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Ketamine inhibits serotonin uptake in vivo
KETAMINE
INHIBITS
SEROTONIN
UPTAKE
IN
L’ZVO”
L. L. MARTIN~$, RENA L. BOUTHAL and D. J. SMITHS Departments
of Ph~rmac~~io~y
and Toxicology and An~sthcsiolo~y, West Virginia Mor~anto~n. WV 26506. U.S.A.
C!niversity
Medical
Ccntet..
Summary Anesthetic (120 and 160 mgkg. i.p.) and subanesthetic (80mg’kg) doses of kctamine HCI were found to prevent completely the depletion of whole brain serotonin (5-HT) by p-chloronmphctamine (PCA). Furthermore, ketaminc HCI (160 m&kg) completely blocked the depletion of 5-HT by PC.4 in every individual brain region studied (Midbrain-thalamus. hypothalamus. strtatum. hippocampus and cortex). Administration of ketamine alone had no effect on brain 5-HT Irvcls. Ninlnmidc (;I monoamine oxidase (MAO) inhibitor) and fluoxetine (a selective 5-HT uptake inhibitor) also prevented the depletion of 5-HT by PCA. However. of these three agents. only nialamide prevented the dcplctron of 5-HT by reserpine. These results suggest that ketamine blocks PCA-induced 5-HT depletion by inhibiting 5-HT uptake and not by inhibiting MAO. Ketamme only weakly atrected either [‘HIS-HT or [~tilspiropcridol binding to 5-HT, and S-HTz receptors respectively even at concentrations as high a< t mM. These data support the contention that the primary direct effect of ketamine on ser(~t~~i~er~ic systems is the blockade of 5-HT uptake and that hlocksde of 5-HT uptake may mediate some of the b~h~~~ior~il etfects of ketamine. such as analgesia.
K&amine is a general anesthetic agent which provides good analgesia for surgery (McCarthy. Chcn, Kaump and Ensor, 1965), but unlike most anesthetics, does not depress the cardiovascular or respiratory systems (Lanning and Harmel. 1975: Virtue. Alanis, Mori, Lafargue, Vogel and Metcalf. 1967). On the negative side. ketamine is also unique because emergence from ketamine anesthesia is often associated with extrapyramidai movements. unpleasant dreams, hallucinations and other psychological disturbances (Albin and Dresner, 1970). Ketamine has previously been demonstrated to be a moderately preferential inhibitor of the high affinity uptake of 5-hydroxytryptamine (5-HT) into crude synaptosomal preparations of the rat brain (Azzaro and Smith, 1977). The ICsO for the inhibition of 5-HT uptake hy ketamine was reported to be approximately 50k~M. Cohen. Chan, Way and Trevor (1973) have reported that the recovery of the righting reflex in rats occurs when brain ketamine levels are approximately 0.03 mg/g tissue or about 130 PM (assuming the brain is a single compartment). When considered together. these data suggest that anesthetic and subanesthetic doses of ketamine should produce brain levels of the
* This wsork was supported by research funds from the U.S. Public Health Service Grant S-T32-GM07039 and the West Virginia University Anesthesiology Research Fund. t The work presented here is in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Pharmacology and Toxicology. West Virginia University. 1 Present address: Tennessee Neurophyschi~tric Institute. 1501 MLlrfreesboro Road. Nashville. TN 37217, U.S.A. $ To whom reprint requests should be sent.
drug which would be in the range necessary to inhibit the uptake of 5-HT in c$it\o. Thus. there existed a strong possibility that blockade of 5-HT uptake might mediate some of the behavioral a&or physiological effects of ketamine. Many of the behavioral phenomena like those produced by ketamine in patients have been suggested to be associated with alterations in serotonergic function. These include analgesia (Messing and Lytle, 1977; Samanin, Miranda and Mennini. 1978; Yaksh and Wilson. 1979), hallucinations (Aghajanian. Foote and Sheard, 1977) and motor disturhantes (Holman, Seagraves. Elliot and Barchas. 1976; Trulson, Ross and Jacobs, 1976; Sloviter. Drust and Conner, 1978). For these reasons. it was decided to determine whether ketamine could inhibit the uptake of 5-HT in view. To accomplish this, the ability of ketamine to prevent the depletion of brain 5.HT by ~-chIoroamphetamine (PCA) was studied. Fuller. Perry and Molloy (1975) have shown that 5-HT uptake inhibitors can be distingLlished iit riro by their ability to prevent the depletion of 5-HT by Pf4. p-Chloroamphetaminc requires a neuronul high atfinity 5-HT uptake process to produce its effect. Some agents such as quipazine and 6-chloro-2-( lpiperazinyl)-pyrazine (MK-212). which are known to inhibit 5-HT uptake (Hamon. Bourgoin. Enjalbcrt, Bockaert. Henry, Ternaux and Glowinski. 1976: Clineschmidt, Totaro. Pflueger and McGuffin. 1978) have also been shown to inhibit the binding of 5-HT to its receptor sites in the brain (Nelson. Bourgoin, Gfowinski and Hamon, 197X). Therefore. it would also be impl~rtant to study the effects of ketaminc on 5-HT receptor binding. The interaction between ketamine and both 5-1-11-t and 5-HTz receptors was
II4
L. I..
studied using [‘H]5-HT and [“Hlspiroperidol spectively as ligands. as described by Peroutka Snyder (1979).
MARTIS CI trl.
rea~nd
\IWHODS
Malc, Sprague Dawley rats (Hilltop Lab Animals. Inc., Scottsdale. PA) weighing 250 300g were used in all experiments.
