- NEUROPSYCHOPHARMACOLOGY 1990-VOL. 3, 0.2 137
Lysergic Acid Diethylamide (LSD) Administration Selectively Downregulates Serotonin, Receptors in Rat Brain Neil S. Buckholtz, Ph.D., * Dongfeng Zhou, M,D./ Daniel X. Freedman, M.D., and William Z. Potter, M.D., Ph.D.
A dosage regimen of lysergic acid diethylamide (LSD) that hours, but not 96 hours, after the last administration. The reliably produces behavioral tolerance in rats was indole hallucinogen psilocybin [1.0 mJ?lkg (3.5 IJ-mollkg) evaluated for effects on neurotransmitter receptor binding for 8 days] also produced a significant decrease in 5HT2 in rat brain using a variety of radioligands selective for binding, but neither the nonhallucinogenic analog bromo- amine receptor subtypes. Daily administration of LSD LSD [1.3 mglkg (2.4 IJ-mollkg) for 5 days] nor mescaline [130 IJ-glkg (0.27 IJ-mollkg) intraperitoneally (IP)] for 5 [10 mglkg (40.3 IJ-mollkg) for 5 or 10 days] affected 5-HT2 days produced a decrease in serotonin, (5- binding. These observations suggest that LSD and other hydroxytryptamine2, 5-HT2) binding in cortex (measured indole hallucinogens may act as 5-HT2 agonists at 24 hours after the last drug administration) but did not postsynaptic 5-HT2 receptors. Decreased 5-HTz binding affect binding to other receptor systems (5-HTIA' 5-HTIB' strikingly parallels the development and loss of behavioral f3-adrenergic, CXl- or cx2-adrenergic, D2-dopaminergic) or to tolerance seen with repeated LSD administration, but the a recognition site for 5-HT uptake. The decrease was decreased binding per se cannot explain the gamut of evident within 3 days of LSD administration but was not behavioral tolerance and cross-tolerance phenomena among demonstrable after the first LSD dose. Following 5 days of the indole and phenylethylamine hallucinogens. LSD administration the decrease was still present 48 [Neuropsychophannacology 3:137-148, 19901
KEY WORDS: Lysergic acid diethylamide (LSD); Repeated administration at 24-hour intervals of d-Iy- Hallucinogens; Mescaline; Psilocybin; Serotonin receptors; sergic acid diethylamide (LSD)produces tolerance to 5-Hydroxytryptamine receptors; Ketanserin certain behavioral and physiologic effects of the drug both in humans and animals (reviewed in Freedman 1984, 1986) and to psychedelic effects in humans. Tolerance appears to be a pharmacodynamic phe- From the National Institute of Mental Health, Laboratory of nomenon because shifts in drug metabolism and Clinical Science, Section on Clinical Pharmacology, Bethesda, MD 20892 (N.S.B., D.Z., W.Z.P.), and the Department of Psychiatry clearance are not accountable (Winter 1971).Whether and Biobehavioral Sciences, School of Medicine, University of Cal- a salient neurochemical or receptor basis for toler- ifornia, Los Angeles, CA 90024 (D.X.F.). ance can be identified is, however, still unclear. We Address reprint requests to: Dr. Neil Buckholtz, NIMH, Park- lawn Building, Room 9C-26, 5600 Fishers Lane, Rockville, MD report here on our search for receptor correlates of 20857. tolerance. Received June 15, 1989; revised October 26, 1989; accepted No- Effects of a single administration of LSD in the vember 1, 1989. • Present address: Nationallnstitute of Mental Health, Parklawn rat are fairly selective to brain serotonin (5-hydroxy- Building, Room 9C-26, 5600 Fishers Lane, Rockville, Maryland tryptamine, 5-HT) metabolism and include a de- 20857. creased 5-HT release and increased nerve ending and t Visiting Fellow under the United States-Peoples Republic of China Joint Health Agreement. Present address: Institute of vesicular storage or retention, with a consequent in- Mental Health, Beijing Medical University, Beijing, P.R. China. crease in whole brain 5-HT concentration, a prompt
0893-133X/90/$0.00 138 N.S. Buckholtz et al. NEUROPSYCHOPHARMACOLOGY 1990-VOL. 3, NO.2
