednarczyk, Reichenberg and Rokosz, 1980). In the present study, however, experimental Iimitations, such as thickness of tissue and focal diffusion of the applied drug(s), severely limited the ability to determine drug doseresponse curves. It was found that application of low6 M d-LSD to the surface of the slice did not elicit a neuronal depolarization, regardless of the depth within the slice at which the neuron was impaled, suggesting that concentrations less than 1O.-6M also did not stimulate S-I-IT2 receptors. Future studies are
Effects
of d-LSD on cortical neurons
needed to further analyze the neuronal effects of d-LSD at nanomolar concentrations. Agonist stimulation of 5-HT, receptors has been suggested to mediate hallucinations produced by compounds such as d-LSD, dimethyltryptamine and the phenalkylamine hallucinogens. This theory is supported largely by two observations. First, hallucinogenic agents display an affinity for the 5-HT, receptor that correlates significantly with their potencies in humans (Glennon et al., 1984; Titeler, Lyon and Glennon, 1988). Secondly, d-LSD and other hallucinogens produce similar effects in drugdiscrimination studies (Glennon et al., 1986; Appel and Cunningham, 1986) and specific S-HT, antagonists block these discriminative cue effects (Glennon et al., 1983; Colpaert et al., 1985; Nielsen et al., 1985). Physiological and biochemical studies indicate that phenalkylamine hallucinogens act as partial agonists at 5-HT, receptors (Heller and Baraban, 1987; Pierce and Peroutka, 1988). In contrast, these same studies, along with the present data, indicate that the indolealkylamine hallucinogen d-LSD acts as a 5HT, receptor antagonist. Therefore, while the 5-HT, receptor might mediate some CNS effects of hallucinogenic agents, it is not clear that hallucinations are derived from direct activation of 5-HT, receptors. In summary, the mechanism of action of hallucinogenic agents in the CNS remains unknown. However, the effects of d-LSD on sensory perception during hallucinations might be a reflection of the unique dual interaction of this drug with 5-HT receptors in the somatosensory cortex. Further study on the effects of d-LSD in this region of cortex may elucidate the mechanism of action of this potent hallucinogen. Acknowledgements-This work was supported in part by the McKnight Foundation and NIH grants 23560-03 and 12151-15. REFERENCES Aghajanian G. K. and Lakoski .I. M. (1984) Hyperpolarization of serotonergic neurons by serotonin and LSD: studies in brain slices showing increased K+-conductance. Brain Res. 305: 181-185. Andrade R., Malenka R. and Nicoll R. A. (1986) A G protein couples serotonin and GABA, receptors to the same channels in hippocampus. Science 234: 1261-1265. Appel J. B. and Cunningham K. A. (1986) The use of drug discrimination procedures to characterize hallucinogenic drug actions. Psychopharmac. Bull. 22: 959-967. Bedard P. and Pycock C. J. (1977) “Wet-dog” shake behaviour in the rat: a possible quantitative model of central 5-hydroxytryptamine activity. Neuropharmacology 16: 663670. Cerletti A. and Doepfner W. (1958) Comparative study on the serotonin antagonism of amide derivatives of lysergic acid and of ergot alkaloids. J. Pharmac. 122: 124136. Cohen M. L., Schenck K. W., Colbert W. and Wittenauer L. (1985) Role of 5-HT2 receptors in serotonin-induced contractions of nonvascular smooth muscle. J. Pharmac. exp. Ther. 23% 770-774.
