Ketamine inhibits serotonin synthesis and metabolism in vivo

KETAMINE

INHIBITS SEROTONIN SYNTHESIS METABOLISM IN 1/11/O* L. L.

Departments

of Pharmacology

MARTINt:

AND

and D. J. SMITHY

and Toxicology. and Center, Morgantown.

4nesthesiology. West Vlrginla WV 26.506. U.S.A.

Unlvcrsity

Medical

Summary~~~Ketamine (160 mg/kg. i.p.) was found to reduce the accumulation of 5-hydroxytryptophan (S-HTP) in whole brain following Inhibition of I.-aromatic amino acid dccarboxylase with 50 mpkg of 3-hydroxqbenrylhydraline (NSD-1015). Smaller doses of ketaminc did not affect whole brain 5-HTP accumulation. However. in regional studies, X0 mg/kg of ketamine significantly reduced 5.HTP accumulation in the spinal cord and the midbrain-thalamus. A dose of 160mglkg ketamine also reduced 5-HTP accumulation in the spmal cord and midbrain-thalamus and In the medulla-pans. striatum and cortex as well. No significant changes m 5-HTP accumulation were observed in the hypothalamus or hippocampus. Ketamine (160 mg;kg) also reduced whole brain S-hydroxyindoleacctlc acid (S-HIAA) levels and slightly elevated whole brain S-hydroxytryptamine (S-HT) levels. Smaller doses did not afl’ect tither S-HIAA or 5-HT levels. Ketaminc did not affect whole brain tryptophan levels nor did it mhiblt [‘Hltryptophan uptake or conversion to [‘H]5-HT it1 ritrr~. These data demonstrate that ketamine reduced both 5-HT synthesis and metabolism irk riro. Since ketamine did not alTcct brain trqptophan levels nor did It inhibit 5-HT in ritro. the reduction of S-HT turnover following ketamine administration appears to be a neuronal. adaptive phenomenon possibly occurring in response to a blockade of 5.HT

uptake by ketamine

Ketamine has been demonstrated to inhibit the uptake of serotonin (5-HT) both in rirro (Azzaro and Smith, 1977) and irl lit?o (Martin, Bouchal and Smith, 1982). In addition, various 5-HT uptake inhibitors have been demonstrated to reduce the rate of turnover of 5-HT in the brain (Marco and Meek, 1979). Therefore, the present authors thought it would be of interest to determine whether ketamine affected 5-HT turnover in a similar manner. Such a finding would support the hypothesis that by inhibiting 5-HT uptake, ketamine should produce alterations in serotonergic function which may mediate certain behavioral effects of ketamine such as analgesia, Previous reports on the effects of ketamine on S-HT turnover have been very conflicting. Such studies have reported 5-HT turnover to be either decreased (Ylitalo, Saarnivaara and Ahtee, 1976; Sung, Frederickson and Holtzman, 1973) or increased (Biggio. Proceddu and Vargiu, 1974: Kari, Davidson, Kohl and Kochhar. 1978; Vargiu. Steffani, Musinu and Saba, 1978) following ketamine administration, In a number of these reports, only one dose of ketamine was studied and. in a few. the times chosen for study were not representative of the time-course of * This work was supported by research funds from the U.S. Public Health Service Grant 5-T32-GM07039 and the West Virginia Universitv Anesthesiologv Research Fund. t The work presented- here is in part% fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Pharmacology and Toxicology. West Virginia University. 1 Present address: Tennessee Neuropsychiatric Institute. 1501 Murfreesboro Road. Nashville. TN 37217. U.S.A. g To whom reprint requests should be sent.

the behavioral effects of ketamine. Therefore, in the present investigation. the effects of ketamine on S-HT turnover in whole brain and in various brain regions with respect to both dose and time have been examined. The effects of ketamine on 5-HT turnover were evaluated through measurements of brain 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels and the rate of accumulation of 5-hydroxytryptophan (5HTP) following the inhibition of L.-aromatic amino acid decarboxylasc. In addition, the effects of ketamine on brain tryptophan levels and on tryptophan uptake and conversion to S-HIT in crude synaptosomal preparations of rat brain were also studied in order to determine whether ketamine afl‘ects 5-HT turnover directly as opposed to indirectly through neuronal adaptive mechanisms. METHODS

Anitnds

Male. Sprague- Dawley rats (Hilltop Lab Animals, Inc., Scottsdale, PA) weighing 2%300 g were used in all experiments.

