Mechanisms of general anesthesia: Brain regional ersponses to baclofen

Physiology& Behavior,Vol. 46, pp. 3-8. ©Pergamon Press plc, 1989. Printed in the U.S.A.

0031-9384/89 $3.00 + .00

Mechanisms of General Anesthesia: Brain Regional Responses to Baclofen J O H N N Y E L. L E W I S , V E R N E R S. W E S T E R B E R G 1 A N D F R A N K S. L A B E L L A

Department of Pharmacology and Therapeutics, Faculty of Medicine University of Manitoba, Winnipeg, Manitoba

LEWIS, J. L., V. S. WESTERBERG AND F. S. LABELLA. Mechanisms of general anesthesia: Brain regional responses to baclofen. PHYSIOL BEHAV 46(1) 3-8, 1989.--The GABAB agonist baclofen is reported to produce general anesthesia when administered either centrally into the lateral ventricles of rats or peripherally to mice. Previously we demonstrated that 13-endorphin given intracerebrally produces anesthesia in rats, a response localized to sites in or adjacent to the inferior third and fourth ventricles. In order to compare the anatomical localization of these two anesthetic responses, we administered baclofen into the inferior or superior lateral or third ventricles, the aqueduct, or fourth ventricle in rats. Although 10 ~g baclofen infusions into several regions caused loss of the righting reflex, in no case did animals exhibit an unconscious state which satisfied strict criteria of anesthesia. Infusions of 20 p.g into the inferior third and fourth ventricles elicited seizures followed by a postictal depression. Although unresponsive to some stimuli, these animals showed no impairment in the corneal reflex. Since this dose was often lethal, higher doses were not tested. Baclofen, given to mice intraperitoneally at doses of 25, 50, or 75 mg/kg, failed to elicit strictly defined anesthesia, although, to varying degrees, animals exhibited analgesia, loss of the righting reflex, and loss of behavioral responses to loud sounds. Animals continued to show motor responses when handled and retained corneal reflexes. Baclofen does not evoke an unconscious anesthetic state when administered centrally or systemically, emphasizing the need for strict criteria to define general anesthesia and to categorize drugs that promote this state. Mechanisms of anesthesia Baclofen Postictal depression Motor activity

GABA Analgesia

GABAB Intracerebroventricular Consciousness Corneal reflex

THE mechanism of action of general anesthetics remains controversial. Theories of mechanisms include nonspecific membranelipid effects based upon the lipophilic nature of clinical agents (18,29) and specific interactions at protein sites (6,16). One currently popular theory postulates the GABA-ionophore complex as a molecular site of action (4,14). Pentobarbital, a known anesthetic agent, binds to this complex and enhances chloride conductance (28). In addition, enhanced chloride conductance in various neuronal preparations has been demonstrated for other classes of anesthetic agents including the steroid alphaxalone (1), diethyl ether, ethanol and enflurane (19). However, some investigators point out that increased chloride conductance occurs only at concentrations far greater than those required to produce anesthesia (2), but that clinical concentrations hyperpolarize neurons by increasing potassium conductance (2,22). The role of GABAergic transmission in anesthesia is also unclear. GABA infused into the cerebral ventricles or cisterna magna of cats produced an anesthetic state defined by loss of righting, corneal, and foot withdrawal reflexes (5). Whether this response is mediated by GABA A or GABA B receptors was not determined, but, in mice, peripheral administration of the GABA analog THIP, an agonist more selective for the GABA A receptor,

Seizure Righting reflex

has since been claimed to cause anesthesia characterized by loss of righting reflex and analgesia (3). The GABA B agonist baclofen is reported to elicit anesthesia after peripheral administration in mice (27) and when administered intracerebroventricularly (ICV) into the lateral ventricles of rats (30). We have observed general anesthesia in rats with ICV administration of the opioid peptide I$-endorphin (10), although our criteria for general anesthesia were much more stringent than those used in either baclofen study. Our criteria include loss of righting, foot withdrawal, and corneal reflexes combined with loss of behavioral and EEG responses to loud sound, surgical incision and suturing. Criteria for baclofen "anesthesia" have included only analgesia and loss of righting reflex (27,30). Therefore, whether the anesthetic-like state elicited by GABA analogs comprises the other characteristics of general anesthesia produced by 13-endorphin was uncertain. Both baclofen and 13-endorphin act potently at specific receptor sites and axe unlikely to cause anesthesia by nonspecific membrane effects. Hyperpolarization of neurons by enhanced potassium conductance is a proposed mechanism of action for both baclofen (12,21) and 13-endorphin (23,25), as well as for certain clinical anesthetics (2,22). If these compounds elicit similar response

1Present address: Department of Psychology, University of New Mexico, Albuquerque, NM 87131.