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No. 8, pp. 705-712, rights reserved
1990 Copyright
0
0028-3908/90 $3.00 + 0.00 1990 Pergamon Press plc
d-LYSERGIC ACID DIETHYLAMIDE DIFFERENTIALLY AFFECTS THE DUAL ACTIONS OF 5-HYDROXYTRYPTAMINE ON CORTICAL NEURONS PAMELAA. PIERCE and S. J. PEROUTKA* Department of Neurology, Stanford University Medical Center, Stanford, California 94305, U.S.A. (Accepted 5 February 1990)
Summary-S-Hydroxytryptamine (S-HT; 10m4M) produces an initial depolarization, followed by a long-lasting hyperpolarization, when focally applied to pyramidal neurons in the somatosensory cortex of the rat. Application of the selective S-HT,, agonist, 8-hydroxy-2-(di-N-propylamino)tetralin (g-OHDPAT; 10m6M) or d-lysergic acid diethylamide (d-LSD; 10m6M), produced only a hyperpolarizing response which was larger than the response to 5-HT. Application of I-OH-DPAT (10e6 M) and S-HT (10e4 M) together produced an initial depolarizing response, similar to the response with 5-HT alone, followed by a hyperpolarizing response which was 23 f 3% larger than with S-HT alone. By contrast, the application of d-LSD (10m6M) and 5-HT (10m4M) together produced either no depolarization (7 of 13 cells) or a significantly smaller depolarizing response (36 If:4% of the response to 5-HT alone), as well as a hyperpolarizing response which was 33 k 4% larger than with S-HT alone. Therefore, d-LSD displayed a unique pharmacological ability to both mimic and block the effect of 5-HT on single neurons in somatosensory cortex of the rat. Key words-d-LSD,
S-HT, S-HT,, receptor, S-HT, receptor, electrophysiology, cortex.
The neurophysiological effects of the neurotransmitter 5-hydroxytryptamine (5-HT) consist of both hyperpolarizing and depolarizing responses. The inhibitory effect of 5-HT on neurons in the CNS appears to be due to an increase in K+ conductance, mediated by 5-HT,, receptors (Segal and Gutnick, 1980; Aghajanian and Lakoski, 1984; Andrade, Malenka and Nicoll, 1986; Colino and Halliwell, 1987). The 5-HT-induced excitation, in contrast, is accompanied by a decrease in membrane K+ conductance (VanderMaelen and Aghajanian, 1980, 1982; Colino and Halliwell, 1987; Davies, Deisz, Prince and Peroutka, 1987) and has been attributed to activation of 5-HT, receptors (Roberts and Straughan, 1967; Penington and Reiffenstein, 1986; Davies et al., 1987). Both responses have been observed in individual neurons (Roberts and Straughan, 1967; Penington and Reiffenstein, 1986; Colino and Halliwell, 1987; Davies et al., 1987), indicating that both 5-HT,, and 5-HT, receptors can be present on single cells. The 5-HT, receptor has also been implicated in the CNS effects of hallucinogenic agents. The rank order potency of various hallucinogens at the 5-HT, receptor has been shown to correlate with dose levels of these agents in humans (Glennon, Titeler and McKenney, 1984). This correlation, together with findings from behavioral cue studies (Glennon, Young and Rosecrans, 1983; Colpaert, Meert, Niemegeers and Janssen, 1985; Nielsen, Ginn, Cunningham *To whom correspondence
should be addressed.