Ketamine hydrochloride was donated by WarnerLambert,‘Parke Davis. Ann Arbor. MI. t),L-TryptoS-hydroxy-D,L-tryptophan, 5-hydroxytryptphan, amine creatinine sulfate and S-hydroxyindoleacetic acid dicyclohexylammonium salt were obtained from Sigma Chemical Co., St Louis, MO. 3_Hydroxybenzylhydrazine dihydrochloride (NSD-101.5) was obtained from Aldrich Chemical Co.. Milwaukee. WI. All doses 119

120

L. L. MARTIN and D. J. SMm

are expressed in terms of their free acid or base forms and were administered in 0.9”,, saline.

The rate of scrotonin synthesis in riw was estimated using the method described by Carlsson. Davis, Kehr, Lindqvist and Atack (1972). L-Aromatic amino acid decarboxylase was inhibited by an injection of 50 mg.‘kg of NSD-1015 (3-hydroxybenzylhydrazine). Fifteen minutes later, the animals were sacrificed for determination of the tissue content of 5-HTP as described below. The accumulation of 5HTP was maximal following a dose of 50 mg/kg NSD-1015 and was linear for 30 min. suggesting that tryptophan hydroxylase activity was not affected by NSD-1015 during this period of time (Carlsson et rrl.. 1972).

Rats were sacrificed by decapitation and the brains and spinal cords quickly removed. Some brains were dissected WI an ice-cold glass plate into the cortex, cerebellum. hippocampus. hypothalamus. midbrainthalamus. striatum and medulla-pons as described by Glowinski and lversen (1966). The extraction and column separation of tryptophan and the 5-hydroxyindoles were performed as outlined by Atack and Lindqvist (1973): tryptophan was measured fluorometrically by the method of Bedard, Carlsson and Lindqvist (1972). The 5-hydroxyindoles were assayed fluorometrically by the method of Atack and Lindqvist (1973) except that the standards and reagent blanks were made up using l.Oml portions of a reagent blank column eluate. Separate internal standards for each sample were not performed. Values were always corrected for recoveries which were approx. 70”,, (tryptophan). 65”,, (5-HTP) and 60”,, (5-HIAA and 5-HT). These recoveries were obtained after correction for the water remaining in the tissue residue after a single extraction and taking into account the water content of brain tissue which WIS assumed to be 75”,, of brain weight (Bertler, Carlsson and Rosengren. 195X: Kehr, Carlsson and Lindqvist. 1972). Recoveries were calculated from the difference between fluorometric determinations performed on two aliquots taken from a pooled homogenate of two control brains. to one aliquot of which was added 0.01 ml of each of the following solutions: 100 /lgiml tryptophan, IO !lg;ml 5-HTP, 10 pg,/ml 5-HT and 10~lg:ml 5-HIAA.

Tryptophan uptake activity was determined by a modification of the method described by Knapp and Mandell (1972). Rats were decapitated and their brains quickly removed and dissected on an ice-cold glass plate. A crude synaptosomal fraction of the rat forebrain was then prepared as described by Whittaker, Michaelson and Kirkland (1964). A lO”J, (w/v)

