Autoanalgesia: Opiate and non-opiate mechanisms

Neuroscience & BiobehavioralReviews, Vol. 4. pp. 55--67.Printed in the U.S.A.

Autoanalgesia: Opiate and Non-Opiate Mechanisms W I L L I A M T. C H A N C E

Department o f Surgery, University o f Cincinnati Medical Center Cincinnati, OH 45267 R e c e i v e d 19 S e p t e m b e r 1979 CHANCE, W. T. Autoanalgesia: Opiate and non-opiate mechanisms. NEUROSCI. BIOBEHAV. REV. 4(1) 55-67, 1980.--Auteanalgesia (behaviorally-activated antinociception) was elicited by lesion-induced hyperemotionality or the classical conditioning of fear to the environmental stimuli associated with measuring antinociception. Both hyperemotionality and antinociception exhibited parallel decline in septal-lesioned rats with daily handling and in VMH-lesioned rats following treatment with diazepam. Autoanalgesia elicited by conditioned fear was blocked by spinal cord transection but not by diazepam. Although opiate binding experiments suggested the involvement of endorphins as mediators of autoanalgesia, hypophysectomy, morphine tolerance or very high doses of opiate antagonists failed to reduce the antinociception. Electrolytic lesions of the nucleus raphe magnus, a descending serotonergic system, did cause a significant reduction in autoanaigesia. Therefore, endorphin systems may be activated by the stress involved in autoanalgesic paradigms as a parallel system, whose functional integrity is not necessary for the expression of behaviorally-induced antinociception. Autoanalgesia VMH lesion

Pain Endorphin Raphe magnus lesion

Naloxone Opiate tolerance Hypophysectomy Conditioned fear Spinal cord transection

Septal lesion

Autoanalgesia Secondary to Lesion-Induced Hyperemotionality

A L T H O U G H endogenous mechanisms of pain control have previously been intimated by general observation [6,39] or theory [46] specific behavioral investigation of these mechanisms is a recent event. Thus, electrical stimulation of central gray areas has been reported to elicit analgesia comparable to a moderate dose of morphine [2,53]. The mechanisms of behavioral activation of this centrifugal inhibitory system, however, were unknown. From the postulates of descending control of pain exposed by the gate control theory of pain [45,46] we hypothesized that manipulation of CNS activity or past experience might activate descending systems in experimental animals to influence the perception of pain [13,54]. We chose to increase CNS activity by creating lesions known to elicit hyperemotional states of behavior. Past experience was manipulated by the classical conditioning of fear to the procedure of assessing antinociception. The radiant heat tail-flick test [24] was selected as our measure of antinociception. Since the tail-flick reflex is mediated at spinal levels [37], it is an ideal test for studying the effects of descending inhibition. We have labelled the antinociception resulting from the above manipulations as autoanalgesia, since it is behaviorally-induced and therefore must result from the incipient neuronal activity of endogenouslysynthesized molecules.

Lesions of the septal area in rats have long been known to produce a syndrome of hyperemotionality, which dissipates with handling [10]. Electrolytic lesions of the septal area were created in 12 adult, male, albino rats by passing 3 mA of direct current through a stainless steel electrode inserted 2.0 m m anterior to bregma, _+0.7 mm lateral to the midline and 6.5 mm below the top of the cranium. An additional 12 rats received sham surgeries. Beginning 2 days after surgery each rat was rated for emotionality by subjectively judging its response to being picked up and held for 15 see by a gloved hand. The ratings were assigned the following numerical values: 0=normal response, 1=increased startle and vocalization, 2=increased startle, vocalization and intermittent biting, 3=increased startle, vocalization and constant biting, 4=increased startle, vocalization, constant biting and explosive behavior upon return to home cage. Immediately after the emotionality rating tail-flick latencies were determined for each rat. The intensity of the heat source was adjusted to elicit response latencies of 3 to 4 see in normal rats and an 8 see cut-off criterion latency was maintained. Figure 1 illustrates the significant increase in emotionality, F(1,22) = 72.99, p <0.01, and antinociception, F(1,22) = 11.16,

1Supported in part by NIH Grant DA-00296, NIDR Grant DE-00116, NIAAA Grant AA-03157 and General Medical Research Grant RR-0S026 to Northeastern Ohio Universities College of Medicine. 2Presented at a Workshop on Stress and Environmentally Induced Analgesia, Eastern Psychological Association annual meeting, Philadelphia, PA, April 1979.

55

56

CHANCE • • scptals o ~ controls

n-12 nuldt

20.0

o emotionality

i4,0

18.0

•

•ntinocieeptlon 16.0 w

~:~ ~ ' :~:..= 3.0

14,0 ,......

12,0 2,0

10.0 8,0 . _L -=.

6.0

"::::

i.0

4.0 2.0

0.0

0,0

SIYTt~.S D~YS

ArTIta

sIJaGllaY

FIG. 1. Mean ratings of emotionality and concomitant tail-flick lateneies of sept•I-lesioned and control rats.

p <0.01, across the 8 days of testing. The significant (p <0.01) positive correlations between emotionality and antinociception as well as the decrease of both of these factors across Days 2 to 5 snggest that the antinociception was secondary to the hyperemotionality. Thus, as the hyperemotionality dissipated with repeated daily handling, the antinociception was also attenuated. In a second experiment, designed to assess the potency of autoanai___gesia elicited by hyperemotionality-producing lesions, septal lesions were created in 9 adult, male, albino rats with an additional 6 rats serving as sham operated controls. In this experiment, the rats were first handled on the day following surgery, and the cut-off criterion latency was increased to 20 sec to examine the full extent of antinociception elicited by hyperemotionality. From Fig. 2 one may observe the extremely potent nature of autoanali~la elicited by septal lesion,induced hyperemotionality. The mean tailflick latency of sept•l-lesioned rats was extremely long, with the reflex being inhibited well past the point of tissue dama~. Destruction of the ventromedial hypothalamic (VMH) nuclei also produces the hyperemotionality syndrome in rats [33]. In our initial invesfiptions [16], we observed that the hyperemotionality elicited by VMH lesions was much more resistant to the effects of daily handling than were the septal lesions. Thus, the VMH-lesioned rat exhibited emotionality ratings of at least 2 with concomitant tall-flick latencies ~ e a t e r than 6 sec after 8 days of handling. Since tnmqullizins drugs had been reported to be effective in reversing these hyperemotional states [5], the effect of pretreatment (30 win) with ~ (2.5 w4/ks, IP) on hyperemotionality and antinociception induced by VMH lesions was investigated in 36 adult, male, albino rats. VMH lesions were created in 18 rats by passing 4.0 mA of direct current for 30 sec through a stainless steel electrode inserted 6.2 mm anterior to the inte~ line, ±0.8 mm lateral to the ~ n e and 9.8 men below the top of the cranium [49]. The a d ~ 18 rats received control sham operations. One week after the surgeries, half of each group of rats was ~ e d with diazepam or vehicle, rated for emotionadty and tested for antinc~iception. From Fig. 3 one may observe that the emotionniity rating of the diazepam-treated VMH Stoup was significantly reduced, t(16)---5.16, p
(N -

9)

6ROUPS

(N • 6)

FIG. 2. Mean (+ SEM) ~ of emotianality ( s ~ bars) and tail-flick laten~es of septal-lesiold ~ control rats. To exa~ne the potency of h y p e r e ~ ~ - i ~ antin~on, the cut-off criterion latency of the tail-flick test was set at 20 sec.