and Appel, 1985; Appel and Cunningham, 1986; Glennon, Titeler and Young, 1986), led to the hypothesis that hallucinogens act as direct agonists at the 5-HT, receptor. In the present study, intracellular recording techniques were used to determine the electrophysiological effects of 5-HT, the 5-HT,, agonist, 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT), and d-lysergic acid diethylamide (d-LSD) on pyramidal neurons in somatosensory cortex of the rat. By observing the neuronal membrane effects, mediated by the 5-HT,, and 5-HT, receptors, an attempt was made to determine agonist/antagonist actions of d-LSD on 5-HT1, and 5-HT, receptor sites in the cortex. No intracellular studies on the effects of d-LSD on the cortex have been reported to date. METHODS
Sprague-Dawley rats (100-l 50 g) were anesthetized with sodium pentobarbital (0.1 cm3/100 g, i.p.). The brains were then dissected. Slices from the somatosensory cortex were cut 400 p M thick using a Vibratome and transferred to an interface recording chamber. The slices were perfused with an artificial cerebrospinal fluid (CSF) saturated with 95% O,-5% CO2 and maintained between 33-34°C containing (in mM): NaCl 124, KC1 3, MgCl, 2, CaCl, 2, NaHCO, 26, NaH,PO, 1.2, glucose 10. Slices were allowed to equilibrate for one hour prior to recording. Microelectrodes were pulled, filled with 3 M potassium acetate and bevelled so that the resistance of 705
706
PAMELA A. PIERCE and
the electrode was 100-150 MO. Recordings were obtained from physiologically identified non-bursting cortical pyramidal neurons, located 60&900 p M below the surface of the pia, corresponding to lower layer 2/3 and upper layer 4. Input resistance was monitored by applying depolarizing or hyperpolarizing intracellular current pulses ( < 0.8 nA; 200 msec) through a bridge circuit and measuring the membrane voltage deflection. Drugs were dissolved in artificial CSF, injected into a microelectrode and focally applied to the slice by means of a timed N2 pulse, applied to the back of the pipette (15 psi). The duration of the pulse was adjusted for each electrode to produce a droplet approximately 100pm in diameter. Concentrations of drugs were chosen to be at least IOO-fold greater than the Ki value for the subtype of 5-HT receptor of interest (Peroutka, Lebovitz and Snyder, 1981; Peroutka, 1986). For antagonism experiments, application of both drugs was accomplished by mixing the solutions before injection into the puffer electrode. The following drugs and sources were used: 5-HT creatinine sulfate (Si~a), S-hydroxy-2-(di-~propylamino)tetralin hydrobromide (8-OH-DPAT) (Research Biochemicals, Inc.) and d-lysergic acid diethylamide tartrate (d-LSD) (National Institute for Drug Abuse). RESULTS
The electrophysiological effects of 5-HT, 8-OHDPAT and d-LSD were determined on 65 pyramidal neurons, located in lower layer 2/3 and upper layer 4 in slices of cortex using intracellular recording techniques. Cells displayed an average resting membrane potential of -73 & 1 mV (mean + SEM) and an average membrane resistance of 42 _t 2 Ma. In 6 out of 9 cells, pressure application of 5-HT (IO-” M) produced an initial depolarization, followed by a long-lasting hy~rpolarization which was present throughout the duration of the recording (up to 15 min; Figs 1A and 3). In the remaining 3 cells, 5-I-IT produced either a depolarizing response only (n = l), a hyperpolarizing response only (n = I), or no response (n = 1). Depolarizations induced by 5-HT averaged 4.5 f 0.4 mV and usually were accompanied by a decrease in membrane conductance averaging 24+ 9%, although a slight increase in conductance was observed in some cells (Fig. 1A). The 5-HT-induced depolarizations lasted an average of 1.4 + 0.3 min, with the longest depolarization lasting 2min (Fig. IB). A current-voltage (Z-I’) relationship graph before appIication of 5-HT and during the 5-HT-induced depolarization is shown in Figure l(C). The plots intersect at approximately - 90 mV, suggesting that a resting K+ conductance was decreased. The hyperpolarizing response to 5-HT averaged 3.9 + 0.5 mV and was accompanied by a 16 & 0.5% increase in membrane