homogenate of tissue was prepared in 0.32 M sucrose (4’C) with a teflon-glass homogenizer. The homogenate was centrifuged at 1000 g for IO min. The supernatant was then centrifuged at lO.OOOg for 20min and the pellet containing the synaptosomes was rcsuspended to produce a 40”. (w.V) homogenate with Krebs-Henseleit buffer at 4 C (Krebs and Henseleit, 1932) which also contained 0.04mM Na,EDTA and 11.1 mM glucose. Incubations were performed in plastic centrifuge tubes in a shaking water bath at 37 C. Each tube received lOO/ll of the tissue suspension. Following a 5 min preincubation with drug. 50111 of L-[“HI-tryptophan (5.4 Ci./mM, New England Nuclear. Boston. MA) in buffer was added to produce a final concentration of 5 /tM and ;I final volume of 2.0ml. Tubes were incubated for an additional 1 min. Control tubes were not preincubated and were maintained at 4 C throughout the entire procedure. The incubation was terminated (including control tubes) by the addition of X.0 ml of ice-cold 0.32 M sucrose to each tube followed immediately by centrifugation at 10,OOOg for IO min. The supernatants were decanted and the pellets waashed with ice-cold Krebs-~Henseleit buffer. The final pellets were acidified with 2.0 ml 2 N HCI, homogenized and centrifuged at IO.000 g for 10 min. Tissue-quantities of [“HI-tryptophan were assayed by liquid scintillation spectrometry in a Packard Tri-carb liquid scintillation spectrometer equipped with automatic standardization. A 0.5 ml aliquot was counted in a 4.5 ml liquid scintillation cocktail consisting of 7.5 g 2,5-diphenyloxazole (PPO). 0.75 g 1,4-bis-2-(4-methyl-S-phenyloxazolyl)benzene (Dimethyl POPOP). 333 ml Triton-X-100 and toluene in a total volume of 1 I. Total protein in each pellet was determined by the Biuret method (Layne, 1967). Uptake was linear with time for 1.5 min and linear with 50 200~11 homogenate. The Km with respect to tryptophan was 16.6~tM which agrees well with results of others (Belin and Pujol, 1973). Srrotonin

qnthesi.5

in vitro

A synaptosomal-rich. 10,OOOg pellet of rat forebrain prepared as described above was resuspended in buffer to produce a 4O:, (w,:v) homogenate. Serotonin synthesis was determined by the evolution of 14C0, from r.-[side-chain-l-‘4C]-tryptophan (Ichiyama, Nakamura, Nishizuka and Hayaishi, 1970). Since this method couples tryptophan hydroxylase with r_-aromatic amino acid decarboxylase. it is possible to detect inhibition of 5-HT synthesis by drugs which inhibit either of these enzymes. Each tube contained 100 ~1 homogenate, drug, L-[side-chain-I-‘4C]-tryptophan (39.4 Ci/M, New England Nuclear, Boston. MA) to produce a final concentration of 4~tM and buffer (as described above) to produce a total volume of 0.7 ml. Before incubation, the mixtures were sealed in glass tubes with rubber caps from which were suspended plastic wells (Kontes Glass Co., Vineland, NJ) containing 100 /II of NCS tissue solubilizer (Nuclear-

Ketamine

121

and S-HT turnover

1

(A)

IZO------(W

:

6

_-

r L

-I-

______

t-

* T 6

70

70

60

60

0”

KETAMINE

(mg/kg)

15

MINUTES

AFTER

160mg/kg

KETAMINE

Fig. 1. (A) Saline or ketamine (40. 80, 120, or lhOmg/kg. i.p.) was administered and 5 min later, NSD-1015 (50 mg/kg. i..p.) was administered to inhibit L-aromatic amino acid decarboxylase. (B) Ketamine (160 mgikg. i.p.) was administered and NSD-1015 (50 mg/kg. i.p..) was administered 5. 20, 50 or 1lOmin later. Animals were sacrificed 15 min after NSD-1015 administration for determination of whole brain 5-HTP levels, Each value is the mean + SEM of Cl2 rats and is expressed as a percentage of control levels of 5-HTP (68.8 f 4.52 ng/g). *Significantly different from saline-treated animals (P < 0.01).