decrease in 5-hydroxyindoleacetic acid (5-HIAA), ferent from those seen in vitro, where LSD is approx- and a later period of decreased synthesis and de- imately equipotent in its effects on 5-HTI and 5-HT2 creased metabolism (reviewed in Freedman 1981, receptor binding (Peroutka and Snyder 1982). The 1986; Freedman and Boggan 1981; Halaris and finding of selectivity for 5-HT2 binding was consis- Freedman 1977; Jacobs and Trulson 1979; Hamon tent with a variety of recent data indicating a selec- 1984). Most measured neurochemical effects com- tive involvement of 5-HT2receptors with the behav- mence and terminate (coincidentally with drug half- ioral effects of LSD (Jacobs 1987). life) during the first 60 minutes post-LSD, the period The LSD dose (260 J.Lg/kg)selected for our initial during which there is an abrupt "pause" and cessa- studies (Buckholtz et a1. 1985) was toward the upper tion of bar pressing by rats for a food reward induced limit of those that can demonstrate behavioral toler- by the drug or by its psychoactive congeners ance on operant schedules. The reliable minimal dose (Freedman et a1. 1964). This effect in the rat (and my- [130 ug/kg 0.27 (umol/kgl] and the temporal param- driasis in the human and animals) shows tolerance or eters required to demonstrate behavioral tolerance to cross-tolerance after appropriate dosage regimens. LSD in the rat had been studied first for rope Repeated administration of LSD produces a climbing behavior (Freedman et a1. 1958) and exten- somewhat diminished magnitude and duration of sively with operant schedules for food reinforcement the brain 5-HT, 5-HIAA, and, strikingly, of trypto- (Freedman et a1. 1964; Appel and Freedman 1965), phan effects (Freedman and Boggan 1974) and also and a degree of acute tolerance with these measures affects the drug-induced changes in brain catechol- had been observed as well (Freedman and Agha- amines and tyrosine (Smith et a1.1975)or plasma cor- janian 1959; Freedman et a1. 1964). The temporal ticosterone (reviewed in Freedman 1981; Freedman parameters of development and loss of tolerance and Boggan, 1981). In general, the rate of change in with this dose in the rat roughly paralleled those ob- both 5-HT and catecholamines observed during the served with the appropriate dosage in the human first 60 minutes after LSD is accelerated after multiple (Freedman 1984). daily doses. The exact relationship of behavioral tol- Therefore, to more precisely relate the receptor erance to these "60-minute" measures and to enzy- response to the temporal parameters for the acquisi- matic modifications noted 18 to 24 hours post-LSD tion and loss of behavioral tolerance, we used the re- after weeks of daily dosage (Diaz and Huttunen 1971; liable lower dose of LSD (130 J.Lg/kg)anda more spe- Peters and Tang 1977) is, however, obscure. The se- cific 5-HT2 receptor radioligand ([3H]ketanserin). To lective and potent effect of LSD on the electrophysio- further examine the specificity of the 5-HT2receptor logic activity of dorsal raphe serotonergic neurons response, we also used relatively selective radioli- also implicates the serotonergic system, but shows gands for catecholaminergic receptor subtypes. Be- no correlation with behavioral tolerance (Trulson and cause other hallucinogens such as psilocybin and Jacobs 1979a; Trulson et a1. 1981). mescaline show behavioral cross-tolerance to LSD in LSD clearly affects postsynaptic serotonergic re- the rat (Appel and Freedman 1968;Rech et a1. 1975), ceptor binding in vitro (Peroutka and Snyder 1982). we also evaluated whether they induced parallel Trulson and Jacobs (1979b) administered LSD [100 types of receptor changes. J.Lg/kg(0.21J.Lmollkg)]torats every 6 hours for 4 days and reported a reduction in [3H]5-HT and [3H]LSD METHODS binding in forebrain and in brainstem plus spinal cord 24 hours after the final administration. How- Animals ever, since both [3H]5-HTand [3H]LSDlabel multiple 5-HT receptor subtypes (Peroutka and Snyder 1982; Male Sprague-Dawley rats (275 to 350 g; n = 213) Peroutka 1986), a selective change in 5-HT receptor were obtained from Taconic Farms. They were subtypes could not be defined. housed four to six per cage under standard labora- Because 5~HTsubtypes are differentially affected tory conditions with ad libitum access to food and by treatments with drugs or lesions (Dumbrille-Ross water and a 12 hour light-dark cycle. and Tang 1983; Quik and Azmitia 1983), we sought such distinctions following systemic administration Drugs of LSD. We initially examined the effect of daily ad- ministration of LSD [260 ug/kg (0.54 umol/kg)] for 10 The following drugs were purchased from Sigma: days and found a decrease in 5-HT2 but not 5-HTI isoproterenol, 5-HT oxalate, alprenolol, norepineph- binding [measured with [3H]LSD using 5-HT and rine, and sulpiride. LSD tartrate, psilocybin, mesca- cinanserin to estimate 5-HTI and 5-HT2receptors, re- line HeI, and bromo-LSD hydrogen tartrate (BOL) spectively (Buckholtz et a1. 