Evaluation of the periaqueductal central gray (PAG) as a morphine-specific locus of action and examination of morphine-induced and stimulation-produced analgesia at coincident PAG loci

Brain Research, 124 (1977) 283-303

283

© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands

EVALUATION OF THE PERIAQUEDUCTAL CENTRAL GRAY (PAG) AS A MORPHINE-SPECIFIC LOCUS OF ACTION AND EXAMINATION OF MORPHINE-INDUCED AND STIMULATION-PRODUCED ANALGESIA AT COINCIDENT PAG LOCI

VAHN A. LEWISand G. F. GEBHART Department of Pharmacology, University of lowa, Iowa City, Iowa 52242 (U.S.A.)

(Accepted July 14th, 1976)

SUMMARY Experiments were carried out in rats to (1) elaborate upon the specificity of drug action in the periaqueductal gray matter (PAG), and (2) to evaluate the possible congruence of PAG sites of morphine-induced and stimulation-produced analgesia (SPA) applied at virtually identical PAG loci. It was demonstrated that the effect of morphine intracerebrally (i.e.) administered into the PAG was not duplicated by other centrally acting agents (chlorpromazine, chlordiazepoxide, pentobarbital or naloxone) administered i.c. at the same PAG site. This selective action of morphine in the PAG was further demonstrated not to be test-bound since morphine significantly altered responding in all four of the analgesiometric tests employed. Thus, multiple i.c. injections of drugs at the same PAG locus were useful in demonstrating site specificity of drug action where behavioral and electroencephalographic methods alone had previously provided ambiguous information. Morphine-induced analgesia and SPA, evaluated at virtually coincident PAG sites, revealed only a general congruence of efficacious loci. The most effective PAG loci for morphine-induced analgesia were not the same as those for SPA; analgesia effected by one analgesia-producing manipulation did not reliably predict that analgesia would also be produced by the other analgesia-producing manipulation at the PAG sites examined. In general, the more efficacious analgesia-producing PAG loci were localized in the ventral-ventrolateral PAG.

INTRODUCTION Recent evidence has suggested a role for the periaqueductal gray matter (PAG) in the mediation of morphine-induced analgesia. The evidence implicating the PAG arises from several different experimental approaches: intracerebral microinjection

284 studies, focal electrical stimulation studies, biochemical studies and electrophysiologic studies. Analgesia can be induced by the direct injection of as little as 3 #g of morphine into the PAG 72. Antinociceptive effects have also been reported after injection of morphine into other brain areas, including the hypothalamus, periventricular gray and 4th ventricle21,2s,62,72,82; the most efficacious region with respect to morphineinduced analgesia, however, appears to be the ventrocaudal PAG. Electrical stimulation of the PAG has also been found to produce profound increases in the response thresholds of animals in various analgesiometric tests 3,1a,48,57,58,~. Although the animals in these studies were unresponsive to a noxious stimulus, they were not immobilized by the electrical stimulation and reportedly responded normally to touch, visual and auditory stimuli. Occasionally, hyperreactive responses were observed to non-noxious stimuli. A comparison between morphine-produced and stimulation-produced analgesia is unavoidable. Stimulation-produced analgesia (SPA) has been reported to be equal to or greater than the analgesia produced by 10 mg/kg of morphine (intraperitoneally) in the rat 4s and 4 mg/kg of morphine (subcutaneously) in the cat 14. Stimulationproduced analgesia has also been demonstrated to be independent of intracranial reward mechanisms and has been observed following focal electrical stimulation at both rewarding and non-rewarding brain loci 3,48. Regional analysis for stereospecific opiate agonist and antagonist binding has further implicated the PAG as a potential central locus of opiate effects since significant amounts of opiate binding sites have been demonstrated there. Initial work by Goldstein et al. 15 and subsequent work by many other workers 22,39,6°,61 have demonstrated that there is an uneven distribution of high affinity stereospecific binding sites for opiates in various regions of the brain. The PAG, while not possessing the highest levels of opiate binding sites within the brain, appears to have perhaps the most behaviorally relevant opiate binding sites. Marked changes in behavior do not result following morphine administration into other brain areas containing larger amounts of opiate binding sites, such as the caudate nucleus and amygdala7,z9,62,85. It is known that analgesiometric tests employing animals are not always specific for analgesic agents per se25,53,54,86. Electrophysiologic studies have demonstrated that the PAG is sensitive not only to narcotic agents, but also to the effects of nonnarcotic agents. Several workers 41,51,7s have reported that morphine as well as pentobarbital, administered peripherally, depress gross evoked potentials in the PAG. In the present experiments, we endeavored to determine the specificity of the PAG as a central analgesic locus by intracerebrally administering not only the prototypical analgesic, morphine, but also other centrally acting agents: the tranquilizer, chlorpromazine ; the depressant, pentobarbital; the benzodiazepine, chlordiazepoxide: and the narcotic antagonist, naloxone. These other non-analgesic agents were used as pharmacologic controls to test the hypothesis that only morphine, when injected into the PAG, would produce a behaviorally demonstrable "analgesic" effect. A second area of interest in these investigations was to evaluate the congruence of efficacious sites of SPA and morphine-produced analgesia within the PAG. In these experiments, cannula and electrode combinations were employed to evaluate both procedures at