Chicago Corporation, Des Plaines, IL) for the collection of “COZ. Samples were incubated for 30min in a shaking water bath at 37’C. The reaction was stopped by placing the tubes on ice and immediately injecting 0.5 ml I N perchloric acid through the rubber cap. The 14C0, was collected in the NCS during a 3 hr incubation at 37°C in a shaking water bath. Controls were performed using homogenate that had been boiled for 5 min. When the reaction was completed, the wells containing NCS were placed directly into counting vials containing 1Oml of scintillation cocktail consisting of 5 g PPO, 0.1 g dimethyl POPOP and 5ml ethanol/l toluene (Lees, 1977) and counted as above. Aliquots of homogenate were assayed for protein as described above. Serotonin synthesis was linear with time for 40 min and between 50 and 200~1 of homogenate. The K, with respect to tryptophan was 20.2pM which agrees well with the results of Ichiyama et a/. (1970). Drrtrr amlysis

rrnd statistics

Kinetic constants were determined by linear regression analysis. 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 were determined using the Tukey Multiple Comparison Procedure. RESULTS Efl>c.ts qf!fetctmine

on 5-HT synthesis

in vivo

As can be seen in Figure l(a), a dose of 160 mg/kg (i.p. anesthetic dose) ketamine significantly reduced

the accumulation of whole brain 5-HTP after the inhibition of L-aromatic amino acid decarboxylase. Smaller doses (40, 80 and 120 mg/kg, i.p.) had no effect on 5-HTP accumulation. Figure l(b) demonstrates that a single injection of 160mg/kg of ketamine depressed 5-HT synthesis in the brain for as long as 1 hr. Figure 2 shows the regional effects of 80 (subanesthetic dose) and 160 mg/kg of ketamine on the accumulation of 5-HTP in the brain and spinal cord. Since 5-HTP accumulation in the cerebellum was too small to measure accurately. this region is not shown. A dose of 160 mg/kg of ketamine significantly reduced the synthesis of 5-HT in the cortex, midbrainthalamus. medulla-pons, striatum and spinal cord, but the reductions in the hippocampus and hypothalamus were not significantly different from control. Serotonin synthesis was also reduced in all regions following a dose of 80mg/kg of ketamine; however, this effect was significant only in the midbrain-thalamus and the spinal cord.

IZficts qf ketamine

on bruin levels of tryptophan,

S-HT

and 5-HIAA

Ketamine did not affect brain levels of tryptophan at any dose (Fig. 3a) or at any time (Fig. 3b) after its administration. Only a minor, but significant increase in brain 5-HT content was observed (Fig. 4a) following the largest dose of ketamine studied (160 mg/kg). The increase in 5-HT levels could be observed at 15. 30 and 60 but not at 120 min after the administration of 160 mg/kg of ketamine (Fig. 4b). Figure 5(a)

* ** 1I In * 1,

J

t 120 I

*

I

LiJ

60

*

40 20 0~

~

Cortex

Hypoth

Hippoc

Midbrain

Medulla

SALINE

0

KETAMINE

180mg/kgi

m

KElPMINE

(160mg/kg)

Cord

PO05

Tholamus

0

Spmai

isaturn

a

Es

Fig. I!. Saline or ketamine (X0 or 160mg. k g. i.p.) sxs administered to rats. Five minutes later. NSD-101.5 (50 mg’kg, i.p.) was ~~drnin~st~r~d to inhibit L-aromatic ammo acid decltrbonylase. Levels of S-HTP were determined 15 min after the ~~drnii~i~tr~ti~~~~ of NSD-1015. Each \,alue is the mean rt SEM of 7--14 rats. *Signific;tntly d~tfcrent from saline-treated animnis (P < 0.05).

demonstrates

lcvcls but not after the administration of ketnmine. Howcvcr, following :I dose of 160mg, kg of ketaminc (Fig. 5b). a Ggnifcant rcduction in brain 5-HIAA content was observed at 30 min but not at other times (15. 60 or I20 min).

altered

at

that

u

time

brain

5-HIAA

(15 min) shortly

1~‘f~~t.s01’Letmine on trptophn to .i-WT in vitro

uptrtkr

and

Ketamine, in ~~n~~ntr~tions as high as i mM had no effect on tr~pt~ph~in uptake (control. 316 I 13.3 pmol trypt~ph~~n~rn~ proteinimin: I mM ketamine.