1985)]. The receptor re- were obtained from the National Institute on Drug sponses after systemic administration were thus dif- Abuse. The following drugs were donated by the re- NEUROPSYCHOPHARMACOLOGY J990-VOL. 3, NO.2 LSDDownregulates5-HTz Receptors 139 spective companies: methysergide maleate (Sandoz), using a Brandel manifold, and the filters were cinanserin (Squibb), fluoxetine (Lilly), and norzimeli- washed three to four times with 5 ml cold homogeni- dine (Astra). zation buffer. For 3H ligands, filters were placed in glass scintillation vials, 10 ml scintillation fluid was added, and vials were counted in a liquid scintilla- Radioligands tion spectrometer at approximately 29% efficiency. The radioligands used, their sources, and representa- For the ICYP binding, filters were placed in glass tive specific activities are as follows: [3H]8-hydroxy- tubes and counted directly in a gamma counter at N,N-dipropyl-2-aminotetralin (8-0H-DPAT), Amer- 73% efficiency. sham, 160 CiJmmol; [3H]ketanserin, New England The drugs used to define specific binding are in- Nuclear (NEN), 70 CiJmmol; [3H]prazosin, NEN, 80 dicated in Table 1. Representative average percent Ci/mmol; [3H]rauwolscine, NEN, 75 Ci/mmol; specific binding in cortex for the various radioligands [3H]para-aminoclonidine (PAC), NEN, 40 CiJmmol; at the concentrations shown in Table 1 are as follows: [3H]spiperone, NEN, 25 Ci/mmol; [3H]dihydroal- [3H]8-0H-DPAT, 68%; [3H]ketanserin, 85%; prenolol (DHA), NEN, 50 Ci/mmol; [3H]imipramine, [12SI]cyanopindolol,59%; [3H]prazosin, 87%; [3H]rau- NEN, 75 Ci/mmol: [3H]paroxetine, NEN, 25 Cilmmol; wolscine, 83%; [3H]PAC, 59%; [3H]spiperone, 70%; [12SI]cyanopindolol(ICYP), NEN, 2200 Cilmmol. [3HlDHA, 72%; [3Hlimipramine, 70%; [3Hlparoxe- tine, 92%. Protein concentration in the homogenate was as- Drug Administration sayed according to Lowry et al. (1951). LSD, bromo-LSD, mescaline, and psilocybin were administered intraperitoneally (IP) at a volume of 1.0 Data Analysis mlIkg body weight. Drugs were dissolved in 0.9% saline. Rats were killed by decapitation 24 hours after Scatchard analysis was done by linear regression of the last drug administration, except in the time bound ligand versus boundlfree. Control versus ex- course of recovery study, where periods of 48 and 96 perimental group differences were analyzed by Stu- hours were also examined. dent's t test and control versus multiple experimental groups in the LSD dose-response study by Dun- nett's t test (Winer 1971). Tissue Preparation Brains were dissected on ice into various parts (in- cluding cortex, hippocampus, striatum, midbrain, RESULTS hypothalamus, and brainstem), frozen on dry ice, and stored at - 70°C until assayed. Tissues were ho- Following administration of 130 f.Lg/kgof LSD for 5 mogenized in ice-cold Tris-HCl buffer, centrifuged at days, there was a 22% decrease in Bmax for [3H]ketan- 40,000 g for 10 minutes, the pellet washed two to four serin binding in cortex (mean ± SO = 279 ± 59 times by rehomogenization and recentrifugation, and fmol/mg protein for LSD, n = 10, and 358 ± 82 frnol/ the final pellet taken up in incubation buffer (Table mg protein for saline, n = 9; P < 0.05) without any 1). For 5-HTIA' 5-HTIB'and 5-HT2binding, there was change in Kd (mean ± SO = 0.34 ± 0.03 for both an intermediate preincubation step (10 minutes at LSD and saline). 