S. J.
RROUTKA
conductance. An I-V graph before S-HT and during the hyperpolarizing response to S-HIT is shown in Figure l(D). The plots intersect at approximately -9OmV, suggesting that a resting K* conductance was increased. Pressure application of the 5-HTIA agonist 8-OHDPAT (10e6 M) produced only a hyperpolarizing response, along with an increase in conductance in 12 of 18 cells (Figs 2A and B). The duration of the hype~olarizing response to 8-OH-DPAT was shorter than for 5-HT, lasting less than 3 min. In 3 cells, only an increase in membrane conductance was observed, while 8-OH-DPAT produced no response in the remaining 3 cells. The 8-OH-DPAT-induced hyperpolarization averaged 5.9 _+ 1 mV with an average increase in membrane conductance of 14 + 3%. Lysergic acid diethyIamide (10e6 M) was pressureapplied onto a total of 9 cells. d-Lysergic acid diethylamide produced only a hyperpolarizing response, averaging 6.0 + 1.1 mV in 7 of the 9 cells. accompanied by an increase in conductance of 21 + 6% (Figs 2C and D). The duration of the hyperpolarizing response to d-LSD usually lasted the length of the recording (up to 15 min). however the cell shown in Figure 2(D) was the one exception. Two of the 9 cells showed no response to d-LSD. Application of 5-HT ( 10m4M) and 8-OH-DPAT ( lO-6 M) together produced an initial depoiarizdtion, followed by a hy~rpola~zation in 9 of 13 cells tested (Figs 3A and B). In the remaining 4 cells, either a hyperpolarizing response was produced (n = 1) or an increase in conductance was observed (n = l), while 2 cells showed no response. The combination of 5-HT and 8-OH-DPAT produced a membrane depolarization averaging 4.2 & 0.6 mV and lasting different from 1.I & 0.2 min. not signj~cantly the depolarizing response to 5-HT alone (P > 0.05; t-test). However, the hyperpolarizing response, due to the combination of 5-HT and 8-OH-DPAT averaged 4.8 + 0.7 mV, 23 + 3% larger than with 5-HT alone. In contrast, appIication of 5-HT (t0-4 M) and d-LSD (10m6 M) together produced either no depolarization (n = 7; Fig. 3C), or a significantly smaller depolarization than with 5-HT alone (1.6 k 0.2 mV; n = 6, P < 0.005). Hyperpolarizing responses to the combination of 5-HT and d-LSD were present in all of these cells (n = 13; Figs 3C and D). The 5-HT/d-LSD-induced hyperpolarization averaged 5.2 + 0.6 mV, 33 f 4% larger than with 5-HT alone. A total of 16 cells was tested; the remaining 3 cells showed no response. A summary of the membrane effects of 5-HT, 8-OH-DPAT and d-LSD is presented in Table I. The effects of 8-OH-DPAT and d-LSD on the depolarizing response to 5-HT are also displayed in Figure 4. These data demonstrate the ability of d-LSD to antagonize 5-HT-induced depolarizations. Six of the 13 cells responding to the combination of 5-HT and d-LSD displayed a significantly reduced membrane
707
Effects of d-LSD on cortical neurons 5-HT
(10-4M)
A
_______
C
D
fj-HT-lnduced
Depolarization
5-HT-hduced
Hyperpolarizetion
0
AfterJ-HT(lO-%i)
Fig. 1. The effects of 5-HT (low4 M) on pyramidal cells in rat somatosensory cortex of the rat. 5Hydroxytryptamine (5-HT) was applied at the time indicated by the black triangles. The values for the resting membrane potential are in mV and are marked by a dashed line (----). (A) Application of S-HT typically caused a depolarization, lasting approximately I min in duration, followed by a longer-lasting hy~rpola~zation. Depolarizing current pulses of 0.66 nA were used. (B) 5-Hydroxytryptamine produced a long-lasting depolarizing response (2 min), followed by a slight h~~la~zation in this pyramidal cell. The hy~rpolarizing current pulses (0.48 nA) allowed measurement of the 24% decrease in membrane conductance, during the depolarizing response. Due to the length of this response, the speed of the chart recorder was decreased 20-fold, twice during the trace (*). All action potentials present in these and the following figures were truncated by the chart recorder. (C) The current-voltage relationship, before S-HT (0) and during the initial depolarization to application of 5-HT (e). (D) Current-voltage relationship, before 5-HT (0) and during the hyperpolarizing response to application of S-HT (a). depolarization, compared to S-HT alone. Furthermore, 7 of the 13 cells displayed no depolarization to S-HT/d-LSD. No depolarization in the presence
of 5-HT occurred in only l/8 cells with 5-HT alone and l/10 cells with 5-HT in combination with 8-OH-DPAT.