4.On

.n2 &0,

i|-

n=9

1.01

IM

U

m VMH-P

VMH-V

S-P

~V

GROUPS FIG. 3. Mean (+ SEM) emotionality rat&q8 of ve~tromedlal hypothalam/c-lesioned (VMH) and s ~ ~ ($)~ rats following (30 min) the it~ction liP) of 2.SmlPq~ ~ (V) or saline (p).

noavlesion control values. S'milarty, a ~lp~ficant, t(6)= 3.27, p<0.01 reduction in tail-flick l a t ~ v,~s also observed (Fig. 4) in the VMH-lesioned rats followiq the/ejection of diazepam. Therefore, as in the septal.ha/emed rat, the mUmmlmsia extdbited result en,m enmeomaty rm/ was diazepam, the antinocice

OPIATE AND NON-OPIATE MECHANISMS OF AUTOANALGESIA

n=9

Autoanalgesia Elicited by Classically Conditioned Fear As a means of manipulating CNS-ANS arousal, in terms of past experience, use was made of a conditioned emotional response (CER) paradigm. According to this procedure, the rats are conditioned to fear a specific stimulus, and the effect of this fear response upon a behavioral variable are assessed. Using this procedure, significant arousal and fear can be conditioned to the environmental stimuli associated with the fear-producing stimulus [44]. Thus, pairing the stimuli associated with the tail-flick procedure with footshock results in the classical conditioning of fear to the tail-flick procedure, and allows assessment of the effects of this fear-induced arousal on antinociception. This procedure also avoids the possible confounding influence of the hyper-reactivity to handling observed in septal-lesioned rats. In this experiment [13], 24 adult, male, albino rats were randomly assigned to 3 groups of 8 each for differential conditioning of fear to the stimulus cues associated with the tall-flick procedure. Each rat in the experimental group (E) was removed from the home cage, held on a grid platform (21 x21 x8 era) and shocked (0.8 mA) for 15 see according to a 2 day on--1 day off-shock schedule for a total of 7 days (5 shock periods). In order to achieve differential conditioning of fear, subjects in the shock control group (S-C) were lowered head-first into a cardboard box that was situated on top of the grid and received the identical parameters of footshock. In addition, each of the S-C subjects was held on the non-electrified grid for 15 see following the administration of footshock in the box. Thus, these animals were receiving the aversive stimulus first, which was associated with the box, and then being held on the non-electrified grid in a manner analogous to that used in determining tail-frick latencies. A third group of rats served as non-shocked controls (C), being held on the non-electrified grid for 15 sec per day. On the day following the last administration of shock, each rat was placed on the non-electrified grid and its tall-flick latency was determined. In order to assess extinction, tail-flick latencies continued to be determined across the next 8 test days with no further shocks being administered. On the 6th extinction day the differences in the stimuli associated with the tail-flick procedure were minimized by testing the rats in a neutral room, removing them from their cages with a bare hand instead of a gloved hand, as in the acquisition phase of the experiment, and substituting a solid platform for the grid apparatus normally used to support the subjects during the tail-flick tests. From Fig. 5 one may observe that the significant, F(2,21)=8.18, p<0.01, difference between groups was distributed in an order corresponding to the pairing of relevant stimulus conditions of the tail-flick procedure with footshock. Thus, Group E, which had the most direct association of footshock with the tail-flick procedure by being handled in the same manner and shocked on the same grid that was used for the antinociception test, exhibited significantly longer latencies than Group S-C, t(14)=2.19, p<0.05, or Group C, t(14)=4.01, p<0.01. Group S-C, which had been shocked on the grid and might be expected to exhibit more generalized fear, also showed significantly longer iatencies than Group C, t(14)= 1.88, p<0.05. The mean tail-flick latencies for each of the groups across the 9 extinction trials is presented in Fig. 6. The increased latencies of Group E were very resistant to extinction, being reduced to the point of nonsignificance (as compared to Group C) only on Day 6 when the tail-flick latencies were determined under a different set

57

&O"

" ~"

7.0'

N: 9 •

i n ----'9

6.0 n

5.0. 4.0"

~

~.oo

--

,.<<

0

0

2.0.

o ~

o //

1.0'

~'J tj

I/ //

I

|

I

|

VMH-P

VMH-V

S-P

S-V

GROUPS FIG. 4. Mean (+ SEM) tail-flick latencies of VMH-lesioned and control rats following (30 min) the injection (IP) of 2.4 mg/kg diazepam (V) or saline (P).

6

=

,-1 N

(nEs)

S-C (n:s)

C (n:s)

GROUPS FIG. 5. Effects of conditioned fear on antinociception. Fear was conditioned to the stimuli associated with the tail-flick procedure in Group E. Group SC served as a shock control, while Group C was a control for the effect of handling. Each bar represents a mean (+ SEM) tail-flick latency following 5 fear-conditioning or control trials.

58

CHANCE

@ bl ~

S.

O-G E 8=8 S-C n=8 ~-~ C nf8

i"

0

9" >,

o

8"

o

7-

t~

.,1 4,

.j

6-

.x

5-

0

t

L

L I

I

4I --

i

2'

i~•• ¸.

t

3-

).- 2 t-

N

,..:.,.-

O

IgxTINCTION ~ I A L 8

CC

(n=7)

CS

(n=O)

EC

(n~7)

ES

(n:v)

FIG. 6. Mean tail-flick latencies of fear conditioned (E), shock, control (S-C) and handling control (C) groups across 9 days of extinction trials. On Day 6 the tail-flick tests were conducted in the presence of neutral environmental stimuli.

FIG. 7. Mean (+ SEM) taft-flick tatencies 24 hr following (stippled bars) transection of the spinal cord of fear conditioned (ES) and control (CS) rats or sham surgery in fear c o n d i t i ~ (EC) and control (CC) rats. Each group's ~l-flick latency on the preceding day is also shown (open bars).

of stimulus conditions. This experiment demonstrates that past events can elevate antinociceptive activity, as measured by the taft-flick test. Furthermore, this autoanalgusia was not accompanied by the hyper-reactivity observed in the septaland VMH-lesioned rats. In order to test the generality of autoanalgusic phenomena, use was made of the flinch-jump test [27]. According to this procedure, the degree of analgesia is determined by assessing the sensitivity to footshock. Group comparisons may then be made on the basis of the current threshold for the elicitation of a flinch (any noticeable response to shock that is not a jump) or a jump response. The jump threshold is defined as that current intensity which elicits removal of both hind limbs from the grid 50~ of the time. Classical ~ n i n g of fear to the stimulus cues associated with the flinch-jump test was accomplished by individually-placing 12 adult, male, albino rats in Plexiglas grid boxes (30x30x30 cm) that also served as the test apperams for determining the thresholds. Six of these rats were shocked (0.8 mA) for 5 rain per day (l 5 sec intershock interval) for 4 days. The other 6 rats were also placed in the boxes but were never shocked. On the 5th day the rats were a n i n placed in the boxes for the determination of flinch and jump thresholds. These thresholds were determined by rating the responses to a series of 5 alternating ascending and descending current intensities of footshock, which changed in 0.1 mA steps and ranlled from 0.1 mA to 1.0 mA. Although there was no difference in the flinch thresholds, the group that had previously been shocked in the test box exhibited a significantly, t(10)ffi2.6, p<0.05, higher jump threshold (0.54 mA) than the control group (0.38 mA). Thus, the prior experience with shock in the flinch-jump test apparatus apparently rendered the animals more analgasic in the test situation. Therefore autommigusia may also be observed in this more complex antinociceptive procedure, suggesting a degree of generality of phenomena. If autoanalgesia does result from inhibition or~inatin 8 in the brain and descending to spinal cord levels which meldate

the taii-fl/ck reflex, spinal cord transection should remove this inhibition and bio~k automZSlllesia. This type of experiment is reasonal~, since the tail-flick reflex is med/~ted at caudal spinal levels with transection as low as Tn not affecting normal reflex activity [37]. To achieve stable autoanalgesia, 14 adult, male, albino rats were given daily fear conditioning treatment for I0 days, while 13 additional rats were subjected to daily handti~ and placement on the nonelectrified grid as control nmnipehdions. On the 1lth day, 7 rats from each group were anesthetized (sodium pentoharbi. ted 40 msJkg; ~ and the spinal cords were exposed at midthoracic level. The cords were severed by pinching (and holding for 15 sec) the intact dura at levels T8 and TIo with forceps. The rentainin~ rats from each group were stlbjected to identical surllical Wocedures, except that the cord was not pinched. On the following day, tail-flick iatencies were determined for each rat as in the acquisition phase. Figure 7 presents the mean tall-flick latencies both prior to (Day 10) and 24 hr following (stippled bars) spinal cord transection in fear conditioned and control animals. Following the 10 days of fear c o ~ , silgnificlmt autoanalllesia was acquired, t(I6)ffi4.82, p<0.0I. Although sham surgery in the fear conditioned group (EC) was without effect, transection of the cord in this group (ES) yielded a significant reduction in analguala, t(13)ffi7.00, p<0.01, Group EC vs ET. Tail,flick lntencies were also sisaificantly, t(12)=3.17, p<0.05, Group CC vs CS, reduced in the control group foliowiq cord trans c ~ t m to yield mean W ~ ~--t~,roXimmiy ~ to ES. Tiros, r e m o v ~ the ~ ~ s of the brain from the ~ aeurom ~ the tall-flick response effectively blocked autoanallesia. These data further support the hypothems that autoUalsasia is being mediated by chanles in the brain which m e l t from the classical condit i o a i ~ of fear and activation of spinal pathways to block the perception of pain. If ~ r&~aits from fear cond/tioned to the tailflick procedure, one should be able to demonstrate gradual

OPIATE AND NON-OPIATE MECHANISMS OF AUTOANALGESIA

8.0"

o

?.0-

~,

6.0

0

0 C (n=io)

5.0 .J a~

4.0

7

~.o

j.

