European Journal of Pharmacology, 184 (1990) 173-177

173

Elsevaer EJP 20668

Short communication

Effects of urethane and ketamine on substance P- and excitatory amino acid-induced behavior in mice Nelson L Dal61 and Ahce A Larson Unwerslty of Mmnesota, Department of Veterinary Btology, 295 Ammal Scwnce/ Vetermary Medzcme Budding, 1988 Fttch Avenue, St Paul, MN 55108, USA

Recewed 22 May 1990, accepted 5 June 1990

Urethane and ketanune were tested for their ablhty to alter the caudally directed binding and scratching response ehcited by the mtrathecal (1 t ) mjecUon of excitatory amino acids (EAAs) or substance P (SP) EAAs, such as N-methyl-D-aspartate (NMDA), kamate acid and quxsquahc acid, but not SP, were inhibited by subanesthetlc doses of urethane In contrast, SP was more sensitive than NMDA to the lnlubltory effect of (+)-ketamlne (-)-Ketarmne produced much less inhibition of the SP-mduced behaviors than the (+)isomer These results have important lmphcatlons regarding the use of urethane and ketamlne as anesthetacs for studies in which these excitatory compounds are potentml medmtors Substance P, Exotatory armno acids, Urethane, Ketamlne

1. Introduction Ketanune and urethane are commonly used as injectable anesthetics m neuropharmacologlc studles m the spinal cord The choice of these compounds as anesthetics has generally been justified on the basis that they preserve spinal reflexes a n d / o r muscle tone and would therefore not slgmficantly alter the pharmacology of spinal neurotranslmtters The neurotransrmtter systems mediating the actions of ketamme and urethane, however, are not fully understood Urethane appears to alter or mtubit the effects of some armno acids xn the CNS Antagonism of excxtatory armno acid (EAA)-mduced depolarization of frog and lmma-

1 Present address Umversldad Centroccldental, Barqms~meto 3001, Venezuela Correspondence to A A Larson, Umverslty of Minnesota, Department of Veterinary Biology, 295 Animal Scwnce/ Veterinary Medicine Bmldmg, 1988 Fitch Avenue, St Paul, MN 55108, U S A

ture rat ventral root preparations by urethane was first reported by Evans and Smith (1982) Ketanune is also thought to interact with a variety of neurotransnutters, but, hke phencychdlne (PCP), ketamlne is also believed to act primarily through binding at a PCP site located within the Nmethyl-D-aspartate ( N M D A ) receptor complex When ketanune occupies the PCP binding site the result is a voltage-dependent inhibition of neuronal translmSSlOn If these proposed mechamsms for urethane and ketamlne are correct, then the use of these anestheUcs for neuropharmacologlcal studies, especially those revolving the study of EAAs, may be a comphcatmg factor m the analysis of such data Based on these reports, we felt it was important to test the hypothesis that urethane or ketarnlne alters EAA or SP actlwty in the CNS, particularly in hght of the importance that has been placed on substance P (SP) (Plercey et al, 1986) and more recently on EAAs (Aanonsen and Wilcox, 1987) m pare transnusslon To test tlus hypothesis we ex-

0014-2999/90/$03 50 © 1990 Elsevaer Soence Pubhshers B V (Blome&calDlvaston)

174 analned the effect of urethane on the caudally directed biting and scratchang behavior (CBS) induced in wvo by the lntrathecal (1 t ) injection of either EAA agomsts or SP The CBS behavior induced by EAAs or SP, i t , has been hypothesized to be assooated with noclceptlon in nuce (Aanonsen and Wilcox, 1986, Piercey et al, 1986, Delander and Wahl, 1989) or with a spinal convulsive-hke state in rats (Frenk et al, 1988) The physiologic slgmflcance of this behavior, however, is unknown Whale its physiological importance is unclear, behavioral studies involving i t injection of EAAs or SP (Hylden and Wilcox, 1981) have proven to be a useful approach to stud3ang drug interactions in neuropharmacology Tbas in vivo behavioral model has the advantage that it does not require the use of any anesthetic that may interfere with the behavioral response Behavior induced by various EAA agomsts can be inhabited by EAA antagomsts, resulting in unique profiles of activity Selective lnlubitlon of NMDA-lnduced CBS was reported using 2-armno-4-amlnophosphonoheptanolc acid (APH) (Delander and Wahl, 1989) and 2-anuno5-phosphonovalerlc acid (APV) (Aanonsen and Wilcox, 1987) Slnularly, SP-mduced behavior is selectively blocked by the SP antagomst, [DPro2,D-Trp7'9]Sp (Hwang and Wilcox, 1986, Delander and Wahl, 1989)