37°C). The next study began an evaluation of the time dependency and specificity of the receptor change using a one-point radioligand concentration analysis. Receptor Binding As shown in Table 2, [3H]ketanserin (5-HT2)but not The conditions for binding are shown in Table 1. For [3H]8-0H-DPAT (5-HT1A),binding was significantly Scatchard analysis of 5-HT2 binding, [3HJketanserin decreased within three days in hippocampus and concentrations were 0.1 to 3.0 nmol/L, and ethanol midbrain. Within 5 days, decreases in [3Hlketanserin was used to dilute the radioligand and the methyser- binding were also significant in cortex and brain- gide (Leysen et al. 1982). Ethanol was not used in stem. Not shown in the Table 2, there was only a analyses where only one [3H]ketanserin concentra- nonsignificant 8% reduction in cortex 24 hours after tion was used, and it had no effect on binding. one administration of 130 f.LglkgLSD(LSD = 161 ± Following incubation, the solution was vacuum 13 fmollmg protein, n = 6; saline = 175 ± 15 fmoll filtered over GF/B filters (presoaked with 0.3% mg protein, n = 6). polyethyleneimine [PEl] for [3H]ketanserin and In separate groups that received 130 f.Lg/kgLSD [3H]imipramine binding and 0.05% PEl for [3H]par- or saline for 5 days and were killed 24, 48, or 96 oxetine binding) or GF/C filters for ICYP binding hours after the last administration, there was a sig- 140 N.S. Buckholtz et al. NEUROPSYCHOPHARMACOLOGY 1990-VOL. 3, NO.2
Table 1. Incubation Conditions for Receptor Binding Studies
Total Radioligand, Buffer, Time/Temp Volume Nonspecific Receptor Reference Concentration pH at 23°C (min/°C) (ml) Binding 5-HTIA Hall et al. (1985) [3H]8-0H-DPAT, 50 mmol/L Tris, 10/37 0.5 10 umol/l. 1.0 nmol/L pH 7.6 serotonin 5-HTIB Hoyer et al. [l25I]cyanopindolol, 10 mmol/L Tris, 90/37 0.25 10 umol/L (1985) 125 pmol/L 0.9% NaCl, serotonin + 10 umol/L 30 urnol/L pargyline, isoproterenol pH7.7 5-HT2 Leysen et al. [3H]ketanserin, 50 mmol/L Tris, 30/37 4.4 1.0 urnol/L (1982) 0.5 nmol/L pH 7.6 methysergide (Xl-adrenergic Vantini et al. [3H]prazosin, 50 mmol/L Tris, 40/23 2.0 0.1 mmollL (1984); 0.1 nmollL 0.5 mmollL norepinephrine Quennedey et EDTA, pH al. (1984) 7.4 (Xz-adrenergic Vantini et al. [3H]rauwolscine, 50 mmollL Tris, 30/23 1.0 0.1 mmollL (1984); 1.0 nmollL 0.5 mmollL norepinephrine Quennedey et EDTA, a!. (1984) pH 7.4 (Xz-adrenergic Vantini et aI. [3H]PAC, 50 mmollL Tris, 30/23 1.0 0.01 mmollL (1984); 1.0 nmol/L 0.5 mmol/L norepinephrine Quennedey et EDTA,pH al. (1984) 7.4 dopamine, Meller et al. pH]spiperone, 50 mmol/L Tris, 30/37 2.0 20 umol/L (1985) 0.4 nmol/L 120 mmollL sulpiride + NaCl, 0.1 urnol/L 5 mmol/L cinanserin KCl, 2 mmollL CaClz,l mrnollL MgClz, pH 7.6 j3-adrenergic Bylund and PH]DHA, 50 mmollL Tris, 30/23 1.0 0.1 umol/L Snyder (1976) 0.5 nmollL pH8.0 (- )alprenolol 5-HT uptake Marcusson et al. [3Hjimipramine, 50 mmollL Tris, 60/0 0.5 1.0IJ-mollL (1985) 2.5 nmollL 120 mmollL norzimelidine NaCl, 5 mmollL KCl, pH 7.0 5-HT uptake Habert et al. [3H]paroxetine, 50 mmol/L Tris, 60/23 2.0 10 umol/L (1985) 0.5 nmollL 120 mmollL fluoxetine NaCl,5 mmollL KCl, pH 7.4 nificant 23% decrease in [3H]ketanserin binding in kg), 32.5 ug/kg (0.068 umol/kg), 65 j.lg/kg (0.135 cortex at 24 hours (LSD = 108 ± 3 fmollmg protein, urnol/kg), and 130 j.lglkg (0.27 umol/kg) LSD and sa- saline = 142 ± 13 fmollmg protein, n = 3 per group; line (n = 8 per group) on [3H]ketanserin binding in p < 0.02) and a modest but significant 15% decrease cortex. The overall analysis of variance indicated a at 48 hours (LSD = 123 ± 7 fmollmg protein; saline significant dose effect (F = 5.98, dt = 4/35, P < 0.01), = 144 ± 15 fmollmg protein, n = 6 per group; p < and subsequent comparisons at the p < 0.01 level of 0.01), but no difference at 96 hours (LSD = 111 ± 10 significance indicated that there was a significant fmol/mg protein, saline = 115 ± 5 fmol/mg protein, 19% reduction after 130 j.lglkg (LSD = 107 ± 11, sa- n = 6 per group). line = 132 ± 13 fmol/mg protein), small but nonsig- A dose-response study compared the effects of nificant 8% reductions after 65 and 32.5 j.lg/kg (121 ± administration for 5 days of 16.25 j.lglkg (0.034 umol/ 11 and 122 ± 9 fmol/mg protein, respectively), and NEUROPSYCHOPHARMACOLOGY 1990- VOL. 3, NO.2 LSD Downregulates 5-HT2 Receptors 141
Table 2. Effects of Daily Administration of LSD (130 ug/kg) for 3 or 5 Days on [3H]Ketanserin (5-HT2) and [3H]8-0H-DPAT (5-HT1A)Specific Binding in Various Brain Regions of Rats Killed 24 hr After the Last Administration