2.0

t.. q i)

1.0-

0.0

I

DAYS

FIG. 8. Mean ( - SEM) tail-flick latencies of fear conditioned (E) and control (C) rats across 7 consecutive test days. This acquisition curve was generated by daily antinociception tests conducted 10 sec prior to the administration of footshock and concurrent conditioning of fear to the test procedure.

acquisition of the phenomenon. Thus, daily increments of antinociception, to an asymptote, would be hypothesized to occur with each conditioning trial. In the present experiment, [19], daily acquisition of antonalagesia was assessed in 30 adult, male, albino rats by measuring tail-flick latencies 10 sec prior to the administration of footshock (0.8 mA, 15 see/day). An additional 20 rats served as controls, being held on the non-electrified grid for 25 sec after the daily determination of their tail-flick latencies. The mean tail-flick latencies of fear-conditioned (E) and control (C) groups for each of the daily test periods are illustrated in Fig. 8. Although there was no difference between groups in basal taft-flick latencies (Day 1), 24 hr following the first shock the subjects of Group E exhibited significantly longer latencies, t(48)=5.45, p<0.01. The latencies of these fear conditioned rats increased across the subsequent conditioning days to an asymptote of almost 6 sec on Day 6, while the non-shocked control rats maintained their responses at approximately 3 sec. Therefore, the fear-conditioned rats exhibited a pattern of acquisition of antinociception that reflected a learning process. Thus, these data again implicate higher CNS areas in the mediation of autoanalgesia.

Autoanalgesia and Opiate Binding Studies The possible neurohumoral mediators of autoanalgesic phenomena could be the recently-discovered endogenous opiate-like peptides [35, 48, 58]. These endorphin compounds have been shown to possess opiate-like activity in in vitro tests [36] and to inhibit the binding of naloxone to rat brain homogenate [55], Since the small endorphin compounds (enkephaiins) have also been reported to produce transient analgesia following intracerebral administration in the rat [7] and mouse [12], a plausible hypothesis of their normal function was as endogenous mediators of behaviorally-activated antinociception. To assess the involvement of endorphins in the mediation of autoanal-

59

gesia, radioreceptor binding of labelled etorphine and NLeu-enkephalin to rat brain homogenate was determined in 10 fear conditioned and 10 control rats from the preceding experiment. These rats had never received any drug treatments and had been subjected to 12 tail-flick-shock pairings. On the day following the last shock, the tail-flick latencies were again assessed and each rat was immediately sacrificed by decapitation. Each brain was rapidly removed and frozen to await the determinations of opiate and opioid binding according to previously described procedures [28,56]. Frozen brains were individually-homogenized in 10 volumes of 0.05 M Tris buffer (pH=7.3 @ 2°C), containing 0.32 M sucrose. The homogenates were cleared of nuclei and cellular debris by centrifngation (l,000xg) for 60 rain at 0*C, and the supernatants were indivudally-sedimented at 37,000xg (60 min @ 0°C). The resulting pellets were then resuspended in 6 ml of 0°C Tris buffer (0.05 M). To assess stereospecific opiate binding, 1 ml allquots containing 3.1 nM (15,16(n)-3H) etorphine (30 Ci/mmole; Amersham/Searle) were incubated at 0°C both with and without an excess of levorphanol (10-~M). After 60 min, incubation was terminated by the addition of an excess of cold Tris buffer. The samples were then vortexed and centrifuged (37,000xg, 15 min, 0°C), with the supernatants being discarded. We have found that this rapid dilution of the sample is equivalent to repeated washing-centrifugation techniques, as significant radioactivity is not removed by subsequent washing [63]. After centrifngation, the pellets were solubilized overnight in Aquasol-2 (New England Nuclear) and bound radioactivity was determined by liquid scintillation. Total binding capacity following degradation of endngenous ligands was determined by assessing stereospecific binding of etorphine following preincubation of the samples at 25°C for 40 min [30]. To assess enkephalin binding capacity of each brain, 1 ml aliquots of the above suspensions, containing 2.0x 10-SM 3H-N-Leu-enkephalin5 (9.4 ci/mmole) were incubated at 0°C for 60 rain. The labelled enkephalin was a gift from Drs. Day and Freer at the Medical College of Virginia and its synthesis has been described [25]. Bound radioactivity was collected and prepared for liquid scintillation as indicated above. Protein levels were determined [40], for each sample and the results for both etorphine and enkephalin are expressed as DPM/mg I)2 protein. Mean stereospecitic binding of 3H-etorphine by fearconditioned (E) and control (C) groups is presented in panels A and B of Fig. 9, while the respective mean tail-flick latencies are illustrated in panel C. As may be observed (panel A), there is significantly less binding in Group E than in Group C, t(16)--2.44, p<0.05. The observation that this difference was eliminated when the samples were preincubated at room temperature (panel B) suggested the involvement of an endogenous heat-labile ligand as the source of the binding difference. There was also a significant, t(18)=6.46, p<0.01, difference in tail-flick latencies between Groups E and C, which inversely paralleled the binding difference to yield a significant negative correlation, r(16)=0.65, p<0.01, between overall binding and antinociception. Although there was no significant difference (C=4.99x102 DPM; E=3.76= 102 DPM) in N-Leu-enkephalin binding (Fig. 10), the trend of less binding in the fear conditioned animals was in the same direction as noted for etorphine. Also similar to that observed with etorphine, was a significant negative correlation, r(7)=-0.87, p<0.01, between binding and antinociception in the control group. In the fear conditioned group, however, this relationship was not observed.

60

CHANCE A

12.0'

S.O-

-8.0 g

c -

~-lL

I0.0,

.+:..

0

~"

-7.0

_L. !:i:(

-6.0

c~ -I

,

S.O.

~

7.0,

/ //

=,6 6.0.

+

5.0

U

6.0.

.4,0

/:7

~

T s.o.

r

..2" 4.0"

4.0-

3.0

-

-2.0

,<

O.C

C

E C

E

/

@

C

I

m- 3.0, M

o

2.0,

-I.0

x -

1.0,

0.0

=..

2.0

E

/

O

r-

x

~

-"

~ O

EO c C)

-~ 0.0

T R E A T M E N T S

Tail-Flick

FIG. 9. Mean (+ SEM) sterospecific binding/ms P~ protein of aHetorphine to brain homngenate (P~ fraction) of fear conditioned (E, n=9) and control (C, n--9) rats under 3 conditions of incubation. When the suspensions were incubated for 60 min at 0°C (A), significantly less etorphine was bound in Group E subjects. Preincubation at room temperature (B), however, equalized the binding capacity of both groups. Group E also exhibited significantly longer tall-flick latencies immediately prior to sacrifice (C).

The result of these binding experiments are suggestive of an involvement of endorphin in the mediation of autoanalgesia. Thus, animals with shorter tail-flkk latencies also exhibited greater binding of radio-labelled opioids. Those rats in which antinociception was behaviorallyactivated by the fear conditioning procedure bound significantly less of the etorphine, suggesting that in these analgesic rats a larger proportion of the opiate receptors were occupied by endogenous ligands. Although differential binding of N-Leu-enkephalin did not reach statistical significance, our previous research has demonstrated significant differences between fear conditioned and control rats for binding of both N-Leu-enkephalin [21] as well as etorphine [20]. Additional evidence that decreased binding in the fear conditioned rats was due to endogenous peptida activity was the observation of increase binding after preincubation at room temperature, a procedure that hydrolyzes endogenous peptides and frees the receptors to bind the exogenous opiate. These data are similar to other reports of increased opiate activity following a chronic schedule of footshock [1,42]. Although these results are indicative of changes in central opiate peptide activity followin8 fear conditioning or chronic footshock, they are only correlational measures and do not demonstrate that these changes in endorphin levels cause autoanalgesia. In an attempt to obtain such proof, we next employed several experiments investigating the endocrinology and pharmacology of autoanalgesia.