The following companies donated drugs used in this paper: ketamine hydrochloride. Warner Lambert,‘Parke Davis. Ann Arbor. Ml: and fluoxetine. Eli Lilly and Co.. Indianapolis IN. Other drugs and chemicals were obtained from the following companies: 5-hydroxytryptamine crcatininc sulfate. U.L-pchlorc7umphetamine hydrochloride and nialamide, Sigma Chemical Co.. St Louis. MO: and reserpine, Ciba Pharmaceuticals Co.. Summit, NJ. Except where indicated. all doses arc cxprrssed in terms of their free acid or base forms and were administered intraperitoneally in 0.9”,, saline. S~~roror7ir7frprtrl\c in vivo The ability of 5-HT uptake inhibitors to block the depletion of brain 5-HT by p-chloroamphetamine (PCA) has been described by Fuller (‘f trl. (1975). This method was used to determine the effects of drugs on 5-HT uptake ir7 r,iro. Drugs were administered either prior to or simultaneously with p-chloroamphetamine HCl (IO mg kg). One hour after the administration of PCA. the animals were decapitated and the brains were removed for determination of their 5-HT content. The ability of an agent to prevent the depletion of brain 5-HT by PCA was suggestike of. but not proof for. inhibition of 5-HT uptake by that agent in I~ilV. Since ketamine is a short-acting agent, it was necessary in these studies to administer supplementary doses to maintain a relatively steady concentration of the drug in the brain. The smallest dose of ketamine which blocked the righting reflex in rats was 120 mg:kg. (i.p.) It was found that additional injections of ketamine (equal to 20”,, of the initial dose) at 15. 30 and 45 min were adequate to maintain the loss of the righting reflex for 1 hr. Assuming that the pharmacokinetics of other doses (40. 80 and 160mg kg) were the same, this dosing regimen was used in all cases.
Rats were decapitated and their brains and spinal cords removed. Some brains were dissected on an icecold glass plate into cortex, cerebellum, hippocampus. midbrain-thalamus, striatum hypothalamus. and medulla-pons as described by Glowinski and Iversen (1966). Whole brain 5-HT levels were determined using the method of Atack and Lindqvist (1973) with
a slight modification as described elsewhere (Martin and Smith. 1982). The method of Jacobowitz and Richardson (1978) was used for the regional analysis of 5-HT in the rat brain and spinal cord. This assay is more scnsitibc than the assay of Atack a~nd Lindqvist (1973) and is therefore better suited for the analysis of 5-HT in small tissue samples. Only fresh tissues wcrc used for 5-HT determination.
Specific [‘Hlligand binding to 5-HT receptors on membrane preparations of the rat frontal cortex was performed according to the method of Peroutka and Snyder (1979). The [‘Hlligands used were [“HIS-HT (28.9 Ci,‘mmol) for the 5-HT1 receptor and [“Hlspiroperidol (25,7Ci,‘mmol) for the 5-HT, receptor. Both [“Hlligands were obtained from New England Nuclear, Boston. MA. The concentrations of the [-‘H]ligands were 2.OnM ([“HIS-HT) and 0.3 nM ([-‘Hlspiroperidol). All assays were performed in triplicate. Specific binding of the [“Hlligands was defined as the binding in excess of blank values obtained in the presence of 300nM 5-HT (higher concentration werewjithout further affect on binding of([3H]5-HT) or 300 /tM 5-HT ([“Hlspiroperidol), Specific binding varied between 55 and 65”,, of total binding for both [“Hlligands. From the preliminary experiments. to establish the conditions of the assay. it was dctermined that the Kn and B,,,,, for [“HIS-HT binding were 4.4 nM and 7.4 pmol g tissue respectively. The binding were Kd and B,,,,, for [“Hlspiroperidol 1.6 nM and 21.1 pmol g tissue. These values agree well with those reported by Pcroutka and Snyder (1979).
To determine the level of significance of treatment effects. data were analyzed by a one-way analysis of variance for groups of unequal size. Significant differences between groups w’ere determined using the Tukey Multiple Comparison Procedure. RESLILTS
A dose of 20mg,‘kg of ketamine produced only a mild degree of agitation lasting less than 15 min. A brief period of excitation (approx. 25 min) was produced by 40 mg:kg ketamine. The most characteristic effect of ketamine at this dose was a stereotyped, sideto-side movement of the head referred to as “headweaving”. A dose of 80 mgkg also produced headweaving and a much greater degree of ataxia as well. Doses of 120 and 160mgkg of ketamine induced anesthesia (loss of righting reflex) lasting approximately 25 and 40 min respectively. Following anesthesia, ataxia and head-weaving became apparent with behavior returning to normal within 2 hr. A dose of 10 mg,‘kg (i.p.) of PCA-HCI reduced whole brain 5-HT levels to 5.5”,, of control in one
5-HT uptake
L
_
Fig. 1. p-Chloroamphetamine-HCI (10 mg, kg. i.p.) w’as injected simultaneously wtth saline or ketamine-HCI (40, 80. 120. or 160 mg/kg. i.p.). Additional injections of ketamine (cqual to 20”,, of the initial injection) were administered at 15. 30 and 45 min in order to maintain the initial behavioral effect of the drug (head ueaving. loss of righting reflex. etc.) Values for the dose of ketamine-HCI refer to the initial injection, Levels of whole brain 5-HT were determined at 60 min. Each value is the mean f SEM of 447 rats and is expressed as a percentage of control 5-HT (375 & 12 ng.gr). *Stgnificantly different from saline-treated animals (P < 0.05).
115
and ketamine
hour. Ketamine-HCl (160 mg/kg, 1 hr) and fluoxetine (10 mg/kg, 1 hr), had no effect on the depletion of brain 5-HT by reserpine. In contrast, both agents were able to prevent completely the 5-HT-depleting effect of PCA. Neither ketamine nor fluoxetine alone. however, had any effect on brain 5-HT levels in control animals. Treatment of animals with nialamide (100 mgikg. 3 hr) increased brain 5-HT content to 152”,, of control (Fig. 3). Reserpine and PCA were administered 2h after the administration of nialamide and. 1 hr later, the animals were sacrificed for determination of brain 5-HT content. Neither reserpine nor PCA prevented the rise in brain 5-HT content following the administration of nialamide. Therefore, nialamide pretreatment completely blocked the depletion of 5-HT by both PCA and reserpine.