[3HlKetanserin" PHlS-OH-DP ATb 3 days 5 days 3 days 5 days Brain % % % % Region Saline LSD change Saline LSD change Saline LSD change Saline LSD change fmol/mg protein fmol/mg protein Cortex 197 157 -20 192 146 -24d 168 168 0 158 150 -5 ± 49C ± 38 ± 37 ± 12 ± 48 ± 49 ± 52 ± 36 Hippocampus 37 27 -27d 39 28 -27d 282 282 0 266 251 -6 ± 6 ± 8 ± 9 ± 6 ± 37 ± 15 ± 40 ± 18 Midbrain 31 21 -32' 31 20 -36' 185 168 -9 170 172 +1 ± 13 ± 7 ± 14 ± 6 ± 28 ± 31 ± 27 ± 23 Striatum 107 102 -5 102 91 -11 108 112 +4 106 106 0 ± 25 ± 19 ± 16 ± 25 ± 28 ± 39 ± 19 ± 22 Hypothalamus 29 23 -21 25 20 -17 153 162 +6 154 146 -5 ± 14 ± 8 ± 6 ±7 ± 43 ±44 ± 48 ± 45 Brainstem 15 14 -6 16 13 -20' 111 112 +1 114 115 +1 ± 2 ±2 ± 3 ± 2 ± 25 ± 16 ± 21 ± 22 • pH]Ketanserin concentration, 0.5 nmollL. b ['H]8-0H-DPAT concentration, 5.0 nmollL. C Values are mean ± SD for seven animals per group. d Significantlydifferent from saline control, p < 0.05, two-tailed test. e Significantlydifferent from saline control, p < 0.05, one-tailed test. no change after 16.25 f.Lglkg(132 ± 14 fmol!mg pro- hallucinogens mescaline and psilocybin were evalu- tein). ated after daily administration. Neither BOL nor To further evaluate whether effects were specific mescaline altered [3H]ketanserin binding in cortex, to 5-HT2receptors, 130 f.,LglkgLSDwas administered but psilocybin produced a 38% reduction in binding for 5 days and binding to a variety of receptors was (Table 4). Following 10 mglkg (40.3 urnol/kg) mesca- determined. Among the ten radioligand binding sites line administration for 5 days and 1.0 mg/kg (3.5 investigated, only that associated with 5-HT2binding urnol/kg) psilocybin for 8 days, binding was also was affected (Table 3). evaluated for other brain regions and other re- In the last study, the effects of the nonhallucino- ceptors. After mescaline, no changes were seen for genic LSD analog bromo-LSD (BOL) as well as the [3H]ketanserin binding in midbrain, or for [3H]DHA,
Table 3. Effects of Daily Administration of LSD (130 ug/kg) for 5 Days on Specific Binding to a Variety of Receptors in Cortex (and Striatum for Dopamine, Receptors) of Rats Killed 24 hr After the Last LSD Administration Specific Binding"
Receptor Radioligand Saline LSD % Change (fmol/mg protein) 5-HTIA [3H]8-0H-DP ATb 46 ± 6 46 ± 6 a 5-HTlB [125I]cyanopindolol 24 ± 2 24 ± 3 a 5-HT2 [3H]ketanserin 173 ± 12 134 ± 15 -23c (cpm/mg protein) 5-HT uptake [3H]imipramine 42 ± 5 39 ± 3 -7 5-HT uptake [3H]paroxetine 4.6 ± 0.3 4.4 ± 0.2 -4 • Values are mean ± SD for six animals per group. b Concentration of radioligands shown in Table 1. C Significantlydifferent from saline control, p < O.OS . ._------142 N.S. Buckholtz et al. NEUROPSYCHOPHARMACOLOGY 1990- VOL. 3,NO.2 Table 4. Effects of Daily Administration of Various Drugs on Specific Binding of [3H]Ketanserin in Cortex of Rats Killed 24 hr After the Last Drug Administration Specific Binding Drug Dose Duration Saline Drug % Change (mglkg) (days) (fmol/mg protein) Mescaline 10 5 162 ± 18" 152 ± 12 -6 Mescaline 10 10 157 ± 8 160 ± 11 +2 Bromo-LSD 1.3 5 157 ± 24 169 ± 8 +8 Psilocybin 1.0 8 147 ± 17 91 ± 10 -38b a Values shown are mean ± SD for six animals per group. b Significantly different from saline control, p < 0.002. [3H]prazosin or [3H]rauwolscine binding in cortex, tration of approximately its Kd value. Although the hippocampus, or striatum. After psilocybin, no reduction in eH]ketanserin binding seen with LSD changes were seen for [3H]DHA, [3H]prazosin or was only 17% to 25%, this change was highly reliable [3H]rauwolscine binding in cortex, but there was a in cortex, and was seen in every study we did. This significant 16% reduction in [3H]8-0H-DPAT binding magnitude of change is also in the range seen with in cortex (psilocybin = 34.0 ± 4.0, saline = 40.6 ± chronic administration of antidepressant drugs (e.g., 4.7 fmoVmg protein, n = 6/group; p < 0.05). Grigoriadis et aI. 1989). In order to further identify regional variations and to be able to measure poten- tially larger changes in more specific regions, the DISCUSSION next logical step in subsequent studies would be to use autoradiographic analysis. Overall, these results show that behavioral tolerance The highest amount of 5-HT2binding was found to LSD is associated with a decrease in 5-HT2 re- in cortex, confirming other results (Leysen et al. ceptor binding. The time course of the receptor 1982; Peroutka and Snyder 1981; Blackshear et al. change strikingly parallels that for the development 1981). The largest percent decreases in 5-HT2binding and loss of behavioral tolerance in rats (Freedman et with LSD occurred in cortex, midbrain, and hippo- a1. 1958, 1964;Winter 1971;Rech et a1. 