1

12c

(A)

(B)

8,

I IO

IIC

I*0

IOC

t

________--_

I_-_.

1

----6---

T

L

go-

90 J ::

$

EO-

s ,\” 7O-

60

60

L

60 DOSE

OF

KETAMINE

img/kgi

mxvr-

sior7

60 MINUTES

AFTER

KETAMINE

1160 mg/kg)

Ketamine 120

123

and S-HT turnover

(A)

*P<.05

*p<.o5

i

T

II0

T

L

T 100

70

60

I5

160 DOSE

OF

KETAMINE

40

50

TIME

(mg/kg)

120

(nun)

Fig. 4. Whole brain serotonin levels were determined (A) 15 min after the administration of saline or ketamine (20, 40. 80 or 160mg/kg, i.p.) and (B) IS, 30, 60 and 120min after the administration of 160 mg/kg (i.p.) ketamme. Each value is the mean + SEM of&l I rats and is expressed as a percentage of control levels of serotonin (381 f 12.9 ngig).

345 f 33.4; n = 5) or tryptophan conversion to 5-HT (control, 49.7 + 5.1 pm01 CO,/mg protein/30 min; 1 mM ketamine, 49.6 f 5.8, n = 5) by crude synaptosomal preparations of rat forebrain.

uptake inhibitors reduce the rates of synthesis and metabolism of 5-HT in riw, it was interesting to determine whether ketamine also reduced 5-HT turnover. The effects of ketamine on 5-HT synthesis in cico were determined by the rate of accumulation of 5-HTP following inhibition of L-aromatic amino acid decarboxylase (Carlsson et al., 1972). It was found that a dose of 160mgikg of ketamine could significantly reduce the rate of 5-HT synthesis in most brain

DISCUSSION

Ketamine has been shown to block the uptake of 5-HT in tlioo (Martin et al., 1982). Since 5-HT

(6)

* PC 05 IIO-

,oo__-

I._

______

r

L

L

70-

70-

60

60-

/

;

0

1

120

(A)

160

20 DOSE

OF

KETAMINE

(mg/kg)

T

0-L

L 13

30 TIME

-

@a” (mm1

Fig. 5. Whole brain 5-HIAA levels were determined (A) 15 min after the administration of saline or ketamine (20. 40, 80 or 160mg/kg, i.p.) and (B) 15. 30, 60 and lZOm1n after the administration of 160 m&kg (Lp.) ketamine. Each value is the mean + SEM of Cl 1 rats and is expressed as a pcrccntage of levels of control 5-HIAA (340 f 17.1 ng/g).

124

L. L. MARTIN and D. J. SMITH

regions and that 80mg/kg of the drug produced significant reductions in 5-HT synthesis in the midbrainthalamus and spinal cord. In order to characterize further the effects of ketamine on serotonin metabolism, brain 5-HT (Fig. 4) and S-HIAA (Fig. 5) levels after ketamine administration were determined. Ketamine (160 mg/kg) produced only a small, but significant, increase in brain 5-HT during the first hour after its administration. In contrast, 5-HT metabolite levels, measured one-half hour after this dose of ketamine. were reduced by 152, compared to saline-injected controls. This reduction in 5-HT metabolite levels does not appear to be related to the weak ability of ketamine to inhibit monoamine oxidase (MAO) in vitro (Azzaro and Smith, 1977) since ketamine does not behave like an MAO inhibitor in vivo (Martin er al., 1982). Thus, it appears that 5-HT metabolism as well as 5-HT synthesis is reduced following administration of ketamine. In order to rule out direct effects of ketamine on 5-HT synthesis, the effects of ketamine on brain tryptophan content and on tryptophan uptake and conversion to 5-HT in vitro were evaluated. Biggio et (I/. (1974) found an increase in brain tryptophan content 2 hr after the administration of 100 mg/kg (i.p.) of ketamine. In the present studies. no effect of ketamine on brain tryptophan levels was observed at any dose or time tested including 2 hr (Fig. 3). In addition, no effect of ketamine on tryptophan uptake or conversion of tryptophan to 5-HT was observed in vitro. Thus, ketamine does not appear to depress 5-HT synthesis in tire by affecting precursor supply or by directly inhibiting enzymes involved in 5-HT synthesis. It appears that ketamine decreased 5-HT turnover by inhibiting 5-HT uptake (Martin et al., 1982). This conclusion is supported by the fact that other 5-HT uptake inhibitors also decreased 5-HT turnover. Furthermore, the present authors have ruled out other possible effects of ketamine on serotonergic systems. Ketamine did not directly affect 5-HT synthesis, did not inhibit MAO in civo, did not affect 5-HT receptor binding (Martin et al., 1982) and did not induce 5-HT release in viva, which is suggested by the fact that brain 5-HT levels were not reduced by ketamine (Fig. 4) despite a reduction in 5-HT synthesis (Fig. I). Inhibition of 5-HT uptake by ketamine should result in enhanced intrasynaptic concentrations of 5-HT available for interaction with both pre- and postsynaptic 5-HT receptors. Ketamine may, thereby, reduce 5-HT turnover by activation of presynaptic 5-HT receptors which inhibit 5-HT release (Cerrito and Raiteri, 1979), by activation of a postsynaptic neuronal feedback loop (Carlsson and Lindqvist, 1963) or by enhancing neurotransmission across inhibitory serotonergic rapheraphe connections (Haigler and Aghajanian, 1974; Mosko. Haubrich and Jacobs. 1977). An unexpected finding was that ketamine did not