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LEWIS, WESTERBERG AND LABELLA

patterns at common neuroanatomical sites (either directly or via neural projections), they may be useful for the elucidation of molecular mechanisms of anesthesia. Elsewhere, we reported that 13-endorphin is most effective in eliciting general anesthesia when administered into either the inferior third or the fourth ventricle (Western Pharmacology Society Annual Meeting, Tucson, 1988). To determine whether baclofen evokes a comparable anesthetic state at a common brain site, we have characterized the responses to ( - ) b a c l o f e n administered into different regions of the cerebroventricular system. METHOD

Intracerebral Cannulations Fourteen male Sprague-Dawley rats (University of Manitoba Central Animal Care Colony) weighing from 250-350 g at time of surgery were used. Guide cannulae (Plastic Products) were implanted in rats under sodium pentobarbital anesthesia (50 mg/kg IP) into the following ventricular locations: right inferior lateral ventricle (AP - 3 . 8 ; ML +4.5; DV - 7 . 0 ) , n = 2; right superior lateral ventricle (AP - 0 . 8 ; ML + 1.5; DV - 2 . 8 ) , n = 2; inferior third ventricle (AP - 3 . 0 ; ML 0.0; DV - 8 . 5 ) , n = 5; superior third ventricle (AP - 4 . 2 ; ML 0.0; DV - 3 . 6 ) , n = 1; cerebral aqueduct (AP - 8.3; ML 0.0; DV - 4.0), n = 2; or fourth ventricle (AP - 12.8; ML 0.0; DV - 8.0), n = 8. Coordinates were referred to bregma at a stereotaxic plane of - 3.3 (24). A screw electrode was fixed in the skull anterior to the olfactory bulbs to serve as a recording ground, and bilateral screw electrodes were fixed in the skull at AP - 6.0, ML +_2.5 for recording of monopolar cortical EEGs. Electrodes and guide cannula were secured to the skull with dental acrylic. A dummy cannula was inserted into the guide when drugs were not being infused. Animals were given one week postsurgical recovery.

Drug Infusions Infusions were made between 1100 and 1400 hr. Infusion cannulae extended 1 mm beyond the guide tip. Test solutions were infused at a rate of 1 p,1/min using a Sage infusion pump. Cannulae were left in place for 10 min postinfusion to eliminate backflow into the guide. All animals reported here received infusions of 50 ~g camel [3-endorphin 7 days prior to ( - ) b a c l o f e n infusions. ( - ) B a c l o f e n (gift from CIBA Pharmaceutical Co.) was diluted with saline to a concentration of either 2.5 I~g/p,l or 5 txg/l~l, and aliquots frozen at - 2 0 ° C until use. For ICV infusions, animals received 10 or 20 Ixg ( - )baclofen in 4 p,1 of saline. Behavior was monitored continuously and recorded at 5-min intervals either until anesthetic criteria were met or over the first 30 rain, and at 15-min intervals for the subsequent 90 man or until recovery of the righting reflex. The time of any dramatic changes such as seizures or return of the righting reflex occurring within these intervals was noted. EEG was sampled for at least 1 min at each recording interval for animals in the 10 pbg group and continuously following 20 txg infusions. Three days after ( - ) b a c l o f e n infusions, animals were given an equal volume of saline vehicle.

Criteria for Anesthesia To be considered anesthetized, animals had to exhibit loss of righting, foot withdrawal, and corneal reflexes, and loss of behavioral and EEG responses to a loud sound, an abdominal incision and suturing of that incision. A 1.5 cm incision of the skin on the abdominal wail was attempted only after all other criteria were met. The incision was terminated at the first sign of a response. Time of onset of anesthesia was defined as that time

when corneal reflex was lost in animals subsequently meeting all other criteria. Duration of anesthesia was determined by recovery of the righting reflex. Righting reflex was considered lost when an animal failed to right itself within 5 min after being placed in the supine position.

Criteria for Analgesia Analgesia was assessed by pinching the tail 3/8 in from the end and by pricking the animal with a sharp needle in the buttocks, shoulder, and ear. Only three categories were used in assessing the response: 1) analgesic--absence of any behavioral response; 2) normal--moderate movement away from source of pain; 3) hyperalgesic--exaggerated escape response involving jumping, vocalizing, or attacks directed at source of pain. Degree of analgesic response was not graded.