Figure 4 demonstrates that ketamine in concentrations as high as 1 mM, had very little, if any. effect on [3H]5-HT binding. Similarly, ketamine only weakly antagonized [“H]spiroperidol binding at concentrations greater than 100 PM.
DISCUSSION
hour (Fig. 1). Ketamine-HCI (40 mg/kg) had no effect on the depletion of brain 5-HT by PCA. On the other hand, doses of 80. 120. and 160 mg/kg significantly blocked the depletion of brain 5-HT by PCA. The values for brain 5-HT content following the coadministration of either 80, 120, or 160 mg/kg ketamine-HCl and PCA were not significantly different from control 5-HT values (375 f 12 ngigr). In regional studies. PCA was found to deplete 5-HT in the spinal cord, medulla-pans, and cerebellum to only a small degree (less than 14?;,, data not shown). Conversely, PCA significantly reduced (by 1644”“) 5-HT in the midbrain-thalamus, hypothalamus. striatum, hippocampus and cortex (Fig. 2). Similar regional variations in the depletion of brain and spinal cord 5-HT by PCA have been reported by Sanders-Bush. Bushing and Sulser (1975). KetamineHCI (160 mg,‘kg) significantly prevented the depletion of 5-HT by PCA in every region studied (Fig. 2). In addition. the regional concentrations of 5-HT following treatment with both ketamine and PCA were not significantly different from control values.
Azzaro and Smith (1977) demonstrated that ketamine can inhibit the high affinity uptake of c3H]5-HT into crude synaptosomal preparations of rat brain, The data presented in this paper suggest
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*
Lo
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05 r 03 01 ‘Ii Cortex
~ PPOC
SOllW? q PCA HCI (IOmg/kg) n PCA HCI IlOmg/hg)
Hypoth
0
+ Ketomlne
HCI
(160mg/kg)
Since ketamine is a weak monoamine oxidase (MAO) inhibitor (Azzaro and Smith, 1977) it was necessary to rule out the possibility that it blocked PCA-induced 5-HT depletion by inhibiting 5-HT metabolism rather than by inhibiting 5-HT uptake. As can be seen in Figure 3, reserpine (5 mg/kg) depleted brain 5-HT to 50”” of control levels in one
Fig. 2. Animals were given saline, PCA-HCI (10mg:kg. i.p.) or PCA-HCI and ketamine-HCl (160 mgkg. i.p.) simultaneously. Additional injections of ketamine were administered as described in Figure 1. Levels of 5-HT in various brain regions were determined at 60 min. Each value is the mean f SEM of 446 rats, Hippoc = hippocampus: Hypoth = hypothalamus. *Significantly different from different from PCA-treated rats (P < 0.05). **Significantly saline-treated rats (P < 0.05).
I16
L. L. MARTIN IJI trl.
Ketamlne 160mg/
/J
SolIne HCI
lIOmg/kg)
m
Reserp~ne
i5mg/kg)
q PCA
HCI
Nlalamtde
kg
lOCmg/kg
Fig, 3. Rats lcere treated with saline. PCA-HCI (10 mg,,kg. 60 min) or reserpine (5 mg, kg. 60 min) in combination with saline. ketamine HCI (160 mg’kg. 60 min plus 32 mg’kg.
IS. 30 and 45 min). Ruoxetinc (IO mg kg. 60 min) or nialamide (100 mg kg. 180 min). All drugs were administered intraperitoncally.
The times refer to the duration of time moment the aniof whole hram 5.HT levels. Each value is the mean i SEM of 4 6 rats and i$ expressed as a percentage of control 5-HT (341 i 14ng.gr). *Signilicantly dlffercnt from rats treated uith saline only (P i 0.05).
the drugs were administered prior to the mals v,erc sacrificed for determination
that ketamine can inhibit S-HT uptake in c>ilo as well. based on its ability to prevent the depletion of 5-HT by PCA (Fuller clt rrl.. 1975). This conclusion is based on the assumption that PCA requires the uptake process for entry into the serotonergic nerve to have its neurotoxic action (see introduction). The effects of ketamine appear to be due to a specific binding of ketamine to the 5-HT carrier rather than a nonspecific anesthetic action since ketamine competitively blocks 5-HT uptake in vitro (Azzaro and Smith. 1977). In addition, the ability of ketamine to prevent the depletion of 5-HT by PCA appears to be due to inhibition of 5-HT uptake and not to inhibition of MAO since. unlike nialamide, ketamine did not prevent the depletion of 5-HT by reserpine (Fig. 3). Nialamide prevented the decline in 5-HT levels by blocking the intraneuronal deamination of 5-HT following the inhibition of vesicular storage by reserpine. The ability of nialamide to prevent the depletion of 5-HT by PCA would also appear to be due to MAO inhibition since it is known not to Inhibit 5-HT uptake (Ferris, Howard. and White. 1975). Since ketamine inhibited the uptake of 5-HT. its interaction with 5-HT receptors was examined. This was considered since quipazine and MK-212. which are known to inhibit S-HT uptake (Hamon et trl.. 1976, and Clineschmidt et d.. 1978). have also been shown to bind to 5-HT receptors (Nelson c’t (I/., 1978). Ketaminc. however. only weakly affected either
[“HIS-HT or [3H]spiroperidol binding even at concentrations as high as I mM. Effects of such concentrations are of doubtful physiological significance (Cohen e’t rrl., 1973). Thus. it appears that the behavioral and physiological effects of ketamine are not likely to be related to a direct interaction between ketaminc and either 5-HT, or 5-HTz receptors (Peroutka and Snyder. 1979). The neuronal. high affinity uptake of 5-HT appears to be the primary mechanism for inactivation of 5-HT released into the synaptic cleft. Thus. blockade of 5-HT uptake could produce numerous physiological consequences resulting from enhanced serotonergic transmission that may be relevant to the clinical effects of ketamine. Indeed, certain behavioral effects of ketamine. observed in man and.!or in animals. appear to be mediated, at least in part, by serotonergic systems. For example, in patients, ketamine produces hallucinations (Albin and Dresner. 1970: Reier, 1970) and. in animals, various hallucinatory agents inhibit the tiring rates of serotonergic raphc neurons in rats, (Aghajanian (~1 t/l.. 1970). In addition, ketamine produces lateral head weaving which. among other changes in motor activity, may be related to the extrapyramidal movements observed in patients recovering from ketamine anesthesia (Albin and Dresner. 1970). Lateral head weaving is also part of a syndrome of changes in motor activity in rats produced by various treatments that are known to cnhancc scrotonergic transmission (Holman et trl.. 1976; Trulson et trl., 1976; Sloviter rt trl., 197X). It is not possible. however. to explain how ketaminc could produce hallucinations or lateral head weaving simply on the basis of its ability to inhibit 5-HT uptake since
00 4’
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x 0
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-
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5-HT
-
[3H]
SPlROPEAlDOl
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50
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35
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[KETAMINE]
Fig. 4. Etfects of ketamine on [‘Hlligand binding to serotonin receptors. [‘H]Ligand binding assays were performed as described in Methods. Data shown are the means of three assays run in triplicate. B/B,, is “bound in the presence of ketamine.‘bound in the absence of ketamine”.