significantly reduce the synthesis of 5-HT in all brain regions studied (Fig. 2). This discrepancy does not appear to be due to differences in the distribution of ketamine or to differences in its ability to inhibit 5-HT uptake in various brain regions. Ketamine has previously been demonstrated to block 5-HT uptake in rim in all brain regions studied, including the hippocampus and hypothalamus. in which structures ketamine did not significantly reduce 5-HT synthesis (Martin KI u/., 1982). In contrast to the present results with ketamine. Marco and Meek (1979) reported that selective 5-HT uptake inhibitors such as chlorimipramine and fluoxetine reduced 5-HT synthesis in all brain regions studied. including the hippocampus and hypothalamus. The key to the discrepancy between the effects of ketamine and selective 5-HT uptake inhibitors on regional brain 5-HT synthesis may be that ketamine is not a very selective 5-HT uptake inhibitor. Indeed, in addition to blocking 5-HT uptake, ketamine also blocked the uptake of norepinephrine and dopamine (Azzaro and Smith, 1977). Ketamine is also an opiate receptor agonist (Smith, Pekoe, Martin and Colgate, 1980) and inhibits acetylcholinesterase (Cohen. Chan, Bhargava and Trevor, 1974). Thus ketamine may influence serotonergic cells both directly. by blockade of 5-HT uptake, and indirectly through effects on non-serotonergic systems which innervate the raphe nuclei. The net effect of these various influences on 5-HT neurons may vary from one region to another. Previous reports regarding the effects of ketamine on brain levels of 5-HT and 5-HIAA have been very contradictory. For example. brain 5-HT levels have been reported to be either increased (Kari et cd., 1978) or unchanged (Sung rt LII., 1973; Ylitalo et rd., 1976 and Vargiu rt al., 1978) 3&60 min following ketamine administration. Even more confusing are the findings regarding brain 5-HIAA levels. Levels of 5-HIAA have been reported to be unchanged at I5 min (Ylitalo rt al., l976), either unchanged (Vargiu et al.. 1978) or increased (Kari rt rll., 1978) at 30min. and either unchanged (Ylitalo cut LII., 1976) or increased (Vargiu et al., 1978) at 60min. Some studies have reported changes in 5-HT turnover (Sung et ctl., 1978) or 5-HIAA levels (Biggio et rrl., 1974 and Vargiu et al., 1978) several hours after the administration of ketamine. However, it is difficult to imagine how these results might relate to the behavioral effects of ketamine. since behavior is generally normal within 2 or 3 hr after ketamine administration. It is not possible to explain why the results of these studies differ from each other and, in many cases, with the present authors’ findings. To date, all studies dealing with the effects of ketamine on serotonergic systems in the rat brain have differed from each other in one or more respects (i.e. different rat strains and weights, doses. routes of administration. 5-hydroxyindole assay methods, etc.). Of these studies, the results of only one agree closely with the present ones. The findings of Ylitalo rf rrl. (1976) and of the present study both