PAMELA A.PIERCE and S. J. PEROUTKA
708
6-OH-DPATt10-6M)
d-LSD (10%)
-J
C
IOmV
IOSW
Y_____
-74__-
0
_-I
5llW
4sec s____--
-68 ___
Fig. 2. The effects of 8-OH-DPAT (IO-’ M) and d-LSD (10e6 M) on pyramidal cells of the somatosensory cortex. (A) Application of I-OH-DPAT caused only a hyperpolarizing response and an 18% increase in membrane conductance in this cell (I = 0.49 nA pulses). (B) 8-OH-DPAT caused a short hyperpolarization and a 27% increase in membrane conductance (I = 0.50 nA pulses). (C) Application of d-LSD produced only a hyperpolarizing response. with an increase in membrane conductance of 17% in this cell (I = 0.62 nA pulses). (Dj d-LSD caused a short hyperpolarization and a 10% increase in membrane conductance in this cell (I = 0.41 nA pulses). DISCUSSION
The major finding of the present study was that d-LSD both blocked and mimicked the effects of 5-HT at the single cell level in the somatosensory
cortex. In a large percentage of cortical pyramidal neurons, S-HT causes both a depolarizing response and a hyperpolarizing response. The selective 5-HT,, agonist. 8-OH-DPAT caused only a hyperpolarizing response and did not affect the depolarizing effects of 5-HT. d-Lysergic acid diethylamide was unique in that it inhibited 5-HT-induced membrane depolarization apparently by its antagonism of putative 5-HTz receptors (Roberts and Straughan, 1967; Penington
and Reiffenstein, 1986; Davies et al., 1987) and simultaneously mimicked the hyperpolarizing effects of 5-HT, possibly by activation of putative 5-HT,, receptors (Segal and Cutnick, 1980; Aghajanian and Lakoski, 1984; Andrade er al., 1986; Colino and Halliwell, 1987). Limited intracellular electrophysiological studies on the cortical effects of S-HT have been reported (Nedergaard, Engberg and Flatmen, 1986; Davies et al., 1987: Reynolds, Baskys and Carien, 1988). Relevant to the present study, Davies and colleagues recorded intracellularly from layer 4 of the cortex of the guinea pig and found that 5-HT produced both
Effects of d-LSD on cortical neurons
109
5-HT/8-OH-DPAT
S-W/d-LSD
c b
--_-
4sec
_-I
IOmV
10 set
Fig. 3. Effects of applications of 5-HT (1W4 M) together with either 8-OH-DPAT (1W6 M) or d-LSD (IOe6M) on pyramidal cells of the somatosensory cortex. (A and B) Application of S-HT and II-OH-DPAT together, produced a membrane depolarization, followed by a hyperpola~zation similar to the response with S-HT alone. Depolarizing current pulses of 0.53 and I .OnA were used, respectively. (C and D) Application of 5-HT and d-LSD together produced either a hyperpolarization only, shown in (C), or a significantly reduced depolarization, followed by a hyperpolarization. An example of this response is shown in (D). Depolarizing current pulses of 0.45 and 0.7 nA were used, respectively. Table I. ElTects of 5-HT. 8.OH-DPAT and d-LSD on the resting membrane potential of pyramidal cells in the somatosensory cortex Drug
N
5-HT ( W4 M) 5-HT/&OH-DPAT 5-HT/d-LSD S-OH-DPAT (IO-*Mf d-LSD (IO-” M)
9 I3 I6 18 9
Neuronal Membrane Response (mV) Depolarization Hyperpolarization No response 4.5 + 4.2 + 1.6 2 -
0.4 (7) 0.6 (9) 0.2 (6) (0) (0)
3.9 It:0.5 (7) 4.8 f 0.7 (10) 5.2 + 0.6 (13) 5.9 f l(l2) 6.0 f 1 (7)
(1) (3) (3) (6) (2)
Int~~llular recordings of pyramidal cells in somato~nsory cortex of the rat were performed as described in the text. N is the total number number of neurons tested in each drugapplication group. Changes in membrane potential are listed as the mean -i: standard error in mV for the number of neurons shown in parentheses.