5-HT uptake and ketamine
the administration of selective 5-HT uptake inhibitors does not produce either lateral head weaving (Holman c’t ctl.. 1976) or hallucinations (Johansson and Von Knorring, 1976: Rowe, Carmichael, Oldham. Horng, Bymaster and Wang. 1978). Apparently other effects of ketamine on serotonergic or non-serotonergic systems must be involved in producing these effects (see discussion. Martin and Smith. 1982). On the other hand. numerous studies have provided evidence that serotonergic fibers originating in the nucleus raphe magnus and terminating in the dorsal horn of the spinal cord. mediate at least in part. the analgesic effects of the opiates (Messing and Lytle. 1977: Samanin cr trl., 1978; Yaksh and Wilson. 1979). In fact. 5-HT uptake inhibitors have been shown to produce analgesia in rats (Messing, Phebus, Fisher and Lytle. 1975) and in man (Johansson and Von Knorring. 1979). Thus blockade of 5-HT uptake may be one mechanism by which ketamine produces analgesia. In addition. inhibitors of S-HT uptake have also been shown to potentiate the analgesic effects of morphine (Larson and Takemori. 1977: Sugrue. 1979). Therefore. since ketamine has recently been shown to be an opiate receptor agonist (Smith, Pekoe. Martin. and Coalgate. 1980). it seems likely that its analgesic effect mediated through opiate mechanisms would be potentiated by 5-HT uptake blockade. Evidence that blockade of 5-HT uptake mediates ketamine-induced analgesia is two-fold. First. the dose-response curves for ketamine-induced analgesia and blockade of 5-HT uptake are very similar. A dose of 80 mg:kg of ketamine both inhibits 5-HT uptake (Fig. 1) and produces analgesia (Pekoe and Smith, 1979). whereas 40 mg.kg does not have either effect. Secondly. the administration of either cinanserin or methysergide (putative 5-HT receptor antagonists) blocks ketamine analgesia (Pekoe and Smith. 1979). The action of ketamine to inhibit the uptake of 5-HT in Gro should lead to compensatory changes in serotonergic nerve function (Arzaro and Smith, 1977; Fuller and Wong, 1977; Martin and Smith, 1982) including reduced 5-HT turnover and release (Fuller and Wong. 1977). These changes are assumed to be regulatory mechanisms opposing the higher synaptic levels of transmitter and are thus secondary to the inhibition of uptake. The regulatory processes may partially limit the elevation in synaptic transmitter concentration. However. it would be assumed that a balance is achieved in the direction of increased postjunctional receptor activation. since reduced turnover and release appears to be initiated by the action of uptake inhibitors to increase intrasynaptic 5-HT. Future studies should be designed to evaluate the importance of compensatory changes in the ultimate pharmacological expression of the inhibition of 5-HT uptake. A~l\,loM/4~lyenlrrlts~~~The authors wish to thank Dr Elaine Sanders-Bush for her contributions and helpful in the preparation of this manuscript.
suggestions
117 REFERENCES
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Sloviter, R. S., Drust. E. G. and Connor. J. D. (1978). Specificity of a rat behavioral model for serotonin receptor activation. J. Phurmc~c. rup. Thu. 206: 339-346. Smith, D. J., Pekoe. G. M., Martin L. L. and Coalgate. B. (1980). The interaction of ketemine with the opiate receptor. L#e .%I. 26: 789- 795. Sugrue. M. F. (1979). On the role of 5-hydroxytryptamine in drug-induced antinociception. Br. J. Phtrrmtrc~. 65: 6777681. Trulson. M. E.. Ross. C. A. and Jacobs, B. L. (1976). Behavioral evidence for the stimulation of CNS serotonin receptors by high doses of LSD. P.s~c~hophtrr,ntr(~. Commun. 2: 1499164. Virtue, R. W., Alanis, J. M.. Mori. M.. Lafargue. R. T., Vogel, J. H. K. and Metcalf. D. R. (1967). An anesthetic agent: 2-orthochlorophenyll,2-methylamino cyclohexanone HCI (CI-581). Anu.sthesio/o~q.v 28: 8233833. Yaksh. T. L. and Wilson. P. R. (1979). Spinal serotonin terminal system mediates antinociception. J. Phcrrmtrc~. exp. Thur. 208: 446 453.
INHIBITS
SEROTONIN
UPTAKE
IN
L’ZVO”
L. L. MARTIN~$, RENA L. BOUTHAL and D. J. SMITHS Departments
of Ph~rmac~~io~y
and Toxicology and An~sthcsiolo~y, West Virginia Mor~anto~n. WV 26506. U.S.A.