Endocrinological Investigations of Autoanalgesia One factor which is common to each of the activators of autoanalgesia is stimulation of the release of ~ o r t i c o tropic hormone (ACTH) from the pituitary. Altheelh ACTH, and its incipient induction of steroid release from the adrenal cortex have been elimi~_ted as major factors in elicitinl aut o a r l a l g e s i a b y studies demonstrating that ether stress, lateral

I,~ltcncy

(scc.)

FIG. I0. Regression of nociception on binding of aH-Nleue n k ~ P2 protein to rat brain~ e . ~ was inversely correlated (r= -0.87) with a m i n o c i ~

in the control

group (C), while in the fear conditionedgroup{E) this reiatinMhip was reversed.

shaking [31,32] or the systemic administration of ACTH [15] do not eikit analilema, more recent interest has focused upon the pituitary ~ s . ~S-Lipotropic ~ ~LN4), which has been ~ [38] as a prohormone for the synthesis of O - e ~ (/1-LPH 61-91) and m e t ~ enkephalin ( ~ - ~ 61-65), is synthesized from a common pr-~urso¢ [43] and stored in the same secretory granules [50] as ACTH. Althon~ the pituitm~ has been implicated as a source of opiate pepttdas, with ~S.en~rphin localized in the intermediate and anterior lobes US], receat histochami~ studies have summed that the smaller ~ + ~ s are localized in the brain [34,62]. The demmstmtion of cencomitaat release of ~-endorphin and ACTH by ideB~.al stressful stimuli [29], however, focuses attemtion upon pituitary ~ systems as important putative tors of autmaalllesk ~ . In this e x ~ the effec~ of d+ptee~ ~ endomhins, wtd~e leaVeS brain acute [31,32] as well as chronic [1] footahock have been reported to elk:it ntiaoeteeptt~, the mmtlp~ ~ o[ acute footshoc~ and classically ~ f e a r were i n ~ in hypophysectomized rats. N i n e t y old h ~ tomized and normal, male, albino rats were p u ~ from Charles River Laboratories (Baltimore, MD), Hypophysectomized (Hy; n=20) and normal+(C; n=20) rats w e ~ each randomby ~ to 2 ~roucs to assess a ~ of aut ~ Each of the ~ ~ (Hy-S; n=10) and nomud expettmental (C-S; n = 10)ratl was

o f ~ ~

~ ~

o.~.

we~ ~ ( O ~ S

mA)for 1 5 ~ ,

To as,e. the Ic-~ ~

of ~ ,

ma.~k ~ were ~ ~ ~r an~, t0 see ~ , ~ ~ (Hy-NS; n = ~ a n d C-NS; n = ~ were ~ in a s ~ ~ , e ~ t shocks were eever ~ ~ , O11 the m ~ m ~ys this ~ ~ L~; except that the ~ ¢dl-l~:k

OPIATE AND NON-OPIATE MECHANISMS OF AUTOANALGESIA

0 HYI~OPHY (.-m) (Z) C()NTR()I. (n-lo)

O C--NS(nfto)

o

(u 9-

8-

[ ] C - S (n-- to) H y-NS(nfto) /% H y - S(n--" to)

!0080-

7~ 6" •"~

6!

~60.

5-

4o I

P-

20"

2" I-

:~ 0

0

i

2 TEST

4

i's 3'o

6'0

! 2o

io

Minutes A f t e r Injection

DAYS

FIG. 11. Mean (± SEM) tailflick latencies of hypophysectomized (Hy) and normal (C) rats under baseline conditions (open symbols, Day I) and following (10 sec) acute footshock (S; t'tlledsymbols, Day 1) or control (NS; filled symbols, Day 1) treatments. To assess acquisition of autoanalgesia, the rats continued to be tested for the next 4 days with each test being followed (10 sec) by footshock (0.9 mA, 15 sec).

test following the shock was eliminated. Thus, on each of these days the effects of shock administered 24 hr prior to the determination of antinoeiception was investigated. To compare the effects of hypophysectomy on autoanalgesia with its effects on opiate analgesia, morphine sulfate (6 mg/kg; SC; Mallinckrodt, St. Louis, MO) was administered to the two non-shock control groups one week after the autoanalgesia experiment. Figure 11 presents the latencies of hypophysectomized and normal rats prior to (open symbols; Day I) and following (closed symbols; Day I) footshock as well as during the acquisition of conditioned fear (Days 2-5). The shock treatment of Day 1 was highly effective in acutely-eliciting analgesia, F(1,36)-25.41, p<0.01, with no difference existing between hypophysectomized and normal rats. A similar response pattern was observed in hypophysectomized and normal rats in the acquisition of autoanalgesia (Days 2-5), with the fear conditioned rats showing significantly longer tail-flick latencies across these trials, F(1,36)-88.56, p<0.01. Although there was a significant difference between hypophysectomized and normal rats, F(1,36)-4.81, p<0.05, this difference was toward more analgesia in the hypophysectomized rats. The absence of a shock×group interaction indicated an overall elevation of tail-flick latencies in these rats irrespective of shock treatment. Figure 12 illustrates that the analgesic response to morphine was also potentiated in the hypophysectomized rats, F(1,18)=5.71, p<0.05, with significant differences being observed at 120, t(18)=3.23, p<0.01, and 180, t(18)=3.34, p<0.01, rain after injection of the drug. Thus, a similar pattern of analgesia was observed in hypophysectomized rats for both opiate and behaviorally-induced analgesia, with the only change being toward potentiation.

FIG. 12. Mean analgesic response expressed as percent MPE (maximum possible effect) of hypophysectomized and normal rats following the injection (SO) of morphine sulfate (6 mg/kg). Percent MPEffilT.-F.(Test)-T.-F.(basal)/T.-F.(Cut-off)-T.-F.(basal)] × 100.

Thus, the integrity of the pituitary-adrenal system is not necessary for the expressing of autoanalgesia. Therefore, again the critical changes eliciting these states of autoanalgesia appear to be occurring within the brain.

Pharmacological Investigations of Autoanalgesia In this experiment [19], to allow comparison of fearinduced antinociception with analgesia following hyperemotionality-producing lesions, the effect of pretreatment with diazeparn on autoanalgesia was investigated. Since the previous binding experiment implicated brain endorphins as possible mediators of behaviorally-induced antinocieeption, the effect of pretreatment with the longlasting opiate antagonist, naltrexone, on autoanalgesia was also assessed. The subjects for this experiment were 50 rats in the previous acquisition study. Thirty of these rats had received 7 tail-flick shock pairings, while the other 20 rats had been subjected only to control manipulations. On the day following the last shock, these rats were randomly assigned to 6 groups for the assessment of drug reversal of autoanalgesia. For the fear conditioned rats, group E-N (n=10) was injected with naltrexone (1 mg/kg, IP: Endo Laboratories, Garden City, NJ), Group E-D (n=10) was administered diazepam (2.5 mg/kg, IP) and Group E-S (n=10) was pretreated with saline, 30 rain prior to the assessment of antinociception. Non-shock control rats Group C-N (n=5), C-D (n=5) and C-S (n=10), were given corresponding doses of naltrexone, diazepam or saline and tested at the appropriate times. The effects of these pharmacological manipulations on autoanalgesia are presented in Fig. 13. To allow within group comparison as well as the assessment of residual drug effects, the iatencies on both the days preceding and following the drug tests are presented. As may be observed, neither drug treatment had any significant effect on the tail-flick latencies in fear conditioned nor

62

CHANCE

®

7.0"

r

6.0"

-1

~

5.0-

~

4.0-

C-control E-fear COO~ N-naltrexone D-dlazepam S-sallne

O 4)

I

9 ~ ..... l ,/)-7 ¸

O ¢-

•

if: [

.2

:!~1

; 3.o-

I:?¸

.X

=

7

~" 2.0-

®

.:I

U)"

--

l: ;

C-N In ffi a }

C-D (n = m )

-N (n = *01

E -D (n s Io1

43"

. . . . . ,t -¢ • - t

1

([ ,).,

".:" "..'.1 !.",'L:4

.-..:,

I-:-S (n • go)

c C-S

f~

2 !