1975), and the campus. Our previous study (Buckholtz et a1. 1985) change was specific to the 5-HT2receptor. There are indicated a somewhat larger decrease in striatum, other parallels. Thus, a dose-response effect on but the variability in the current study may have pre- 5-HT2binding was not evident below 130 fJ-g/kg,and cluded seeing a significant change. Interestingly, doses well below 130 fJ-glkgdid not demonstrate con- operant behavior in rats was disrupted by LSD ad- sistently reliable or useful quantitative effects on bar ministered directly into either prefrontal cortex, mid- pressing for food reward (Freedman et a1.1964);with brain raphe, or hippocampus (Mokler et al. 1986). doses of 20 ug/kg (0.04 urnol/kg) no LSD effect was With regard to selectivity to the 5-HT2receptor, it detectable on fixed ratio (FR) schedules (Appel et a1. is, first of all, interesting that although LSD is equi- 1970a). There may also be a relationship of receptor potent in vitro at 5-HT1 and 5-HT2 receptors (Per- change with acute tolerance (Freedman et a1. 1958) outka and Snyder 1982), after its daily administration because 24 hours after a single very high dose of LSD there is a change only in 5-HT2receptors. At the time [650 fJ-g/kg(1.35 fJ-mol/kg)]there was a significant de- we did these studies, we did not evaluate the re- crease in 5-HT2 binding (Buckholtz et a1. 1988). Of cently identified 5-HT subtypes such as 5-HTIC' the other hallucinogens assessed in this study, psilo- 5-HTlD, and 5-HT3' The 5-HT1Creceptor may be par- cybin, an indole like LSD, decreased [3H]ketanserin ticularly relevant because it is pharmacologically and binding, whereas mescaline, a phenylethylamine, structurally similar to the 5-HT2 receptor (Hoyer did not. However, other phenylethylamine halluci- 1988) and LSD has nanomolar affinity to it (Yagaloff nogens such as 001 and DOB do decrease 5-HT2 and Hartig 1986). Second, although LSD interacts binding as shown previously by us (Buckholtz et a1. with dopamine (DA) receptor systems (Kelly and 1988) and others (McKenna et al. 1989b; Pranzatelli Iversen 1975;Creese et a1.1975),we found no change 1988). in O2 type DA receptors in striatum as defined by Because our present studies initially using Scat- [3H]spiperone binding. Trulson and Jacobs (1979b) chard analysis of [3H]ketanserin binding showed no also reported no change in [3H]spiperone binding fol- change in the Kd value after daily LSD administra- lowing chronic LSD. To our knowledge 01 receptor tion, in the subsequent measures we used a one- binding has not yet been evaluated in this paradigm. point binding analysis with a [3H]ketanserin concen- Third, we investigated the o-noradrenergic receptor NEUROPSYCHOPHARMACOLOGY 1990- VOL. 3, NO.2 LSD Downregulates 5-HTz Receptors 143 system because of a report that yohimbine, an it is the result of residual drug in the incubation be- such as components of tactile startle (Geyer et al. cause tissue is extensively washed, there is no effect 1978) and bar-pressing for food (Rech and Commis- at 24 hours after a single administration, and other saris 1982). Tilson and Sparber (1973)concluded that reports indicate that LSD clears from brain within 4 the two drugs, although sharing a cross-tolerance hours, even at doses higher than those used here and similar components of action, acted on two (Rosecrans et al. 1967;Diab et al. 1971).Also, there is different receptors. Neurophysiologic differences no evidence for accumulation of LSD following 3 between LSD and mescaline have been shown on days of administration (Winter 1971). The mecha- suppression of dorsal raphe neurons (Haigler and nism for LSD's agonist action may involve both an Aghajanian 1973),but their actions are similar in po- excess of 5-HT in the nerve ending (by enhanced re- tentiating the postsynaptic effect of monoamines on tention and decreased release, perhaps via a non-5- facial motoneurons (McCall and Aghajanian 1980) HT2autoreceptor effect) and a simultaneous deficit of and in facilitating locus coeruleus activation by pe- 5-HT in the synaptic cleft and thus putatively at the ripheral stimuli (Aghajanian 1980).Unfortunately, no postsynaptic receptor (Freedman 1986). In this envi- tolerance studies on these effects have been re- ronment of decreased 5-HT