2. Materials and methods

Male Swiss-Webster mice weIghang 20-25 g (BioLab, Whate Bear Lake, MN) were used in all experiments All I t injections of SP and EAAs were made as previously described (Hylden and Wilcox, 1981) using a 30-gauge 0 5 inch disposable needle on a 50/~1 luer tip Hamalton syrmge The volume rejected was 5/~1 The number of caudally directed biting and scratcbang responses were counted for 1 nun immediately after each mjecuon NMDA, kalmc acid and qulsquahc acid were obtained from Sigma Chenucal Co (St LOUIS, MO) and urethane from Merck (Rahway, N J) The ( + ) and ( - ) forms of ketamlne were gifts from Dr Anthony J Trevor from the Umverslty

of Cahforma at San Francisco CBS behavior was induced by the i t adrmnlstratlon of N M D A (0 2 nmol/5/~1), kalnlC acid (0 075 n m o l / 5 #1), qulsquahc acid (0 5 n m o l / 5 #1) and SP (5 or 10 n g / 5 #1) Equipotent doses of EAAs and SP were chosen such that between 20 and 50 CBS responses/nun were observed At the doses used the CBS behavioral episode began immediately after injection, was maximal at 30 s and typically lasted 60 s in duration Statistical significance of a drug effect compared to a sahne control value was deterrmned by Student's t-test using a cutoff of P < 0 05 The effects of urethane, (+)-ketamlne or ( - ) ketamlne on SP- or EAA-induced CBS behavior were tested following either systemic (intrapentoneal, i p ) or I t administration at nonanesthetic doses In addition, both ( + ) - and ( - ) ketanune were tested for their effect on the desensitization observed following repeated doses of SP

3. Results

Adnunistration of a mild sedative dose of urethane (0 4 g / k g 1 p ), 30 nun before the i t challenge with an EAA, was sufficient to inhabit behaviors induced by N M D A and kalnlC acid, but not qtusquallC a o d (fig 1A) A larger, but still non-anestheUc dose of urethane (0 8 g / k g i p ), slgmflcantly lrdubited qulsquallC acid-induced as well as N M D A - and katmc aod-lnduced CBS behavior In contrast, behavioral responses to i t rejections of SP were not altered by either close of urethane Injection of either 2 5 or 5/~mol of urethane i t e h o t e d no CBS when adnumstered alone The higher dose of urethane (5/~mol i t ) significantly inhabited the CBS induced by all three EAAs agomsts, however, thas dose of anesthetic also caused a notlcable motor impairment in the band 1Lrnbs (duration 5 to 15 s) In spite of this motor impairment, i t urethane did not affect the response to 5 or 10 ng of SP Unhke urethane, ketanune is commercially avadable as a racenuc mLxture The ( + ) isomer exhibits more potency as an anesthetic than the ( - ) form winch displays a greater incidence of

175

halluonogemc, convulsant and dysphorlc activity Our results mdicate that the ( - ) i s o m e r of ketamme, which has been reported to be only approximately one thard as potent an anesthetic as the ( + ) form, was indeed less effective in its ability to inhabit SP-tnduced CBS than the ( - ) i s o mer (fig 2A) The relatively greater inhibitory effect of ( + ) - k e t a m l n e on SP-lnduced CBS, however, appears to be transient Repeated mjectton of SP in n-ace at 90 s intervals led to a dramatic decrease in the intensity of CBS induced by each subsequent injection of SP, as reported previously

~

50-

10. 0

10

~

Urelh 0 8 g/kg I p

*

0

50.] ; M D "

KA

QUIS

SP

!