• k .

..j::

-" : " , ' , l

)t ,, ,11

0.0

3"

O

i ,

i/i i

6"

:/:'./: ~.. ,. i ....

',.:-t "! : ..-/, :

( n ==t o t

~.-.:::

G R O U P S

SC

FIG. 13. Mean (+ SEM) tail-flick latencies of fear conditioned (E) and control (C) rats 30 rain following(stippled bars) the in~ectiontiP) of naltrexone (N, 1.0 mg/kg), diazepam (D, 2.5 mg/kg) or saline (S). For comparative purposes, the tail-flick latencies for both the preceding and subsequent days are presented. control groups. Furthermore, there was no suggestion of a residual drug effect on the following day in any of the groups. This inability of diazepam to antagonize autoanMgesia elicited by conditioned fear suggested a basic difference between this paradigm and autoanalgesia elicited by hyperemotionality-producing brain lesions. Thus, either daily handling of septal.lesioned rats or pretreatment of VMH-lesioned rats with diazepam effectively antagonized both hyperemotionality and antinociception. In the fear conditioned paradigm, however, diazepam was without effect. This observation suggests that analgesia within the fear-conditioning paradigm is not secondary to competing responses, such as straggling, biting and attempts to escape, as may be the case in hyperemotional states. Furthermore, these competing responses are observed only during or following the actual periods of footshock and not during the tail-flick test preceding the shock, on which all of these data are based. The lack of effect of naltrexone on autoana!gesia presents an apparent paradox in that one expects an opiate antagonist to block analgesic activity of endogenous opiate systems. Differences in affinities for receptor sites may account in part of this lack of antagonism. Recent experiments have suggested that the various endorphin peptides have varying MYmities for a variety of opiate and opioid receptors. Thus, ieu-enkephalin exhibits saturable binding at a highaffinity site that is not antagonized by the opiate antagonist, naloxone, as well as a lower-affinity site where naloxone does reduce binding of the peptide [4, 41, 57]. Conversely, jS-endorphin appears to bind to "typical" morphine sites [41], showing a high affinity for opiate receptors [9]. ~ - ~ also e ~ strong antspnism of 3-H-mtoxeae binding, While l e u ~ i n shows only w e a k ~ [41]. l~mbermore, in tl~ mouse vas deferens test, w h i e h ~ been characterized as similar to brain leu-enkeg~lin mrs [41], 11 times the dose of naloxone is required to antagonize l e u - e n k e ~ as nor.morphine-induced inhibition of contraction. In addition, padoxone has been reported to elevate the EDse ~ s i c doses of morphine ~ ~ e by a factor of 5, while actually reducing the EDso anMIleStC

(n:S)

NC (n

: 9)

SE

(rt :tO)

NE

(n:8)

FIG. 14. Mean (+ SEM) tail,flick iatcncies of rats 10 sec prior to (open bars) and 10 sec after (stippled bars) the administration of footshock (0.9 mA, 15 sec) to Groups SE and NEar control manipulations (Groups SC and NC). Groups NCand NE were pretreated (15 rain) with naloxone (20 mg/kg, lP), while Groups SC and SE received saline. dose of leu-enkephalin [ 12]. Therefore, autoanaigesia may be partially-mediated by endorphin receptors which are relatively-resistant to the antagonistic effects of naloxone and naltrexone. To test the above hypothesis, the effect of a dose of naloxone (Endo Laboratories, Garden City, NJ) much larger than necessary to antallonize morphine-indnced analgesia was investigated [18] within both the acute shock and conditioned fear-induced analgesia ~ s . Thirty-five adult. male, albino rats were randomly assigned to 4 groups. Groups NE (n=8) and NC (n=9) were injected with naloxone (20 mg/kg, IP), while Groups SC (n=8) and SE (n=10) were injected with saline 15 rain prior to the determination of baseline tail-flick latencies. Ten seconds after the basal test each rat in Groups NE and SE received footshock (0.9 mA, 15 see), with tail-flick latencies again being determined 10 sec after the termination of the shock. Latencies were also determined for Groups SC and NC at these times but no shock was administered. On each of the following 4 days the schedule offootshock continued to be administered with antinociception being determined prior to the fontshoek only. To assess the effect of naloxone on conditioned fearinduced antinociception, naloxone (20 ms/kg, IP)was again administered to Groups NC and NE 15 rain prior to the antinncioeptive test of Day 6. Groups SC and SE were also injected with an equal volume of saline, with tail-flick latencies being determined 15 sec later. In this study, a cutoff latency criterion of 9 sec was maintained with baseline responses aver~liq[3 to 4 sex. As may be observed in Fig. 14, 15 sec of footshoek elidted sillnificaat analgesia, t(16)=3.98, p<0.01: SE vs ~ ; t(15)=7.07, p<0.01: NE vs NC in the shocked groups as compared to controls. Neither baseline tail-flick ~ a s nor the acntely-didted anMjesia was signiflc~tly ~ by pmmmlmem with naioxone. Figure I5 presents a c q u i s i ~ of ~ ~ s i a across the next 4 days as well as the c ~ ~ (open symbols)and acute shock (filed symbols) data of Day 1. The rats that had pre-

OPIATE AND NON-OPIATE M E C H A N I S M S OF A U T O A N A L G E S I A

0 N S ( n - - 17) A S(n--18)

¢..)

9-

9" >' O c

63

8'

e=* ..J ...J

6"

"

5"

6~ 5"

22 4 .

v,. I

~-

I-

2"

e"

|_

2

G)

SC

(n=8)

TEST

DAYS

FIG. 15. Acquisition of autoanalgesia by rats, expressed as mean (-+ SEM) tail-flick latencies, for both acute footshock (S, Day 1) or conditioned fear (S, Days 2-5) paradigms. On Day 1 the tail-flick latencies were determined for Group S prior to (open symbols) and 10 sec following (filled symbols) footsbock, Tail-flick latencies continued to be determined for Group S prior to foutshock for Days 2-5. Antinociceptiou was also assessed for Group NS at these times, but shock was never administered. viously (24 hr) been shocked (S) exhibited significantly longer tail-flick latencies on Day 2 than did the non-shocked control (NS) rats, t(33)=4.22, p<0.01. This difference further increased on Day 3 to an asymptote of approximately 7 sec in the shock group. Figure 16 presents the effects of naloxone on autoanalgesia elicited by conditioned fear. As in the preceding drug test, naloxone failed to reduce the tail-frick latencies of control or fear conditioned rats. Thus, increasing the dose of the opiate antagonist to over 20 times that necessary to antagonize morphine-induced analgesia had no effect on autoanaigesia. Naloxone (4 mg/kg) has been reported to block chronic footshock-induced analgesia in mice [22]. Although this analgesic test was the abdominal constriction test induced by the injection of formic acid or prostaglandin E1 and probably does not reflect similar antinociceptive process as the tailflick test, we attempted to replicate this result in mice using autoanalgesic procedures [18]. Forty-six adult, male, albino (ICR) mice were subjected to procedures identical to those of the preceding experiment, except no test of naloxone antagonism of fear-induced antinociception was conducted. Thus, the effects of naloxone (4 mg/kg, IP) on antinociception acutely-elicited by footshock (0.9 mA, 15 sec) was in* vestigated as well as the ability of mice to elicit conditioned fear-induced antinociception. Although the mice demonstrated lower baseline tall-flick latencies and less acute analgesic effects of shock than rats, 15 sec of footshock still elicited significantly increased antinociception under both saline, t(20)=3.70, p<0.01, and naloxone, t(22)=3.70, p<0.01, conditions (Fig. 17). Again, naloxone was ineffective in antagonizing the increased tall-flick response laten-

NC

SE

( n =9)

(n=g)

NE

(n=8)

FIG. 16. Effect of naloxone (20 mg/kg, IP) on conditioned fearinduced autoanalgesia. Fifteen minutes prior to the tail-flick tests of Day 6, naloxone was again administered to Groups NC and NE, while Groups SC and SE received saline. P, O

==

9

>' 8' C

.J

76-

a¢ 5

¢J

'.:'.::.'i :::..'..':

I

:."i?; :'.'72"(;i :;:';'/;.