at the receptor, a partial ported, and these are critical to settle, agonist action of LSD could be facilitated at the 5-HT2 For a fully satisfactory explanation of the behav- receptor site (Sanders-Bush et al. 1988). LSD may ioral-receptor relationship, procedures that atten- also affect other sites such as that labeled by DOB uate the effects of LSD in humans such as chronic (Lyon et al. 1987; Peroutka et al. 1988) that may be (but not acute) monoamine oxidase (MAO) inhibition different from the 5-HT2receptor or may be a 5-HT2 should also downregulate the 5-HT2 receptor (Res- subtype (Schmidt and Peroutka 1989). nick et al. 1964; Peroutka and Snyder 1980). Al- Whatever the actual situation, regulation of the though MAO inhibitors do so, chronic administra- 5-HT2 receptor has been puzzling. The receptor is tion of BOL, which can purportedly "block" the be- downregulated both by a direct agonist and by an- havioral response to LSD in humans (Balestrieri and tagonists (Blackshear et al. 1983, 1986; Leysen et al. Fontanari 1959) or rat (Appel and Freedman 1968), 1986)as well as by indirect agonists such as reuptake did not produce receptor downregulation. Without blockers and monoamine oxidase inhibitors (Per- documenting ambiguities in the older literature, we outka and Snyder 1980). The receptor is, however, believe that for clear inferences in linking receptor upregulated by reserpine (Stockmeier and Kellar change with blocking, attenuation, or enhancement 1986). of the LSD effect, new experiments in humans with The other major aspect of the current results in- careful control of dose and time of pretreatment volves the other compounds studied. The nonhallu- regimens of BOL (or chlorpromazine or MAO inhib- cinogenic LSD analog, bromo-LSD, did not affect itors) and measurements of mydriasis are required 5-HT2binding. This is consistent with the results of (Freedman and Halaris 1978). Trulson and Jacobs (1979b)measuring [3H]5-HTand In humans, pretreatment with reserpine mark- [3H]LSD binding. Because the hallucinogens psilo- edly enhances the effect of LSD (Freedman 1961;Is- cybin and mescaline show cross-tolerance to LSD in bell and Logan 1957), and, in animals variously de- both humans (Wolbach et al. 1962; Isbell et al. 1961) pleted of brain 5-HT, there is a striking fourfold low- and animals (Appel and Freedman 1968;Winter 1971; ering of the threshold dose for LSDbehavioral effects Rech et al. 1975), we thought that they might also (Appel and Freedman 1964;Appel et al. 1970a,b). Al- reduce 5-HT2binding. This was true only for the in- though direct effects on 5-HT2 receptor binding of doleamine psilocybin, which in addition produced a different depleting procedures reportedly differ small but significant decrease in 5-HTlA binding. (Stockmeier and Kellar 1986), it is possible that the Mescaline surprisingly did not affect 5-HT2 LSD receptor effect could be enhanced with these binding whereas structurally related hallucinogens different depletion regimens. Thus, in very prelimi- such as 001, DOB, and DOM did (Buckholtz et al. nary studies, we tested whether reserpine and PCPA 1988;Pranzatelli 1988;McKenna et al. 1989b; Leysen pretreatment could potentiate a reduction in 5-HT2 et al. 1989). It may be important, however, that at a binding by a low dose of LSD (32,5 j.1g/kg)and did binding site labeled by [17Br]DOB,DOB and 001 observe a trend in this direction, were more than 400 times and LSD was almost 100 In the human, tolerance to LSD is observed for times as potent as mescaline (Peroutka et al. 1988). the psychoactive as well as certain autonomic effects There are also a number of differences between the such as mydriasis (Isbell et al. 1961;Freedman 1961). neurochemical effects of LSD and mescaline that Not all effects of LSD show tolerance, although this could contribute to different mechanisms of action has clearly been established only in animals and not (Tilson and Sparber 1972;Freedman et al. 1970;Stolk studied in humans. In animals there is tolerance to et al. 1974) as well as different behavioral effects, food-reinforced effects on FR schedules but not to NEUROPSYCHOPHARMACOLOGY 1990-VOL. 3, NO.2 LSD Downregulates 5-HT2 Receptors 145 shock-reinforced behaviors such as avoidance or Appel JB, Freedman DX (1968): Tolerance