PAMELA
5--KF
S-i-IT/ WH-OPAT
A. PIERCE and S. J. PEROIJTKA
5-H&f d-LSD
Fig. 4. Effects of 8-OH-DPAT and d-LSD on the depolar-
izing response to 5-HT. Only those cells responding to anolication of drugs are induded. (Al 5-HT-induced depokizations were unaffected by S-Ofi-DPAT but were reduced in the presence of d-LSD. Numbers in parenthesis are the fraction of ce& displaying a depolarizing response to 5-HT. fB) The percentage ofcetis which did not depolarize in the presence of 5-HT. Only cells showing a response to S-HT were included in the ~lculations.
a depolarizing elect, which could be blocked by ritanserin and cinanserin and a hy~~ola~~ng effect which was mimicked by &OH-DPAT. Membrane depolarization and increased neuronal excitability induced by 5-HT have been shown to be blocked by 5-HT, receptor antagonists in both cortex and hippocampus (Roberts and Straughan, 1967; Penington and Reiffenstein, 1986: Davies ef al., 1987). Specifically, this response has been blocked by the S-HT, antagonists ritanserin, cinanserin, methy sergide, metergohne, c~roheptadine and ketanserin. In the present study, d-LSD, but not I-OH-DPAT, prevented the depolarizing response to S-FIT in cortical pyramidal neurons, suggesting that d-LSD acts as an antagonist at S-HT2 receptors in the cortex. Moreover, the finding that 8-OH-DPAT and d-LSD did not produce depolarizing responses indicates that, at the applied concentrations, these drugs do not display agonist activity at the 5-HT, receptor in cortex.
The hy~rpola~zin~ effect of 5HT has been extensively studied in the hippocampus (Segal and Gutnick, 1980; Andrade et al., 1986; Colino and HalliwelI, 1987). S-Hydrox~ryp~mine, apparently acting through the S-HT,, receptor, causes an increase in KC conductance, resulting in a hyperpolarization of the cell and a decrease in firing rate. This response has been blocked using the 5-HT,, antagonists spiperone and propranolol and mimicked using the 5-H-I,, agonists &OH-DPAT and S-methoxytryptamine. The present finding that 5-HT, 8-OH-DPAT and d-LSD produced a hyperpolarization of cortical pyramidal cells suggest that these agents act as agonists at the putative 5-HT,, receptor in somatosensory cortex. In addition to the results of the present study, numerous physiological, behavioral and biochenlical data also suggest that d-LSD acts as an antagonist at 5-HT, receptors. Recently, nanomolar concentrations of d-LSD were shown to inhibit S-HTz-mediated hydrolysis of phosphatidyhnositol in the cortex of the rat (Pierce and Peroutka, 1988). In addition, d-LSD antagonizes S-HT-induced contraction of the guinea pig trachea (Heller and Baraban, 1987) and the rat uterus (Gaddum, Khan. Hathway and Stephens, 1955; Cerletti and Doepfner, 1958; Hashimoto, Hayashi, Nakahara, Niwagu~hi and Ishii, 1977), both shown to be 5-HT,-mediated effects (Van Nueten, Leysen, Vanhoutte and Ianssen, 1982; Millar, Facoory and Laverty, 1982; Cohen, Schenck, Colbert and Wittenauer, 1985). Moreover, d-LSD antagonizes 5-HT-stimulated edema of the paw of the rat (Doepfner and Cerletti, 1958) tryptamine induced seizures (Tedeschi, Tedeschi and Fellows. 1959) and head-twitch (Corne, Pickering and Warner, 1963; Gerber, Barbaz, Martin, Neale, Williams and Liebman, 1985). These effects have also been shown to be mediated by 5-HTz receptors (Leysen, Niemegeers, Tollenaere and Laduron, 1978; Peroutka et al., 1981; Ortmann, Bischoff, Radeke, Beuch and Dehni-St&a, 1982). While these data suggest that d-LSD inhibits 5-HTinduced effects through 5-HTz receptors, the possibility of mixed agonist-antagonist or weak partial agonist activity of d-LSD cannot be ruled out. For instance, head-twitch studies indicate