0iIi n't

•

Control

[] []

Ureth 2 5 I~mol i t Ureth 5 p,mol i t

J

NMDA

KA

QUIS

SP

Treatment Fig 1 Inlubltlon by pretreatment with or coadrmmstratlon of urethane (Ureth) of the caudally directed biting and scratching behavior (CBS) produced by exotatory armno a o d agomsts but not by substance P (SP) The intensity of the behavioral response to 0 2 nmol of NMDA, 0 075 nmol of kaimc acid, 0 5 nmol of qulsquahc acid or 10 ng of SP was measured over a 1 roan Ume period following their ~ t mjecuon Statxsueally significant difference from control group tested the same day • ** P < 0 0 0 1 , ** P < 0 0 1 , * P < 0 0 5 (A) Inlubltoryeffect on EAAs but not SP of two subanesthet]c doses of urethane adrmmstered ] p as a 30 nun pretreatment Each bar represents the mean + S E of at least eight mace (B) Selectwe ]nlubmon of EAA- but not SP-mduced behavior by coadmamstration of urethane I t with each excitatory compound Each bar represents the mean + S E of at least six nuce

210

310

B ~

50"

~

40"

t0"

[]

I 10

o0

20"

:

NMDA (0 2 nmolus) NMDA & (+)ketarnll~ NMDA & ( )ketamlne

20"

E ~

O

20

•

30"

Z ~ Control Ureth 0 4 g/kg I p

SP (10 ng) SP & (+)ketamlne SP & ( )ketamlne

o

40 *

•

40I~

.~m 30'

*

A

60-

60. A

30

o

70o

0

0

i 1 0

i 2 0

310

410

Number of Intrathecal ~jec'tlon

Fig 2 Effect of coadnumstratlon with 0 33 ng of the ( + ) or ( - ) isomer of ketamane 1 t on N M D A - and SP-mduced CBS behavior * Indacates a slgrnflcant difference from control group tested the same day (P < 0 05) Each hne represents the means + S E obtamed over time from groups of 8-12 n'ace (A) Decreased intensity of CBS behaviors m response to repeated adrmmstrauon of 10 ng of SP at 90 s intervals Stereoselectlvity of the effects of 033 ng of the ( + ) and ( - ) isomers of ketarmne on SP-mduced CBS when co-injected repeatedly at 90 s intervals (B) Failure of 0 33 ng of ( + ) - or ( - ) - k e t a r m n e to slgmflcantly alter the intensity of CBS ehoted by 0 2 nmol of N M D A when coadmtmstered ~ t

(Larsen, 1988) Whale coadmlmstratton of SP together with ( - ) - k e t a n u n e tended to umformly inhabit responses to each injection of SP, coadnunlstratlon of ( + ) - k e t a n u n e with SP resulted in an mitlal decrease in the CBS response after the first injection but an increase m the number of CBS behaviors induced by each subsequent inJection of these two compounds (fig 2B) In contrast to the selective inhibition of EAA agomsts by urethane, coadtmmstratxon of 0 33 ng of either ( + ) - or ( - ) - k e t a m l n e , I t did not significantly alter the CBS induced by either a single injection or repeated injections of 0 2 nmol of N M D A , as shown in fig 2A InJection of 78 ng of ( + ) - k e t a rmne inhabited both SP- and N M D A - m d u c e d CBS by 64 4 and 65 2%, respectively, confirming its

176 abthty to lnhiblt b o t h N M D A - and SP-mduced activity, but at much larger doses (data not shown)

4. Discussion The results of the present investigation demonstrate that the use of non-anesthetic doses of urethane slgmflcantly lntUblt the behavior induced by i t N M D A , kalnlC acid and qutsquahc acid (fig 1) Doses that dramatically lnhabtt the CBS induced by EAAs, however, do not alter the intensity of CBS induced by SP These data support the imtlal suggestion by Evans and Smith (1982) that the depressant effect of urethane, is likely related to inhibition of EAA activity In their hands, urethane at doses of 980-1247 m g / k g , antagomzed armno actd-evoked responses in the isolated rat spinal cord preparation In comparison, we report that even non-anesthetic doses of 400-800 m g / k g were sufficient to inhabit behavior reduced by the i t injection of the three EAA agomsts tested These studies emphasize that care must be used In the interpretation of in VlVO or in vitro experiments related to the action of EAAs that are performed using urethane as an anesthetic In sptte of ketarmne's proposed interaction w~th the PCP binding site of the N M D A receptor complex, our results indicate that very low doses of (+)-ketamlne produced a greater inhibition of SPthan of NMDA-induced CBS at doses of these exotatory agents that elicit the same intensity of CBS behavior In contrast, PCP, a dissociative anesthetic closely related to ketamane, has been found to have a greater mhabltory effect on NMDA-induced CBS than on SP-lnduced CBS, yet still significantly inhahats the CBS behavior induced by SP, presumably through a dopamlnerglc mechanism (Delander and Wahl, 1989) Unhke the stereoselective effect of ketamlne on SP-lnduced CBS, coadrmmstratlon of the same dose of either ( + ) - or ( - ) - k e t a l m n e with N M D A did n o t appear to significantly alter the behavioral responses to N M D A (fig 2B) These data suggest that while ketamlne inhibits N M D A activity, it exhibits an even more potent inhabltory effect on SP activity in the spmal cord that is not mediated