115

Ketamine and 5-HT turnover demonstrate a lack of effect or only a minor effect of kctamine on 5-HIAA and 5-HT levels at 1.5 and 60min. Although in this study a reduction in 5-HIAA levels at 30min was found, values for 5-HIAA levels at this time were not reported by Ylitalo et al. (1976). Nevertheless. the mesent findine that ketamine decreased brain S-HIAA content, if only at one time, supports the finding of Ylitalo et al. (1976) that ketamine signi~cantl~ decreases brain 5-HT metabolism during the first 2 hr after its administration. To summarize. it has been demonstrated that ketamine reduces 5-HT turnover in the central nervous system. This effect is probably an adaptive change in serotonergic function occurring in response to 5-HT uptake blockade by ketamine. This conclusion is supported by the fact that ketamine did not affect brain tryptophan levels and did not affect S-HT synthesis itz t+fro. The ability of ketamine to alter serotonergic function bv inhibitinr W-IT uutake would suggest that the serotonergic system may mediate some of the behavioral changes, such as analgesia, occurring as a result of ketamine administration. Aikno\~/edgemc~nt--The authors wish to thank Dr Elaine Sanders-Bush for her contributions and helpful suggestions in the preparation of this manuscript.

Cerrito, F. and Raiteri. M. (1979). Serotonin release is modulated by presynaptic autoreceptors. EIW. J. PharMCIC. 57: 427-430. Cohen, M. L., Ghan, S. L., Bhargava, H. N. and Trevor. A. J. (1974). Inhibition of mammalian brain acetylcholinesterase by ketamine. Eiochrm. Phurtt~ctc~. 23: 1647~ 1652. Glowinski. J. and Iversen, L. L. (1966) Regional studies of catecholamines in rat brain. 1. The disposition of E”H]-norepittepllrine. [‘HI-dopamine and [3H]-dopa in various regions of the brain. i. ~~f~~r~~cft~~~i.13: 665 669.

Haigler, H. J. and Aghajanian. G. K. (1974). Lysergic acid diethylamide and serotonin: a comparison of effects on serotonergic neurons and neurons receiving a serotonerpit innut. J. Phftrtnac. rsn. Ther. 188: 6X8--699.

Ichiyami, A.. Nakamura. s.. Nishizuka. Y. and Hayaishi, 0. (1970). Enzymic studies on the biosynthesis of serotonin in mammalian brain. J. hiol. C/I~,,I. 245: 1699 1709. Kari, H. P., Davidson. P. 0.. Kohl, H. H. and Kochhar. M. M. (1978). Effect of ketamine on brain m~~no~irninclevels in rats. Res. Cornmutt. Chetn. Pmth. Phartncrc~. 20: 475 488. Kehr. W.. Carisson, A. and Lindqvist, M. (1972). A method for the determination of 3,4-dihydroxyphcnylalaninc (DOPA) in brain. Naun~tt-S[,fttnieduhrr.8\ Arc,h. Phtrrtnctc~. 214: 273-280. Knapp, S. and Mandell. A. J. (1972). Narcotic drugs: effects on the serotinin biosynthetic systems of the brain. Science 177: 1209-1211. Krebs. H. A. and Henseleit. K. (1932). Unt~rsueh~n uher die harnsto~ildung Physiof.

REFERENCES Atack, C. V. and Lindqvist, M. (1973). Conjoint native and orthophthaldialdehyde condensate assays for the Ruorimetric determination of 5-hydroxyindoles in brain. Naunyn-Schmiedrher(/,

Arch.

Phortnac.

279: 267-284.