that doseresponse curves for d-LSD are bell-shaped with weak partial agonist activity present at small doses and antagonist activity occurring at larger doses (Bedard and Pycock, 1977; Vetulani, Bednarczyk, Reichenberg and Rokosz, 1980). In the present study, however, experimental Iimitations, such as thickness of tissue and focal diffusion of the applied drug(s), severely limited the ability to determine drug doseresponse curves. It was found that application of low6 M d-LSD to the surface of the slice did not elicit a neuronal depolarization, regardless of the depth within the slice at which the neuron was impaled, suggesting that concentrations less than 1O.-6M also did not stimulate S-I-IT2 receptors. Future studies are
Effects
of d-LSD on cortical neurons
needed to further analyze the neuronal effects of d-LSD at nanomolar concentrations. Agonist stimulation of 5-HT, receptors has been suggested to mediate hallucinations produced by compounds such as d-LSD, dimethyltryptamine and the phenalkylamine hallucinogens. This theory is supported largely by two observations. First, hallucinogenic agents display an affinity for the 5-HT, receptor that correlates significantly with their potencies in humans (Glennon et al., 1984; Titeler, Lyon and Glennon, 1988). Secondly, d-LSD and other hallucinogens produce similar effects in drugdiscrimination studies (Glennon et al., 1986; Appel and Cunningham, 1986) and specific S-HT, antagonists block these discriminative cue effects (Glennon et al., 1983; Colpaert et al., 1985; Nielsen et al., 1985). Physiological and biochemical studies indicate that phenalkylamine hallucinogens act as partial agonists at 5-HT, receptors (Heller and Baraban, 1987; Pierce and Peroutka, 1988). In contrast, these same studies, along with the present data, indicate that the indolealkylamine hallucinogen d-LSD acts as a 5HT, receptor antagonist. Therefore, while the 5-HT, receptor might mediate some CNS effects of hallucinogenic agents, it is not clear that hallucinations are derived from direct activation of 5-HT, receptors. In summary, the mechanism of action of hallucinogenic agents in the CNS remains unknown. However, the effects of d-LSD on sensory perception during hallucinations might be a reflection of the unique dual interaction of this drug with 5-HT receptors in the somatosensory cortex. Further study on the effects of d-LSD in this region of cortex may elucidate the mechanism of action of this potent hallucinogen. Acknowledgements-This work was supported in part by the McKnight Foundation and NIH grants 23560-03 and 12151-15. REFERENCES Aghajanian G. K. and Lakoski .I. M. (1984) Hyperpolarization of serotonergic neurons by serotonin and LSD: studies in brain slices showing increased K+-conductance. Brain Res. 305: 181-185. Andrade R., Malenka R. and Nicoll R. A. (1986) A G protein couples serotonin and GABA, receptors to the same channels in hippocampus. Science 234: 1261-1265. Appel J. B. and Cunningham K. A. (1986) The use of drug discrimination procedures to characterize hallucinogenic drug actions. Psychopharmac. Bull. 22: 959-967. Bedard P. and Pycock C. J. (1977) “Wet-dog” shake behaviour in the rat: a possible quantitative model of central 5-hydroxytryptamine activity. Neuropharmacology 16: 663670. Cerletti A. and Doepfner W. (1958) Comparative study on the serotonin antagonism of amide derivatives of lysergic acid and of ergot alkaloids. J. Pharmac. 122: 124136. Cohen M. L., Schenck K. W., Colbert W. and Wittenauer L. (1985) Role of 5-HT2 receptors in serotonin-induced contractions of nonvascular smooth muscle. J. Pharmac. exp. Ther. 23% 770-774.
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