Histological Verification of Cannulae Placements Animals were infused with 2 Ixl of 0,2% cresyl violet, 15 minutes later were given a lethal IP injection of sodium pentobarbital and then perfused by intracardial puncture with phosphate buffered 10% formalin. Brains were stored overnight in formalin solution and transferred to 10% sucrose for at least 6 hr before sectioning on a cryostat. Visual verification of dye location was made from the frozen block while sectioning. Sections were then mounted and stained with cresyl violet for subsequent light microscope analysis. Only animals having accurate placements are reported.

Peripheral Administration of ( - )Baclofen Nine Swiss-Webster mice (15-20 g, University of Manitoba Dentistry Colony) were injected IP with 25, 50, or 75 mg/kg of ( - ) b a c l o f e n (5 mg/ml in saline). Behavioral responses were observed as above for the first two hr, after which time animals were monitored every 30 min until recovery of the righting reflex. Anesthetic criteria were as above with the exception of the EEG criteria. RESULTS

Central Administration Signs of anesthesia. Anesthesia did not occur in response to either 10 or 20 ixg infusions of ( - )baclofen at any ventricular site. Table 1 summarizes the occurrence of signs of anesthesia at each site tested. All but one animal showed loss of the righting response with 10 tJ-g of (-)baclofen. The remaining animal showed a marked slowing in righting with drug infusion in the fourth ventricle, but never completely lost the reflex. Loss of righting occurred most rapidly after infusions into the inferior third ventricle (mean= 13.33 min). Slower loss of the righting reflex occurred with infusions into other ventricular sites, with times ranging from 23 and 25 min in the inferior lateral ventricle to 45 and 60 min in the cerebral aqueduct. With 10 IJ-g infusions, analgesia was observed in only one animal. Hyperaigesia occurred in all but one of the remaining animals tested. No animal showed loss of either the foot withdrawal or corneal reflexes at this dose, although two animals did show loss of behavioral response to sound. Following 20 p,g infusions of ( - ) b a c l o f e n into either the inferior third or fourth ventricles, all animals showed loss of the righting reflex. Mean time for loss of righting was 15.00 min in the inferior lateral and 16.25 min in the fourth ventricle. Initially, all animals exhibited hyperalgesia which progressed to analgesia

BACLOFEN AND ANESTHESIA

5

TABLE 1 ANESTHETIC

RESPONSES

TO ICV INFUSIONS OF ( - )BACLOFEN

Ventricular Infusion Site Signs of Anesthesia* 10 p,g (-)Baclofen Analgesia Loss of Righting Reflex Loss of Foot Withdrawal Reflex Loss of Response to Sound Behavioral EEG Loss of Corneal Reflex

Inferior Superior Inferior Superior Lateral Lateral T h i r d T h i r d Aqueduct Fourth (n=2) (n=2) (n=3) (n=l) (n=2) (n=4)

0 O0

O0

20 I~g (-)Baclofen Analgesia Loss of Righting Reflex Loss of Foot Withdrawal Reflex Loss of Response to Sound Behavioral EEG Loss of Corneal Reflex

000

0

0

O0

000

0

(n = 2)

(n = 4)

0

0000

O0

0000

OOO

OOO OO

*Since cornealreflex was never lost, incisionand suture criteria were not assessed (see the Method section).

of the head and forequarters in all but one animal. Two animals tested in the fourth ventricle later developed analgesia in all body regions tested, but the others remained hyperalgesic in the hindquarters and analgesic in the forequarters. Although some animals tested at the higher dose of baclofen showed loss of foot withdrawal reflex and responses to sound, none experienced loss or impairment of the corneal reflex. With infusions in the inferior third ventricle, one animal died after 34 min and the second after 6 hr. Respiratory effects. Following infusions of 10 I~g ( - ) b a c lofen, animals showed depression of the respiratory rate by up to 80 percent within 15 min of infusions into the inferior third ventricle, but only after 30 min at other sites. Infusions into the fourth ventricle elicited hyperventilation within the first 10 min which later developed into respiratory depression. Animals at this site showed a deep, gasping pattern of breathing, sometimes accompanied by vocalization. Cheyne-Stokes respiration was common. In spite of the low respiration rate, extremities showed only occasional mild cyanosis. With 20 i~g infusions, animals tested in both inferior third and fourth ventricles showed initial hyperventilation followed by respiratory depression. Seizure responses. Following 10 wg baclofen infusions, animals in all groups exhibited spiking in the EEG accompanied by myoclonic jerks. Trains of afterdischarge were recorded in EEG records of animals infused in the inferior lateral, inferior and superior third, and fourth ventricles. This afterdischarge was accompanied in some cases by behavioral seizures including clonic motion of forelimbs and tonic extension of hindlimbs. Since EEG was monitored only at specific intervals in animals receiving