by an mteraction wtth the N M D A receptor complex The effect of ketanune on SP-lnduced CBS was both greater than its effects on NMDA-lnduced CBS and stereoselective ( + )-Ketarmne was a more potent trdubttor of SP-lnduced CBS than ( - ) ketamlne, which correlates well with its greater anesthetic and analgesic effect Whale one could speculate that the ablhty of (+)-ketarmne to s e n s i t i z e the mouse to subsequent m lection of SP may be related to ketamlne-induced seizure activity, excitatory responses and increased muscle tonus are, in fact, more frequently associated with the ( - ) - i s o m e r (Whate et al, 1982) The physiologic significance of behavior produced by the i t injection of compounds producing excitatory effects m the CNS is unclear, but these results clearly Indicate that urethane selectively inhibits EAA- but not SP-induced activity whale ketalmne produces a stereoselectlve inhibition of SP-induced CBS behavior These studies suggest that (1) investigations focusing on EAAs should be interpreted wath caution when utlhzang urethane as anesthetic and (2) electrophyslologlcal and behavioral studies of either SP or EAAs can be similarly comphcated by the use of ketarmne

Acknowledgements This research was supported by Umted States Pubhc Health Servace Grants NIDA00124, NIDA04090 and NIDA04190 The authors would hke to thank Drs Stephen R Slolhng and Chnstropher W Murray for their excellent e&tonal assistance in the preparation of ttus manuscript

References Aanonsen, L M and G L Wilcox, 1986, Phencychdme selectwely blocks a spinal action of N-methyl-D-aspartate m mice, Neuroscl Lett 67, 191 Aanonsen, L M and G L Wdcox, 1987, Nooceptlve aclaon of excitatory armno acids m the mouse Effects of spinally adrmmstered oplolds, phencychdlne and sigma agomsts, J Pharmacol Exp Ther 243, 9 Delander, G E and J J Wahl, 1989, In vtvo characterization of phencychdlne/a agomst-medlated mhlbmOn of noclcepnon, European J Pharmacol 159, 149

177 Evans, R H and A S Smith, 1982, Effect of urethane on synaptxc and amino acid-reduced excltat~on m isolated spinal cord preparaUons, Neuropharmacology 21, 857 Frenk, H , D Bossut and D J Mayer, 1988, Is substance P a primary afferent neurotransmitter for noclceptlve input 9 Valpro~c acid and chlordlazepoxade decrease behaviors ehclted by mtrathecal lnjectton of substance P and excitatory compounds, Brain Res 455, 240 Hwang, A S and G L Wdcox, 1986, Intradermal hypertomc sahne-mduced behavaor as a noc~ceptwe test m mace, Life Scl 38, 2389

Hylden, J L K and G L Wdcox, 1981, Intrathecal substance P ehclts a candally-dlrected biting and scratching behavior m mace, Brain Res 217, 212 Larson, A A , 1988, Desenslt~zatlon to mtrathecal substance P in mace possthle involvement of oplolds, Pare 32, 367 Plercey, M F , M W Moon, J R Bhnn and P J K DobrySchreur, 1986, Analgesic actlxaUes of spinal cord substance P antagomsts ~mphcate substance P as an neurotranslmtter of pain sensation, Brain Res 385, 74 White, P F , W L Way and A J Trevor, 1982, Ketamane - ~ts pharmacology and therapeuUc uses, Anesthesiology 56, 119