C!niversity
Medical
Ccntet..
Summary Anesthetic (120 and 160 mgkg. i.p.) and subanesthetic (80mg’kg) doses of kctamine HCI were found to prevent completely the depletion of whole brain serotonin (5-HT) by p-chloronmphctamine (PCA). Furthermore, ketaminc HCI (160 m&kg) completely blocked the depletion of 5-HT by PC.4 in every individual brain region studied (Midbrain-thalamus. hypothalamus. strtatum. hippocampus and cortex). Administration of ketamine alone had no effect on brain 5-HT Irvcls. Ninlnmidc (;I monoamine oxidase (MAO) inhibitor) and fluoxetine (a selective 5-HT uptake inhibitor) also prevented the depletion of 5-HT by PCA. However. of these three agents. only nialamide prevented the dcplctron of 5-HT by reserpine. These results suggest that ketamine blocks PCA-induced 5-HT depletion by inhibiting 5-HT uptake and not by inhibiting MAO. Ketamme only weakly atrected either [‘HIS-HT or [~tilspiropcridol binding to 5-HT, and S-HTz receptors respectively even at concentrations as high a< t mM. These data support the contention that the primary direct effect of ketamine on ser(~t~~i~er~ic systems is the blockade of 5-HT uptake and that hlocksde of 5-HT uptake may mediate some of the b~h~~~ior~il etfects of ketamine. such as analgesia.
K&amine is a general anesthetic agent which provides good analgesia for surgery (McCarthy. Chcn, Kaump and Ensor, 1965), but unlike most anesthetics, does not depress the cardiovascular or respiratory systems (Lanning and Harmel. 1975: Virtue. Alanis, Mori, Lafargue, Vogel and Metcalf. 1967). On the negative side. ketamine is also unique because emergence from ketamine anesthesia is often associated with extrapyramidai movements. unpleasant dreams, hallucinations and other psychological disturbances (Albin and Dresner, 1970). Ketamine has previously been demonstrated to be a moderately preferential inhibitor of the high affinity uptake of 5-hydroxytryptamine (5-HT) into crude synaptosomal preparations of the rat brain (Azzaro and Smith, 1977). The ICsO for the inhibition of 5-HT uptake hy ketamine was reported to be approximately 50k~M. Cohen. Chan, Way and Trevor (1973) have reported that the recovery of the righting reflex in rats occurs when brain ketamine levels are approximately 0.03 mg/g tissue or about 130 PM (assuming the brain is a single compartment). When considered together. these data suggest that anesthetic and subanesthetic doses of ketamine should produce brain levels of the
* This wsork was supported by research funds from the U.S. Public Health Service Grant S-T32-GM07039 and the West Virginia University Anesthesiology Research Fund. t The work presented here is in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Pharmacology and Toxicology. West Virginia University. 1 Present address: Tennessee Neurophyschi~tric Institute. 1501 MLlrfreesboro Road. Nashville. TN 37217, U.S.A. $ To whom reprint requests should be sent.
drug which would be in the range necessary to inhibit the uptake of 5-HT in c$it\o. Thus. there existed a strong possibility that blockade of 5-HT uptake might mediate some of the behavioral a&or physiological effects of ketamine. Many of the behavioral phenomena like those produced by ketamine in patients have been suggested to be associated with alterations in serotonergic function. These include analgesia (Messing and Lytle, 1977; Samanin, Miranda and Mennini. 1978; Yaksh and Wilson. 1979), hallucinations (Aghajanian. Foote and Sheard, 1977) and motor disturhantes (Holman, Seagraves. Elliot and Barchas. 1976; Trulson, Ross and Jacobs, 1976; Sloviter. Drust and Conner, 1978). For these reasons. it was decided to determine whether ketamine could inhibit the uptake of 5-HT in view. To accomplish this, the ability of ketamine to prevent the depletion of brain 5.HT by ~-chIoroamphetamine (PCA) was studied. Fuller. Perry and Molloy (1975) have shown that 5-HT uptake inhibitors can be distingLlished iit riro by their ability to prevent the depletion of 5-HT by Pf4. p-Chloroamphetaminc requires a neuronul high atfinity 5-HT uptake process to produce its effect. Some agents such as quipazine and 6-chloro-2-( lpiperazinyl)-pyrazine (MK-212). which are known to inhibit 5-HT uptake (Hamon. Bourgoin. Enjalbcrt, Bockaert. Henry, Ternaux and Glowinski. 1976: Clineschmidt, Totaro. Pflueger and McGuffin. 1978) have also been shown to inhibit the binding of 5-HT to its receptor sites in the brain (Nelson. Bourgoin, Gfowinski and Hamon, 197X). Therefore. it would also be impl~rtant to study the effects of ketaminc on 5-HT receptor binding. The interaction between ketamine and both 5-1-11-t and 5-HTz receptors was
II4
L. I..
studied using [‘H]5-HT and [“Hlspiroperidol spectively as ligands. as described by Peroutka Snyder (1979).
MARTIS CI trl.
rea~nd
\IWHODS
Malc, Sprague Dawley rats (Hilltop Lab Animals. Inc., Scottsdale. PA) weighing 250 300g were used in all experiments.