C t~

!

SC

(n=l=)

NC

(n =to')

SE

(n : l o )

NE

(n:t=)

FIG. 17. Mean (+ SEM) tail-flick iatencies of mice 10 sec prior to (open bars) and 10 sec after (stippled bars) the administration of footshock (0.9 mA, 15 sec) to Groups SE and NE or control manipulations (Groups SC and NC). Groups NC and NE were pretreated (15 min) with naioxone (4.0 mg/kg, IP), while Groups SC and SE received saline.

cies acutely-elicited by footshock. Acquisition of autoanalgesia within the conditioned fear paradigm (Day 2-5) as well as the combined data of baseline (open symbols, Day I) and acute shock (filled symbols, Day 1) tests are presented in Fig. 18. Significant antinociception was acquired within one day, t(44)=3.16, p<0.01: Day 2, and continued to increase to approximately 5 sec on Day 5. Thus, mice exhibited a pattern of autoanaigesia that was very similar to that previously observed in rats. These results agree with pre-

64

CHANCE

>' (3 (.. ~)

L)

O N S (n=24) A S(n=z=)

o

v

8

8-

o

7'-

~"

7

/

6

.~

$

/ -g o

5-

--

4-

o .i

4' !

!

I --

3-

(¢ I-

2-

c

9

I--

.... ..%

2-

~.

. • ...

!

0

.., .. , ....,.. • ....

c ,lli

i

iI

,,

3 TEST

!

i

,i DAYS

FIG. 18. Mean ( - SEM) taft-flick laten¢ies of mice prior to (open symbols, Day 1) and following (filled symbols, Day 1) acute shock (S) or control (NS) treatment. On Days 2-5, acquisition of autoanalgesia was assessed by determining tailflick lateneies prior to shock (S) or control (NS) treatment. vious experiments in which naioxone did not antagonize acute shock-induced antinociception [31,32] and only partially blocked chronic shock-induced antinociception [ 1]. A similar array of effects of naloxone has been reported for analgesia elicited by electrical stimulation of midbrain structures, with naloxone totally [47], or partially [3], reversing or not affecting [51,65], stimulation produced analgesia. Similarly, naloxone has been reported to have no effect [26], augment or reduce [11] pain perception in humans. Opiate-autoanalgesic interactions may also be investigated from the aspect of tolerance. Since analgesia induced by classically conditioned fear does not exhibit tolerance, but rather increases to an asymptote by the fourth or fifth day of conditioning, in this experiment [17], auto-analgesia was assessed in morphine-tolerant rats. Evidence of crosstolerance would, therefore, be suggestive of similar mechanism mediating the two phenomena. Thirty adult, male, albino rats were randomly assigned to 3 groups of 10 rats each. Groups MT and MNT were each injected (iP) with 10 mg/k8 morphine sulfate while Group SC was injected (IP) with an equal volume of saline, Taibflick latencies were determined for each rat I hr after the injection. Across the next 8 days the dose of morphine was gradually incremented for Group MT to an asymptote of 60 mg/kg for Days 7-9, while Groups MNT and SC received injections of saline. Tail-flick iatencies continued to be assessed 1 hr after the injections throughout this period. To assess tolerance, 10 n l ~ q of morphine was adafim'stered to Groups MT a ~ MNT on Day 10, while Group SC again received saline. To ~ the tolerance, Group MT received 60 ~ of m o r l ~ n e 1 hr after the tolerance test as well as following ~ s u b ~ l t ~ n t experimental manipulations. On Day I 1 the ~ response to acute footshock (0.9 mA, 15 see) was for Groups MT and MNT, while Group SC served only as a

,=..w.

S C

M T

•

MNT

r'l sm l o / g p ,

FIG. 19. Mean (+ SEMI taibflick latencies of rats before and after chronic administration of morphine sulfate or saline. The analgesic effects of 10 mg/kg of morphine (MT, MNT) or saline (SC) were assessed 60 win after the initial (open bars) as well as following the chronic (MT, 9 days) schedule (stippled bars) of drug administration.

handling control. To assess acquisition of fear-induced anaisesia in the tolerant and non-tolerant rats. tail-flick latancies continued to be assessed on each of the next 6 days for Groups MT and MNT prior to footshock. The mean tailflick latencies 60 mLn ~ g the injection of sahm (SC) or morphine in tolerant (Mr) and non-tolerant (MNT) rats is presented in Fig. 19. To allow comparison, the analgesic response to the initial treatment of Day 1 (open bars) and following the chronic schedule of morphine injection (stippled bars) on Day 10 is presented. Althmlgh there was no difference between Groups MT and MNT foiiowiltg the initial injection of morphine, repeated daily admiaigration of incremented doses of morphine induced a state of tolerance in Group MT, t(18)=3.91, p<0.01: MT vs MNT; t(18)= 1.24, n.s.: MT vs SC. The mean taibflick latencies of morphine tolerant and nowtolerant rats pcior to (open symbols, Day 1) and following (closed symbols, Day 1) footshoek as well as following acquisition of conditioned fear (Days 2-7) are presented in few. 20. Althmtgh there was no differe~e in baseline tail-flick iatencies between the groups, 15 sec of footshock s i r ~ _ ~ l y elevated the response iatencies of both Groups MT and MNT, F(2,27)=15.8, p < 0 , 0 1 , t o the same degree, t(15)=0.20, n.s.: MT vs MNT. Thus, morphine tokranc,e had no effect on ~ acutely-elicited by footshuck. $i!aittdy, there was no d i ~ r e n c e between Groups MT and MIWr across the next 6 days within the fear conditioning ~ with the tail41ick latencies of both of these 8reu~ ~ to an asymptote o f approximately 7 se¢. The total ~ of ¢li~awaee between the toleraat and nontolerant groups on any trial emphasizes the ~ of opiate to attemtate n ~ s i a . Since cross.tolerance has been demonstrated between morphine and various en-

OPIATE AND NON-OPIATE MECHANISMS OF AUTOANALGESIA

65

C) s c ( n = to)

(n= 1o) ~ MT MNT(n=so)

O

8.0-

>,

8-

:~ 7 . 0

7-

6.0

r-

¢

6"'

5-

,,.-.

4-

I

3-

0

~. s.o. 4.0. !

e

3.0-

'g 2.0-

!

2

3 T E ST

4

5

6

7

DAYS

FIG. 20. Mean (-+ SEM) tail-flick latencies of morphine tolerant (MT), non-tolerant (MNT) and control (SC) rats under baseline conditions (open symbols, Day 1) and following acute footshock (MT, MNT; closed symbols, Day 1) or control manipulations (SC; closed symbols, Day 1). To assess acquisition of autoanalgesia in tolerant and non-tolerant rats, taih2ick latencies were determined across the next 6 days with each test of groups MT and MNT being followed by footshock.

dorphin peptides [59, 60, 61], these results deemphasize the role of endorphins as mediators of autoanalgesia.

Lesions of Descending Spinal Systems and Autoanalgesia Since endorphins did not appear to be the primary mediators of autoanalgesia, we next turned to lesioning of descending systems in an attempt to antagonize autoanalgesia. Lesions of the nucleus raphe magnus, a descending serotonergic pathway [23], have been reported to reduce the analgesic effects of opiates [ 14,52]. Therefore, we investigated the effects of electrolytic lesions of this brain area on analgesia elicited by acute shock and conditioned fear. Lesions of the raphe magnus were created in 6 adult, male, albino rats by passing 2 mA of direct current through a nichrome electrode inserted at the midline 2.5 mm posterior to the interaural line and 6.0 mm below stereotaxic zero [49]. An additional 6 rats underwent sham operations. Two weeks following the surgeries, the analgesic effects of acute footshock (0.9 mA, 15 sec) and classically conditioned fear were investigated in both groups. Although there was no difference in baseline tail-flick latencies (Fig. 21), 15 sec of footshock elicited significantly more antinociception in control than in raphe magnus-lesioned rats, t(10)=2.18, p<0.05, one-tailed t-test. There was also a significant difference between the lesioned rats and controls across the 4 conditioning trials, F(1,10)=13.82, p<0.01, again with the lesioned rats exhibiting less autoanalgesia. Therefore, this preliminary experiment suggests that a descending serotonergic system may be important for the full expression of behavorially-induced analgesia. That lesions of this same system also reduce opiate-induced analgesia, indicates that it may represent a common pathway in the expression of inhibitory influences descending from the brain. The incomplete reduction of the antinociception, however, suggests

•

Raphc Madnus (.=,s)

•

Sham (n=e)

0o0 shock shock

Contlilionin~.