Azzaro, A. J. and Smith. D. J. (1977). The inhibitory action of ketamine HCI on [3H]-5-hydroxytryptamine accumulation by rat brain synaptosomal-rich fractions: comparison with [3H]-catecholamine and C3H]-gammaaminobutyric acid uptake. N~u~~~~zur~t~~~/~)~~16: 349-356. Bedard, P., Carlsson, A. and Lindqvist, M. (1972). Eflcct of a transverse cerebral hemisection on 5-hydroxytryptamine metabolism in the rat brain. Naunyn-Schmiedebergs Arch. Pharmuc. 272: l-l 5. Belin. M.-F. and Pujol, J.-F. (1973). Transport synaptosmal du tryptophane et de la tyrosine c&brale. Existence de systtmes de capture d’afiniti diffkrente. E~perie~fi~~ 29: 41 I-413.

Bertler, A., Car&son, A. and Rosengren, E. (19.58). A method for the fluorimetric determination of adrenaline and noradrenaline in tissues. Acta physiol. stand. 44: 273-292.

Biggio, G.. Proceddu. M. L. and Vargiu, L. (1974). Effect of ketamine Rir.

Farm.

(Ketalar)

on

brain

monoamine

metabolism.

Ter. 5: 163-168.

Carlsson, A. and Lindqvist, M. (1963). Effect of chlorpromazine or haloperidol on formation of 3-methoxytyramine and normetanephrine in mouse brain. Acra pharnl1zc.

tax.

20: 14% 144.

Carlsson, A., Davis, J. N.. Kehr, W., Lindqvist, M. and Atack, C’. V. (1972). Simultaneous measurement of tyrosine and tryptophan hydroxylase activities in brain in ril;o using an inhibitor of the aromatic amino acid decarboxylase. Nuun~n-Schmiedehergs Arch. Pharmac. 275: 153--168.

im tierkorper.

~op~(~-S~~~~~~,r~,~ Z.

Chem. 210: 33&&.

Layne. E. (1967). Spcctrophotometric and turbidimetric methods for measuring proteins. In: hlcthod it1 Enzymoloyy (Kolowick, S. P. and Kaplan, N. 0.. Eds). Vol. 3. pp. 447 454. Academic Press, New York. Lees. G. J. (1977). Functional significance of aromatic amino acid: aromatic keto acid aminotransferases in rat brain and liver: competition for tryptophan between aminotransferases and tryptophan hydroxylasc iti ~‘ifro. L+ Sri. 20: I749- 1762. Marco, E. J. and Meek, J. L. (1979). The effects of antidepressants on serotonin turnover in discrete regions of rat brain.

NNNn!‘,l-S~,fttttic,drhrrg~

Arch.

Pfwwctc~.

306:

75m79.

Martin. L. L., Bouchal, R. L. and Smith, D. J. (1982). Ketamine inhibits serotonin uptake in vile. Neurophartnacology, 21: 113--l 18. Mosko, S. S., Haubrich, D. and Jacobs, B. L. (1977). Serotonergic afferents to the dorsal raphe nucleus: evidence from HRP and synaptosomal uptake studies. Brtrin Rcs. 119: 269-290. Smith. D. J., Pekoe, G. M., Martin, L. L. and Colgate, B. (1980). The interaction of ketamine with the opiate receptor. I,#” Sci. 26: 789-795.

Sung, Y. F., Frederickson, E. L. and Holtzman. S. G, (1973). Effects of intravenous anesthetics on brain monoamines in the rat. Anesthesiology 39: 478-487. Vargiu, L.. Steffani. E., Musinu. C. and S&a. G. (1978). Possible role of brain serotonin in the central efTects of ketamine. ~e~r~phurm~cofog~ 17: 4051105. Whittaker. V. P.. MichaelsoIl, I. A. and Kirkland, R. J. (1964). The separation of synaptic vesicles from nerve ending particles (synaptosomes). Bioc,fwtn. J. 90: 293-303. Ylitalo, P., Saarnivaara, L. and Ahtee, L. (1976). Efiect of ketamine anesthesia on the content of monoamines and their metabolites in the rat brain. Actu Anaesth. Surnd. 20: 216-220.