The following companies donated drugs used in this paper: ketamine hydrochloride. Warner Lambert,‘Parke Davis. Ann Arbor. Ml: and fluoxetine. Eli Lilly and Co.. Indianapolis IN. Other drugs and chemicals were obtained from the following companies: 5-hydroxytryptamine crcatininc sulfate. U.L-pchlorc7umphetamine hydrochloride and nialamide, Sigma Chemical Co.. St Louis. MO: and reserpine, Ciba Pharmaceuticals Co.. Summit, NJ. Except where indicated. all doses arc cxprrssed in terms of their free acid or base forms and were administered intraperitoneally in 0.9”,, saline. S~~roror7ir7frprtrl\c in vivo The ability of 5-HT uptake inhibitors to block the depletion of brain 5-HT by p-chloroamphetamine (PCA) has been described by Fuller (‘f trl. (1975). This method was used to determine the effects of drugs on 5-HT uptake ir7 r,iro. Drugs were administered either prior to or simultaneously with p-chloroamphetamine HCl (IO mg kg). One hour after the administration of PCA. the animals were decapitated and the brains were removed for determination of their 5-HT content. The ability of an agent to prevent the depletion of brain 5-HT by PCA was suggestike of. but not proof for. inhibition of 5-HT uptake by that agent in I~ilV. Since ketamine is a short-acting agent, it was necessary in these studies to administer supplementary doses to maintain a relatively steady concentration of the drug in the brain. The smallest dose of ketamine which blocked the righting reflex in rats was 120 mg:kg. (i.p.) It was found that additional injections of ketamine (equal to 20”,, of the initial dose) at 15. 30 and 45 min were adequate to maintain the loss of the righting reflex for 1 hr. Assuming that the pharmacokinetics of other doses (40. 80 and 160mg kg) were the same, this dosing regimen was used in all cases.
Rats were decapitated and their brains and spinal cords removed. Some brains were dissected on an icecold glass plate into cortex, cerebellum, hippocampus. midbrain-thalamus, striatum hypothalamus. and medulla-pons as described by Glowinski and Iversen (1966). Whole brain 5-HT levels were determined using the method of Atack and Lindqvist (1973) with
a slight modification as described elsewhere (Martin and Smith. 1982). The method of Jacobowitz and Richardson (1978) was used for the regional analysis of 5-HT in the rat brain and spinal cord. This assay is more scnsitibc than the assay of Atack a~nd Lindqvist (1973) and is therefore better suited for the analysis of 5-HT in small tissue samples. Only fresh tissues wcrc used for 5-HT determination.
Specific [‘Hlligand binding to 5-HT receptors on membrane preparations of the rat frontal cortex was performed according to the method of Peroutka and Snyder (1979). The [‘Hlligands used were [“HIS-HT (28.9 Ci,‘mmol) for the 5-HT1 receptor and [“Hlspiroperidol (25,7Ci,‘mmol) for the 5-HT, receptor. Both [“Hlligands were obtained from New England Nuclear, Boston. MA. The concentrations of the [-‘H]ligands were 2.OnM ([“HIS-HT) and 0.3 nM ([-‘Hlspiroperidol). All assays were performed in triplicate. Specific binding of the [“Hlligands was defined as the binding in excess of blank values obtained in the presence of 300nM 5-HT (higher concentration werewjithout further affect on binding of([3H]5-HT) or 300 /tM 5-HT ([“Hlspiroperidol), Specific binding varied between 55 and 65”,, of total binding for both [“Hlligands. From the preliminary experiments. to establish the conditions of the assay. it was dctermined that the Kn and B,,,,, for [“HIS-HT binding were 4.4 nM and 7.4 pmol g tissue respectively. The binding were Kd and B,,,,, for [“Hlspiroperidol 1.6 nM and 21.1 pmol g tissue. These values agree well with those reported by Pcroutka and Snyder (1979).
To determine the level of significance of treatment effects. data were analyzed by a one-way analysis of variance for groups of unequal size. Significant differences between groups w’ere determined using the Tukey Multiple Comparison Procedure. RESLILTS
A dose of 20mg,‘kg of ketamine produced only a mild degree of agitation lasting less than 15 min. A brief period of excitation (approx. 25 min) was produced by 40 mg:kg ketamine. The most characteristic effect of ketamine at this dose was a stereotyped, sideto-side movement of the head referred to as “headweaving”. A dose of 80 mgkg also produced headweaving and a much greater degree of ataxia as well. Doses of 120 and 160mgkg of ketamine induced anesthesia (loss of righting reflex) lasting approximately 25 and 40 min respectively. Following anesthesia, ataxia and head-weaving became apparent with behavior returning to normal within 2 hr. A dose of 10 mg,‘kg (i.p.) of PCA-HCI reduced whole brain 5-HT levels to 5.5”,, of control in one
5-HT uptake
L
_
Fig. 1. p-Chloroamphetamine-HCI (10 mg, kg. i.p.) w’as injected simultaneously wtth saline or ketamine-HCI (40, 80. 120. or 160 mg/kg. i.p.). Additional injections of ketamine (cqual to 20”,, of the initial injection) were administered at 15. 30 and 45 min in order to maintain the initial behavioral effect of the drug (head ueaving. loss of righting reflex. etc.) Values for the dose of ketamine-HCI refer to the initial injection, Levels of whole brain 5-HT were determined at 60 min. Each value is the mean f SEM of 447 rats and is expressed as a percentage of control 5-HT (375 & 12 ng.gr). *Stgnificantly different from saline-treated animals (P < 0.05).
115
and ketamine
hour. Ketamine-HCl (160 mg/kg, 1 hr) and fluoxetine (10 mg/kg, 1 hr), had no effect on the depletion of brain 5-HT by reserpine. In contrast, both agents were able to prevent completely the 5-HT-depleting effect of PCA. Neither ketamine nor fluoxetine alone. however, had any effect on brain 5-HT levels in control animals. Treatment of animals with nialamide (100 mgikg. 3 hr) increased brain 5-HT content to 152”,, of control (Fig. 3). Reserpine and PCA were administered 2h after the administration of nialamide and. 1 hr later, the animals were sacrificed for determination of brain 5-HT content. Neither reserpine nor PCA prevented the rise in brain 5-HT content following the administration of nialamide. Therefore, nialamide pretreatment completely blocked the depletion of 5-HT by both PCA and reserpine.
Figure 4 demonstrates that ketamine in concentrations as high as 1 mM, had very little, if any. effect on [3H]5-HT binding. Similarly, ketamine only weakly antagonized [“H]spiroperidol binding at concentrations greater than 100 PM.