Trials

FIG. 21. Mean (- SEM) tail-flick latencies prior to (10 sec) and acutely-following (10 sec) footshoek (0.9 mA, 15 see) in raphe magnus-lesioned and sham operated control rats. Acquisition of autoanalgesia was assessed in these groups across the subsequent 4 days with each antinociceptive test being followed by footshock.

that this pathway is probably only one of a number of descending systems. Recent data [64] indicate that spinal noradrenergic as well as serotonergic systems are important in the expression of descending inhibitory activity of opiates. Therefore, noradrenergic systems may also be important mediators of autoanalgesia.

Conclusions In the present series of experiments, we have demonstrated that a centrifugal pain inhibitory system may be activated in experimental animals by hyperemotional states or classically conditioned fear. Although anecdotal accounts exist, the human correlates of these demonstrations may represent extreme states of CNS activity, such as seizures, and degrees of fear that approach terror. Since it is well known that mild to moderate anxiety actually lowers pain thresholds in humans, the effects of the anxiety-fear continuum on analgesia may be represented by a curvilinear function. The generality of autoanalgesia induced by conditioned fear was shown by its demonstration within the flinch-jump paradigm, while its specificity was indicated by the lack of effect of diazepam in reversing the antinociception as well as its specificity to relevant cues. Although the opiate binding experiments suggested the involvement of an endorphin in the mediation of autoanalgesia, additional experimentation emphasized non-opiate mechanism by demonstrating that hypophysectomy, opiate tolerance and opiate antagonists did not reduce the antinociception. Involvement of endorphins in autoanalgesia may be within parallel pathways that are stress-activated but are not necessary for the expression of autoanalgesia. Alternatively, the antinociception may involve an opioid receptor that has a low affinity for traditional opiate drugs, such as morphine and naloxone. The complete lack of effect of very large doses of naloxone, however, make this explanation less plausible. A descending sereotonergic pathway was implicated as a common pathway

66

CHANCE

for both opiate and behaviorally-induced analgesia. Additional research may also reveal the importance of noradrenergic systems within the spinal cord in the mediation of autoanalgesic phenomena. Elucidation of the roles of the various neurotransmitters in the mediation of behaviorallyinduced antinociception is an important goal in neuropharmacological research. If the systems that normally mediate these behaviorally-activated analgesic states are understood, then our understanding of the dysfunction of these same systems in chronic pain syndromes would be enhanced. This

knowledge could then be used in initiating prevention and treatment programs of these pain problems. ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Glenn M. Krynock for technical assistant in the behavioral experiments, Alice C. White for help in designing and conducting the opiate binding experiments and John A. Rosecrans without whose support this work would not have been possible.

REFERENCES 1. Akil, H., J. Madden IV, R. L. Patrick and J. D. Barhcas. Stress-induced increase in endogenous opiate peptides: concurrent reversal by naloxone. In: Opiates and Endogenous Opioid Peptides, edited by H. W. Kosterlitz. Amsterdam: Elsevier/ North Holland, 1976, pp. 63-70. 2. Akil, H. and D. J. Mayer. Antagonism of stimulation.produced analgesia by p-CPA, a serotonin synthesis inhibitor. Brain Res. 44: 692-697, 1972. 3. Akil, H., D. J. Mayer and J. C. Liebeskind. Antagonism of stimulation-produced analgesia by nalxoone, a narcotic antngonist. Science 191: 961-962, 1976. 4. Audi~er, Y., B. Malfroy-Camine, A. Virion, J. Roy, J.-L. Mor[,at and J.-C. Schwartz. Sites de liaison de la leu-enkephalin-SH darts ie striatum du rat. C. R. hebd. S~anc. Acad. Sci., Paris 284: 73-76, 1977. 5. Beattie, C. W., H. I. Chernov, P. S. Bernard and F. H. Gleeny. Pharmacological alteration of hyperreactivity in rats with septal and hypothalamic lesions. Int. J. Neuropharmac. 8: 365-371, 1969. 6. Beecher, H. K. Measurement of Subjective Responses. New York: Oxford University Press, 1959. 7. Belluzi, J. D., N. Grant, V. Garsky, D. Sarantakis, C. D. Wise and L. Stein. Analgesia induced in vivo by central administration of enkephalin in the rat. Nature 260: 625-626, 1976. 8. Bloom, F., E. Battenburg, J. Rossier, N. Ling, J. Leppaluoto, T. M. Vargo and R. Guillemin. Endorphins are located in the intermediate and anterior lobes of the pituitary gland, not in the neurohypophysis. Life Sci. 20: 43-48, 1977. 9. Bradbury, A. F., D. G. Smyth, C. R. Snell, N. J. M. Birdsall and E. C. Huime. C fragment of iipotropin has a high affinity for brain opiate receptors. Nature 260: 793-795, 1976. 10. Brady, J. V. and W. J. H. Nanta. Subcortical mechanisms in emotional behavior: affective changes following septal and habenular lesions in the albino rat. J. comp. physiol. Psychol. 46: 339-346, 1953. 11. Buchsbaum, M. S., G. C. Davis and W. E. Bunney, Jr. Naloxone alters pain perception and somatosensory evoked potentials in normal subjects. Nature 270: 620--622, 1977. 12. Buscher, H. H., R. C. Hill, D. Romer, F. Cardinaux, A. Closse, D. Hauser and J. Pless. Evidence for analgesic activity of enkephalin in the mouse. Nature 261: 432--435, 1976. 13. Chance, W. T., G. M. Krynoek and J. A. Rosecrans. Antinociception following lesion-induced hyperemotionality and conditioned fear~ Pain 4: 243--252, 1978. 14. Chance, W. T., G. M. Krynock and J. A. Rosecrans. Effects of medial raphe and raphe magnus lesions on the analgesic activity of morphine and methadone. Psychopharmacology 56:133-137, 1978. 15. Chance, W. T., G. M. Krynock and J. A. Rosecrans. Investigation of pituitary influences on autoanalgesia. Psychoneuroendocrinology 4: 199-205, 1979. 16. Chance, W. T., G. M. Krynock and J. A. Rosecrans. Reduction of VMH lesion-induced hyperemotionality and antinociception by diazepam. Fedn Proc. 36: 394, 1977. 17. Chance, W. T. and J. A. Rosecrans. Lack of cross-tolerance between morphine and autoanalgesia. Pharmae. Biochem. Behay. 11: 639--642, 1979.