10 Ixg of baclofen, afterdischarges occurring between recordings would not have been observed. EEG records from animals infused in the inferior and superior lateral, inferior third, and fourth ventricles often showed sharp wave patterns which were accompanied by clonus of forelimbs or paddling and tonic extension of hindlimbs. Early records from some animals also show rhythmic activity at 3 Hz, characteristic of absence or petit mal seizures (see Fig. 1). The higher dose of (-)baclofen (20 t~g) into the fourth ventricle elicited generalized locomotor seizures in all animals. Representative EEG records accompanying various behavioral stages are depicted in Fig. 1. Ataxia with subsequent wobbly circling occurred within 2-3 min. Animals rolled onto their sides with forelimbs extended tonically by 10 min. In two cases there was tonic extension of hindlimbs, lasting 2-5 min and progressing to coordinated locomotor activity. In the early stages only, this "locomotion" could be arrested by tactile stimulation of foot pads. Locomotor activity was accompanied by sharp waves in the EEG. The locomotion became more rapid and developed into running-like activity. Myoclonic jerks were superimposed on this running and, periodically, the animal would flail in an uncoordinated, generalized convulsion of a few seconds duration. Running activity would then resume. Loud sounds or attempts to handle these animals during the next 15-20 min precipitated convulsive episodes. Animals were flaccid between behavioral seizures. Cessation of this behavior was accompanied by the postictal pattern of EEG shown in Fig. 1. During this period, the animals were often unresponsive to external stimuli such as loud sounds or touch but showed no impairment of corneal reflexes.

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LEWIS, WESTERBERG AND LABELLA

A ~/L,t-. j t , , ~ , . . . , , . ~ Lt,., • ,i ,.~... ,u. *,, a~_Li .aidiu.~i_.,d~,.,~tt.L.I.d,.i,, .,..a..,

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r~'--,r,~-~,~'rr'lrl",l"l-Tp-,v,-,',r~

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FIG. 1. (A) EEG record 5 min after 10 jxg infusion of ( -)baclofen into cerebral aqueduct showing rhythmic 3 Hz pattern. (B--G) Responses to 20 p.g infusion into the fourth ventricle. (B) Ten min postinfusion: record during tonic extension of forelimbs; (C) 15 rain postinfusion: arrow indicates transition from tonic extension to locomotor behavior; (D) 23 rain postinfusion: record indicates sharp waves accompanied by running behavior (chart speed × 2.5 at arrow); (E) 76 rain postinfusion: spiking accompanied by flaccidity. Animal on back with no behavioral response to sound; (F) 78 min postinfusion: arrow indicates EEG response to loud sound which elicited no behavioral change and subsequent temporary cessation of spiking; (G) 181 min postinfusion: normal asynchronous, fast EEG activity reverts to rhythmic 3 Hz activity at arrow following tactile stimulation of foot.

Other motor responses. Infusion of 10 Ixg of ( - ) b a c l o f e n produced similar motor responses at all sites tested, although the times of onset varied. In general, the shortest latencies for the responses described below were in the inferior third and fourth ventricles. Following infusion, animals exhibited marked ataxia, sometimes accompanied with hyperactivity and hyperreactivity. Frenzied eating of feces or food occurred following infusions at all sites but the fourth ventricle. Circling often ensued. Some animals curled into a tight ball and exhibited head and neck bobbing, irregular forelimb clonus, and tonic extension of the hindlimbs and digits. Animals infused in the fourth ventricle showed tonic extension of the forelimbs. Often, there was "paddling" of the forelimbs. Animals uncurled from the tight ball position as flaccidity developed (by 20 min with infusions in the inferior third ventricle, 35-37 min in the inferior lateral, 45 min in the superior lateral, and 45-75 min in the aqueduct and fourth ventricles). Similar patterns were seen with infusions of 20 p,g baclofen, but as was pointed out previously, the intensity of the responses was more characteristic of seizure behavior. All animals infused with either dose in the fourth ventricle, but not other sites, exhibited exophthalmos.