and cross-toler- escape (Hamilton 1960) or to drug discrimination ance among psychotomimetic drugs. Psychopharma- procedures or to parasympathetic effects of LSD cologia 13:267-274 (Freedman et al. 1958). Thus the exact link of the Appel JB, Lovell RA, Freedman DX (1970a): Alterations in 5-HT2receptor to mechanisms specifically and selec- the behavioral effects of LSD by pretreatment with p- tively effecting tolerance to mydriasis and psyche- chlorophenylalanine and o-methyl-p-tyrosine. Psycho- pharmacologia 18:387-406 delic and behavioral effects remains to be identified. What is clear is that both tolerance dosage regimens Appel JB, Sheard MH, Freedman DX (1970b): Alterations in the behavioral effects of LSD by midbrain raphe le- and 5-HT2blockade (by ritanserin, for example) block sions. Comm Behav Biol 5:237-241 a selective sequence of effects initiated by LSD. Balestrieri A, Fontanari D (1959): Acquired and crossed tol- Whatever the dose and temporally contingent erance to mescaline, LSD-25, and BOL-148. Arch Gen "counterprocesses" set into motion by LSD that ac- Psychiatry 1:279-282 count for tolerance, one would have hoped that the Barchas JD, Freedman DX (1963): Brain amines: Response sites or mechanisms would respond similarly to mes- to physiological stress. Biochem Pharmacol 12:1232- caline and LSD. The drugs may act through different 1235 mechanisms when they produce not only common Blackshear MA, Steranka LR, Sanders-Bush E (1981): Mul- behavioral and psychedelic effects, but tolerance and tiple serotonin receptors: Regional distribution and ef- cross-tolerance to them. If intersecting common areas fect of raphe lesions. Eur J Pharmacol 76:325-334 or circuits are entailed and identified, this would be Blackshear MA, Friedman RL, Sanders-Bush E (1983): directly useful in determining what neural systems Acute and chronic effects of serotonin (5HT) antago- are critical in "turning on" or diminishing a psyche- nists on serotonin binding sites. Naunyn-Schmiede- bergs Arch PharmacoI324:125-129 delic effect. In sum, we believe that studies of receptor inter- Blackshear MA, Martin LL, Sanders-Bush E (1986): Adap- tive changes in the 5-HT2 binding site after chronic ad- actions with psychedelic drugs can provide initial ministration of agonists and antagonists. Neurophar- leads in the pursuit of the link between behavioral macology 25:1267-1271 tolerance and receptor change and point to pathways Buckholtz NS, Freedman DX, Middaugh LD (1985): Daily critical for their uniquely interesting mental effects. LSD administration selectively decreases serotonin, re- These studies have not, as yet, produced a fully satis- ceptor binding in rat brain. Eur J Pharmacol 109:421- factory explanation for mode of action. Thus, further 425 research, perhaps focusing on processes beyond the Buckholtz NS, Zhou D, Freedman DX (1988): Serotonin, clearly implicated 5-HT2 receptor-processes that agonist administration down-regulates rat brain sero- amplify or attenuate receptor responses such as re- tonin, receptors. Life Sci 42:2439-2445 ceptor-effector coupling and second and third mes- Bylund DB, Snyder SH (1976): Beta adrenergic receptor sengers-should be pursued to account adequately binding in membrane preparations from mammalian brain. Mol Pharmacol 12:568-580 for the neurobiologic processes underlying the be- Chase TN, Breese GR, Kopin IJ (1967): Serotonin release havioral phenomena of tolerance and cross-toler- from brain slices by electrical stimulation: regional dif- ance. ferences and effect of LSD. Science 157:1461-1463 Colpaert FC (1984): Cross generalization with LSD and yo- himbine in the rat. Eur J Pharmacol102:541-544 ACKNOWLEDGMENT Colpaert FC, Meert TF, Niemegeers CJE, Janssen PAJ (1985): Behavioral and 5-HT antagonist effects of ritan- The excellent technical assistance of Emelia Johnson is serin: A pure and selective antagonist of LSD discrimi- gratefully acknowledged. nation in rat. Psychopharmacology 86:45-54 This research was supported, in part, by the Howard Conn PJ, Sanders-Bush E (1987): Central serotonin re- Pack Foundation and the Judson Braun Chair of Psychiatry ceptors: Effector systems, physiological roles and regu- and Pharmacology (D.X.F.). lation. 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