DISCUSSION
hour (Fig. 1). Ketamine-HCI (40 mg/kg) had no effect on the depletion of brain 5-HT by PCA. On the other hand, doses of 80. 120. and 160 mg/kg significantly blocked the depletion of brain 5-HT by PCA. The values for brain 5-HT content following the coadministration of either 80, 120, or 160 mg/kg ketamine-HCl and PCA were not significantly different from control 5-HT values (375 f 12 ngigr). In regional studies. PCA was found to deplete 5-HT in the spinal cord, medulla-pans, and cerebellum to only a small degree (less than 14?;,, data not shown). Conversely, PCA significantly reduced (by 1644”“) 5-HT in the midbrain-thalamus, hypothalamus. striatum, hippocampus and cortex (Fig. 2). Similar regional variations in the depletion of brain and spinal cord 5-HT by PCA have been reported by Sanders-Bush. Bushing and Sulser (1975). KetamineHCI (160 mg,‘kg) significantly prevented the depletion of 5-HT by PCA in every region studied (Fig. 2). In addition. the regional concentrations of 5-HT following treatment with both ketamine and PCA were not significantly different from control values.
Azzaro and Smith (1977) demonstrated that ketamine can inhibit the high affinity uptake of c3H]5-HT into crude synaptosomal preparations of rat brain, The data presented in this paper suggest
*
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+ I
0.7
* *
I c
I
*
Lo
f
05 r 03 01 ‘Ii Cortex
~ PPOC
SOllW? q PCA HCI (IOmg/kg) n PCA HCI IlOmg/hg)
Hypoth
0
+ Ketomlne
HCI
(160mg/kg)
Since ketamine is a weak monoamine oxidase (MAO) inhibitor (Azzaro and Smith, 1977) it was necessary to rule out the possibility that it blocked PCA-induced 5-HT depletion by inhibiting 5-HT metabolism rather than by inhibiting 5-HT uptake. As can be seen in Figure 3, reserpine (5 mg/kg) depleted brain 5-HT to 50”” of control levels in one
Fig. 2. Animals were given saline, PCA-HCI (10mg:kg. i.p.) or PCA-HCI and ketamine-HCl (160 mgkg. i.p.) simultaneously. Additional injections of ketamine were administered as described in Figure 1. Levels of 5-HT in various brain regions were determined at 60 min. Each value is the mean f SEM of 446 rats, Hippoc = hippocampus: Hypoth = hypothalamus. *Significantly different from different from PCA-treated rats (P < 0.05). **Significantly saline-treated rats (P < 0.05).
I16
L. L. MARTIN IJI trl.
Ketamlne 160mg/
/J
SolIne HCI
lIOmg/kg)
m
Reserp~ne
i5mg/kg)
q PCA
HCI
Nlalamtde
kg
lOCmg/kg
Fig, 3. Rats lcere treated with saline. PCA-HCI (10 mg,,kg. 60 min) or reserpine (5 mg, kg. 60 min) in combination with saline. ketamine HCI (160 mg’kg. 60 min plus 32 mg’kg.
IS. 30 and 45 min). Ruoxetinc (IO mg kg. 60 min) or nialamide (100 mg kg. 180 min). All drugs were administered intraperitoncally.
The times refer to the duration of time moment the aniof whole hram 5.HT levels. Each value is the mean i SEM of 4 6 rats and i$ expressed as a percentage of control 5-HT (341 i 14ng.gr). *Signilicantly dlffercnt from rats treated uith saline only (P i 0.05).
the drugs were administered prior to the mals v,erc sacrificed for determination
that ketamine can inhibit S-HT uptake in c>ilo as well. based on its ability to prevent the depletion of 5-HT by PCA (Fuller clt rrl.. 1975). This conclusion is based on the assumption that PCA requires the uptake process for entry into the serotonergic nerve to have its neurotoxic action (see introduction). The effects of ketamine appear to be due to a specific binding of ketamine to the 5-HT carrier rather than a nonspecific anesthetic action since ketamine competitively blocks 5-HT uptake in vitro (Azzaro and Smith. 1977). In addition, the ability of ketamine to prevent the depletion of 5-HT by PCA appears to be due to inhibition of 5-HT uptake and not to inhibition of MAO since. unlike nialamide, ketamine did not prevent the depletion of 5-HT by reserpine (Fig. 3). Nialamide prevented the decline in 5-HT levels by blocking the intraneuronal deamination of 5-HT following the inhibition of vesicular storage by reserpine. The ability of nialamide to prevent the depletion of 5-HT by PCA would also appear to be due to MAO inhibition since it is known not to Inhibit 5-HT uptake (Ferris, Howard. and White. 1975). Since ketamine inhibited the uptake of 5-HT. its interaction with 5-HT receptors was examined. This was considered since quipazine and MK-212. which are known to inhibit S-HT uptake (Hamon et trl.. 1976, and Clineschmidt et d.. 1978). have also been shown to bind to 5-HT receptors (Nelson c’t (I/., 1978). Ketaminc. however. only weakly affected either
[“HIS-HT or [3H]spiroperidol binding even at concentrations as high as I mM. Effects of such concentrations are of doubtful physiological significance (Cohen e’t rrl., 1973). Thus. it appears that the behavioral and physiological effects of ketamine are not likely to be related to a direct interaction between ketaminc and either 5-HT, or 5-HTz receptors (Peroutka and Snyder. 1979). The neuronal. high affinity uptake of 5-HT appears to be the primary mechanism for inactivation of 5-HT released into the synaptic cleft. Thus. blockade of 5-HT uptake could produce numerous physiological consequences resulting from enhanced serotonergic transmission that may be relevant to the clinical effects of ketamine. Indeed, certain behavioral effects of ketamine. observed in man and.!or in animals. appear to be mediated, at least in part, by serotonergic systems. For example, in patients, ketamine produces hallucinations (Albin and Dresner. 1970: Reier, 1970) and. in animals, various hallucinatory agents inhibit the tiring rates of serotonergic raphc neurons in rats, (Aghajanian (~1 t/l.. 1970). In addition, ketamine produces lateral head weaving which. among other changes in motor activity, may be related to the extrapyramidal movements observed in patients recovering from ketamine anesthesia (Albin and Dresner. 1970). Lateral head weaving is also part of a syndrome of changes in motor activity in rats produced by various treatments that are known to cnhancc scrotonergic transmission (Holman et trl.. 1976; Trulson et trl., 1976; Sloviter rt trl., 197X). It is not possible. however. to explain how ketaminc could produce hallucinations or lateral head weaving simply on the basis of its ability to inhibit 5-HT uptake since
00 4’
80 -
IT”
u 2
x 0
60
-
40
-
,m 02 Zs 2 ,’
-
20
-
;;-_
H
[%i]
5-HT
-
[3H]
SPlROPEAlDOl
01 55
50
45 -LOG
40
35
30
[KETAMINE]
Fig. 4. Etfects of ketamine on [‘Hlligand binding to serotonin receptors. [‘H]Ligand binding assays were performed as described in Methods. Data shown are the means of three assays run in triplicate. B/B,, is “bound in the presence of ketamine.‘bound in the absence of ketamine”.