18. Chance, W. T. and J. A. Rosecrans. Lack of effectof naloxone on autoanalgesia. Pharmac. Biochem. Behov. 11: 643-646,

1979. 19. Chance, W. T., A. C. White, G. M. Krynock and J. A. Rosecrans. Autoanalgesia: acquisition, blockade and relationship to opiate binding. Eur. J. Pharmac. 58: 461--468. 1979. 20. Chance, W. T., A. C. White, G. M. Krynock and J. A Rosecrans. Autoanalgeisa: behaviorally.activated antinociception Eur. J. Pharmac. 44: 283-284. 1977. 21. Chance, W. T.. A. C. White, G. M. Krynock and J. A. Rosecrans. Conditional fear-induced antinociception and decreased binding of (~l)N-Leu-enkephalin to rat brain. Brain Res. 141: 371-374, 1978. 22. Chesher, G. B. and B. Chart. Footshock induced analgesia in mice: its reversal by naloxone and cross-tolerance with morphine. Life Sci. 21: 1569-1574. 1977. 23. Dahlstrom, A. and K. Fuxe. Evidence for the existence of monoamine neurons in the central nervous system. II. Experimentally induced changes in the interneuronal amine levels of bulbospinal neuron systems. Acta. physiol scand. (SUppl.247) 64: 1-36, 1965. 24. D'Armour, F. E. and D. L. Smith. A method for determining loss of pain sensation. J. Pharmac. exp. Ther. 72: 74-79. 1941. 25. Day, A. R. and R. J. Freer. Synthesis of an intrinsically radiolabelled enkephalln analog: (p-tritio-phenylalanyl) 4-norleucyP-enkephalin. J. Label. Cpds. Radiopharaceut. 14: 381-391, 1978. 26. EI-Sobky, A., J. O. Dostrovsky and P. D. Wall. Lack of effect of naloxone on pain perception in humans. Nature 263:783-784. 1976. 27. Evans, W. O. A new technique for the investigation of some analgesic drugs on a reflexive behavior in the rat. Psychopharmacologia 2: 318-325, 1961. 28. Gispen, W. H.. J. Buitelanr. V. M. Wiegant, L. Terenius and D. DeWied. Interaction between ACTH ~ n t s . brain opiate receptors and morphine-induced analgesia. Eur. J. Pharmac. 39: 393-397, 1976. 29. Guillemin, R,, T. Vargo, J. Rossier, S. Minick, N. Ling, C. Rivier, W. Vale and F. Bloom. /3-Endorphin and adrenocorticotropin are secreted concomitantly by the pituitary gland. Science 197: 1367-1369, 1977. 30. Hambrook, J. M., B. A. Morgan, M. J. Rance and C. F. C. Smith. Mode of deactivation of the enkephalins by rat and human plasma and rat brain homogenates. Nature 26'2:782-783, 1976. 31. Hayes, R. L., G. J. Bennett, P. Newlon and D. J. Mayer. Analgesic effects of certain noxious and stressful manipulations in the rat. Proc. Soc. Nearosci. 2: 939, 1976. 32. Hayes, R. L., G. J. Bennett, P. G. Newion and D. J. Mayer. Behavioral and physioiogical studies of non-narcotic analgesia in the rat elicited by certain environmental stimuli. Brain Res. 155: 69-90, 1978. 33. Hetherinston, A. W. and S. W. Ranson. Hypothalamic lesions and adiposity in the rat. Anat. Rec, 715: 149-172, 1940. 34. Hokfelt, T., R. Elde, O. Johansson, L. Terenius and L. Stein. The distributionof enkephalin immunoreactive ceHbodies in the rat central nervous system. Neurosci. Lett. 5: 23-31, 1977.

O P I A T E A N D N O N - O P I A T E M E C H A N I S M S OF A U T O A N A L G E S I A 35. Hughes, J. Isolation of an endogenous compound from the brain with pharmacological properties similar to morphine. Brain Res. 88: 295-309, 1975. 36. Hughes, J., T. W. Smith, H. W. Kosterlitz, L. A. Fothergill, B. A. Morgan and H. R. Morris. Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 258: 577-579, 1975. 37. Irwin, S., R. W. Houde, D. R. Bennett, L. C. Hendershot and M. H. Seevers. The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation. J. Pharmac. exp. Ther. 101: 132-143, 1951. 38. Lazarus, L. H., N. Ling and R. Guillemin. /3-Lipotropin as a prohormone for the morphinomimetic peptides endorphins and enkephalins. Proc. hath. Acad. Sci. U.S.A. 73: 2156-2159, 1976. 39. Livingston, W. K. What is pain? Scient. Am. 88: 59-66, 1953. 40. Lowry, O. H., N. J. Rosebrough, A. F. Farr and R. J. Randall. Protein measurement with the folin phenol reagent. J. biol. Chem. 193: 265-275, 1951. 41. Lord, J. A. H., A. A. Waterfield, J. Hughes and H. W. Kosterlitz. Endogenous opioid peptides: multiple agonists and receptors. Nature 267: 495--499, 1977. 42. Madden IV, J., H. Akil, R. L. Patrick and J. D. Barchas. Stress induced parallel changes in central opioid levels and pain responsiveness in the rat. Nature 265: 358-360, 1977. 43. Mains, R. E., B. A. Eipper and N. Ling. Common precursor to corticotropins and endorphins. Proc. hath. Acad. Sci. U.S.A. 74: 3014-3018, 1977. 44. McAllister, W. R., D. E. McAllister, G. H. Weldin and J. M. Cohen. Intertrial interval effects in classically conditioning fear to a discrete conditioned stimulus and to situational cues. J. comp. physiol. Psychol. 87: 582-590, 1974. 45. Melzack, R. and K. L. Casey. Sensory, motivational and cantral control determinants of pain. In: The Skin Senses, edited by D. Kensbalo. Springfield, Ill.: Thomas, 1968, pp. 423-435. 46. Melzack, R. and P. D. Wall. Pain mechanisms: a new theory. Science 150: 971-979, 1965. 47. Oliveras, J. L., Y. Hosobuchi, F. Redjemi, G. Guilbaud and J. M. Besson. Opiate antagonist, naloxone, strongly reduces analgesia induced by stimulation of a raphe nucleus (centralis inferior). Brain Res. 120: 221-229, 1977. 48. Pasternak, G. W., R. Goodman and S. H. Snyder. An endogenous morphine-like factor in mammalian brain. Life Sci. 16: 1765-1769, 1975. 49. Pellegrino, L. J. and A. J. Cushman. A Stereotaxic Atlas o f the Rat Brain. New York: Appleton-Century-Crofts, 1967.

67

50. Pelletier, G., R. Leclerc, F. Labrie, J. Cote, M. Chretien and M. Lis. Immunohistochemical localization of/3-1ipotropic hormone in the pituitary gland. Endocrinology 100: 770-776, 1977. 51. Pert, A. and M. Walter. Comparison between naloxone reversal of morphine and electrical stimulation induced analgesia in the rat mesencephalon. Life Sci. 19: 1023-1032, 1976. 52. Proudfit, H. K. and E. G. Anderson. Morphine analgesia: blockade bu raphe magnus lesions. Brain Res. 98: 612--618, 1975. 53. Reynolds, D. V. Surgery in the rat during electrical analgesia induced by focal brain stimulation. Science 164: A.A~ AA5' 1969. 54. Rosecrans, J. A. and W. T. Chance. Emotionality-induced antinociception. Proc. Soc. Neurosci. 2: 919, 1976. 55. Simantov, R. and S. H. Snyder. Isolation and structure identification of morphine-like peptide "enkephalin" in bovine brain. Life Sci. 18: 781-788, 1976. 56. Simon, E. J. and J. Groth. Kinetics of opiate receptor inactivation by sulfhydryl reagents: evidence for conformational change in presence of sodium ions. Proc. hath. Acad. Sci. U.S.A. 72: 2404-2407, 1975. 57. Terenius, L. Opioid peptides and opiates differ in receptor selectivity. Psychoneuroendocrinology 2: 53-58, 1977. 58. Terenius, L. and A. Wahistrom. Morphine-like ligand for opiate receptors in human CSF. Life Sci. 16: 1759-1764, 1975. 59. Tseng, L. F., H. H. Loh and C. H. Li. /3-Endorphin: cross tolerance to and cross-physical dependence on morphine. Proc. natn. Acad. Acad. Sci. U.S.A. 73: 4178-4189, 1976. 60. Van Ree, J. M., D. De Wied, A. F. Bradbury, E. C. Hulme, D. G. Smyth and C. R. Snell. Induction of tolerance to the analgesic action of lipotropin C-fragment. Nature 264: 792-794, 1976. 61. Waterfield, A. A., J. Hughes and H. W. Kosterlitz. Crosstolerance between morphine and methionine-enkephalin. Nature 260: 624-625, 1976. 62. Watson, S. J., H. Akil, S. Sullivan and J. D. Barchas. Immunocytochemical localization of methionine enkephalin: preliminary observations. Life Sci. 21: 733-738, 1977. 63. White, A. C. The relationship of opioid receptor binding capacity to pharmacologically ard behaviorally induced antinociception. Doctoral Dissertation, Medical College of Virginia, Virginia Commonwealth University, 1977. 64. Yaksh, T. L. Direct evidence that spinal serotonin and noradrenaline terminals mediate the spinal antinociceptive effects of morphine in the periaqueductal gray. Brain Res. 160: 180-185, 1979. 65. Yaksh, T. L., J. C. Yeung and T. A. Rudy. An inability to antagonize with naloxone the elevated nociceptive thresholds resulting from electrical stimulation of the mesencephalic central gray, Life Sci. 18:1193-1198, 1976.