Peripheral Administration of Baclofen No animals became anesthetized at any of the three doses of baclofen tested. The response pattern was similar for all three doses, with onset of responses being shorter and intensity greater with increasing dose. All animals showed analgesia to tail-pinch within the first five minutes of injection. Animals lay flat on their stomachs with limbs splayed out. Twitching of hind, then forelimbs developed into uncoordinated flailing from side to side which progressed to rotations along the longitudinal axis of the body. This rolling differed from classic barrel rotations in that it alternated from side to side and was not accompanied by tonic limb extension. Time of onset of rotations was 17-20 min with 25 mg/kg (-)baclofen, 10-12 rain with a 50 mg/kg dose, and 6-7 min at 75 mg/kg. Within 20-30 min of injection, animals began to breath in the deep, gasping pattern seen in rats after baclofen infusions into the fourth ventricle. Respiration rate dropped from 130-160 breaths per minute immediately postinjection to 16-20 breaths per minute 30 rain later. In spite of the depressed response, animals showed no marked cyanosis in their extremities. Loss of righting reflex followed rotations at the two highest doses only and

BACLOFEN AND ANESTHESIA

7

occurred at 22 and 35 min for the 50 and 75 mg/kg doses respectively. In the animals given 25 mg/kg, the rate of rotation diminished, but rotations were precipitated by external stimuli for at least 3 hr. Animals in the two highest dose groups showed loss of the righting reflex for at least 6 hr, but continued to show coordinated motor responses to handling during that time as well as corneal reflexes. These animals exhibited no behavioral responses to loud sounds. One animal in each of the high dose groups died between four and five hr after injection, but other animals showed no apparent deficits 12 hr after injection. DISCUSSION Animals tested at each of several ventricular sites showed loss of righting reflexes, occasionally accompanied by lack of behavioral responses to loud sounds and loss of foot withdrawal reflexes. At higher doses, some animals developed analgesia. However, severe motor deficits, behavioral and electrographic seizures, and persistence of corneal reflexes suggest postictal depression rather than anesthesia. It is unlikely that the absence of anesthesia reflected insufficient drug. Corneal reflexes were normal even at doses which were lethal to some animals. In addition, all three doses of baclofen given peripherally failed to produce anesthesia, although again, higher doses were lethal. These data stress that definition of anesthesia may be a major confound in studies designed to elucidate underlying mechanisms. There is general agreement that surgical anesthesia is a reversible state associated with analgesia, unconsciousness, and amnesia. It is clear, also, that specific agents are more effective at eliciting certain signs of anesthesia, and that several agents may be required concurrently to produce useful clinical anesthesia. In addition, each agent produces a specific response profile rendering it difficult to determine which molecular actions underly anesthesia rather than side effects. The issue is further complicated by the difficulty of assessing amnesia and unconsciousness, clearly illustrated by problems associated with the one time use of the paralytic agent, curare, as a general anesthetic (17). Distinguishing among paralytic, anesthetic, ictal, and postictal disruptions of consciousness is problematic. Although seizures and anesthesia are sometimes thought of as opposite poles of a

continuum from CNS activation to depression, it is also known that many anesthetic agents evoke spiking in the EEG (31,32). In addition, seizures are most commonly seen in clinical patients during induction or recovery from anesthesia (26). These aspects of anesthetic agents suggest seizures and anesthesia may be adjacent steps on a continuum reflecting activation of similar molecular or anatomical substrates. Winters suggests these processes are divergent, interconnecting branches on a continuum from sleep to death, whereby anesthesia may result from marked stimulation or depression of the CNS (32). In line with this logic is the theory that coma, and presumably anesthesia, may reflect neural activation rather than global depression of the CNS (11). The problems outlined here point to the importance of defining anesthesia by strict and consistent criteria. In the present study, although baclofen failed to elicit anesthesia, introduction of the drug into the inferior third ventricle resulted in the most rapid loss of the righting reflex (approximately 13 min). This brain region is one of two regions we have found to mediate the anesthetic response to 13-endorphin (Western Pharmacology Society Annual Meeting, Tucson, 1988). Since the loss of righting occurs much later with 13-endorphin than with baclofen (approximately 41 min with 50 Ixg), we postulated the recruitment of a secondary system such as the GABA B complex. Administration of [3-endorphin centrally or morphine peripherally alters the rate of GABA turnover (20) and the GABA content in various thalamic nuclei (15,33). GABA B receptors occur in high densities in several thalamic nuclei and in medium density in hypothalamic regions (8). Both the hypothalamus and these thalamic nuclei are within diffusion distance of the inferior third ventricle, and have long been implicated in both anesthesia (7,9) and absence (petit mal) seizures (13). Increased GABA turnover in the globus pallidus has been suggested as a mechanism in catalepsy produced by [3-endorphin (20). Although baclofen does not in itself produce anesthesia, GABA B receptors in these regions may mediate loss of the righting reflex associated with anesthesia produced by other agents such as [3-endorphin. Similarly, the observed seizure responses may reflect interactions between the endogenous opiates and GABAergic system.

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