5-HT uptake and ketamine
the administration of selective 5-HT uptake inhibitors does not produce either lateral head weaving (Holman c’t ctl.. 1976) or hallucinations (Johansson and Von Knorring, 1976: Rowe, Carmichael, Oldham. Horng, Bymaster and Wang. 1978). Apparently other effects of ketamine on serotonergic or non-serotonergic systems must be involved in producing these effects (see discussion. Martin and Smith. 1982). On the other hand. numerous studies have provided evidence that serotonergic fibers originating in the nucleus raphe magnus and terminating in the dorsal horn of the spinal cord. mediate at least in part. the analgesic effects of the opiates (Messing and Lytle. 1977: Samanin cr trl., 1978; Yaksh and Wilson. 1979). In fact. 5-HT uptake inhibitors have been shown to produce analgesia in rats (Messing, Phebus, Fisher and Lytle. 1975) and in man (Johansson and Von Knorring. 1979). Thus blockade of 5-HT uptake may be one mechanism by which ketamine produces analgesia. In addition. inhibitors of S-HT uptake have also been shown to potentiate the analgesic effects of morphine (Larson and Takemori. 1977: Sugrue. 1979). Therefore. since ketamine has recently been shown to be an opiate receptor agonist (Smith, Pekoe. Martin. and Coalgate. 1980). it seems likely that its analgesic effect mediated through opiate mechanisms would be potentiated by 5-HT uptake blockade. Evidence that blockade of 5-HT uptake mediates ketamine-induced analgesia is two-fold. First. the dose-response curves for ketamine-induced analgesia and blockade of 5-HT uptake are very similar. A dose of 80 mg:kg of ketamine both inhibits 5-HT uptake (Fig. 1) and produces analgesia (Pekoe and Smith, 1979). whereas 40 mg.kg does not have either effect. Secondly. the administration of either cinanserin or methysergide (putative 5-HT receptor antagonists) blocks ketamine analgesia (Pekoe and Smith. 1979). The action of ketamine to inhibit the uptake of 5-HT in Gro should lead to compensatory changes in serotonergic nerve function (Arzaro and Smith, 1977; Fuller and Wong, 1977; Martin and Smith, 1982) including reduced 5-HT turnover and release (Fuller and Wong. 1977). These changes are assumed to be regulatory mechanisms opposing the higher synaptic levels of transmitter and are thus secondary to the inhibition of uptake. The regulatory processes may partially limit the elevation in synaptic transmitter concentration. However. it would be assumed that a balance is achieved in the direction of increased postjunctional receptor activation. since reduced turnover and release appears to be initiated by the action of uptake inhibitors to increase intrasynaptic 5-HT. Future studies should be designed to evaluate the importance of compensatory changes in the ultimate pharmacological expression of the inhibition of 5-HT uptake. A~l\,loM/4~lyenlrrlts~~~The authors wish to thank Dr Elaine Sanders-Bush for her contributions and helpful in the preparation of this manuscript.
suggestions
117 REFERENCES
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and Samanin, R.. Eds). pp. 523-541. Raven Press. New York. Sanders-Bush, E., Bushmg, J. A. and Sulscr. F. (1975). Long-term effects of p-chloroamphetaminc and related drugs on central serotonergic mechanisms. J. Phtrrmrrc,. u.upyThrr.
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_
Sloviter, R. S., Drust. E. G. and Connor. J. D. (1978). Specificity of a rat behavioral model for serotonin receptor activation. J. Phurmc~c. rup. Thu. 206: 339-346. Smith, D. J., Pekoe. G. M., Martin L. L. and Coalgate. B. (1980). The interaction of ketemine with the opiate receptor. L#e .%I. 26: 789- 795. Sugrue. M. F. (1979). On the role of 5-hydroxytryptamine in drug-induced antinociception. Br. J. Phtrrmtrc~. 65: 6777681. Trulson. M. E.. Ross. C. A. and Jacobs, B. L. (1976). Behavioral evidence for the stimulation of CNS serotonin receptors by high doses of LSD. P.s~c~hophtrr,ntr(~. Commun. 2: 1499164. Virtue, R. W., Alanis, J. M.. Mori. M.. Lafargue. R. T., Vogel, J. H. K. and Metcalf. D. R. (1967). An anesthetic agent: 2-orthochlorophenyll,2-methylamino cyclohexanone HCI (CI-581). Anu.sthesio/o~q.v 28: 8233833. Yaksh. T. L. and Wilson. P. R. (1979). Spinal serotonin terminal system mediates antinociception. J. Phcrrmtrc~. exp. Thur. 208: 446 453.