285 essentially identical sites in the PAG. Although the term "analgesia" and the use of analgesiometric tests can be problematical, we hoped in this study to establish reliability by employing several non-analgesic agents as well as several analgesiometric test procedures requiring different levels of CNS integration. In this report, the term analgesia is meant to indicate a decrement in the response(s) to noxious stimuli when rats were exposed to various experimental manipulations. METHODS Male Sprague-Dawley rats initially weighing 280-300 g were employed throughout. These animals were implanted with a cannula/electrode assembly consisting of a 30-gauge guide cannula affixed with Epoxylite to a small (127/zm diameter) stainless steel bipolar electrode whose insulation was bared 0.5 mm at the tip. The electrode component of the assembly was employed to evaluate SPA, the guide cannula component to permit intracranial (i.c.) drug administration. The 30-gauge cannula served as a guide for a 35-gauge injection cannula manufactured from 35-gauge stainless steel tubing cemented with Eastman 910 adhesive to the inside of a 30-gauge hub 27. Thirtyfive-gauge injection cannulae were employed in these experiments to both minimize the area of neuronal damage and also to more closely localize and restrict drug placement. Both the guide and injection cannulae were cut electrolytically40; the 35-gauge injection cannula was cut to extend precisely 1 mm beyond the end of the guide cannula so as to coincide with the tip of the adjacent bipolar PAG stimulating electrode. Stereotaxic coordinates for the cannula/electrode assembly were: 5.5 mm posterior to bregma, 1.0 mm lateral to the sagittal suture and 5.0 mm below the dura. These coordinates were selected on the basis of work by Reynolds 66 who demonstrated profound SPA at this site and also to avoid drug injection directly into the aqueduct and ventricles, which has also been reported to produce analgesia 21. A second bipolar stimulating electrode, implanted in the contralateral ophthalmic branch of the trigeminal nerve (5.1 mm anterior to bregma, 1.8 mm lateral and 10.5 mm below the dura), was employed to induce escape behavior subsequent to noxious trigeminal stimulation. The electrode was constructed from 0,31 mm diameter insulated nichrome wires bared for 0.5 mm at their tips. Following a recovery period of at least one week after surgery, the animals were trained to escape noxious trigeminal nerve stimulation (initially 200/~A) by shuttle crossing a plastic chamber to deactivate the stimulus. Trigeminal nerve stimulus pulse trains, each consisting of 4 unipolar square wave pulses, were delivered at the rate of one pulse train/sec for a maximum of 6 sec. Pulse trains are thought to produce a more reliably noxious stimulus than single pulses 67. The individual pulses, delivered from a Grass $4 stimulator through a stimulus isolation unit, were administered as 1 msec duration pulses at 64 Hz. The 6 sec noxious stimulus duration and a 30 sec intertrial interval was controlled electromechanically. Stimulus amperage was measured across a 1 kD resistor using a Tektronic Type 502 dual-beam differential oscilloscope. After training to escape noxious trigeminal nerve stimulation, each animal was allowed to titrate its threshold by the following procedure. When the animal crossed

286 the chamber during the 6 sec stimulation period, the stimulus was terminated and its intensity was reduced by 0.5 V for the next trial. If the animal failed to cross within the 6 sec period, the stimulus intensity was increased without delay by 0.5 V for the next trial. Thus, each animal was able to titrate the level of stimulation delivered. Training was continued until stable titration thresholds were obtained. The mean overall titration threshold over all experimental groups was 62 ± 40 #A (S.D.). Several analgesiometric tests were used to evaluate the effects ofi.c, administered drugs. More than one test was employed to afford an evaluation of both the extent and magnitude of drug effects. The tests employed were the trigeminal nerve stimulation escape test (TG), the hot plate test (HP), the tail flick test (TF) and a pinch-squeal test (PS). The T G test, as described above, is a threshold titration procedure in which the animal is free to respond to noxious stimulation or not. The mean value of l0 maximum and minimum levels of stimulation in the titration procedure was used to compare trials. The HP method of Johannesson and Woods 31 as modified by Kayan et al. ~2 was also used to measure analgesia, analgesia being defined as a significant increase in reaction time on a 55 °C heated plate. If an animal failed to respond by licking its paws within 30 sec, it was removed from the plate to prevent tissue damage. T F response times were evaluated by a modification of the T F technique described by D ' A m o u r and Smith H. TF latencies were sampled 3 times and calculations were based on the mean of the 3 samples. The fourth test employed was a PS test with which we attempted estimate the somatotopic distribution (if any) of analgesia produced at each of the P A G sites examined. Responses were rated on a 4-point scale at each of 5 body sites before and after drug administration. The animals were pinched above the forelimbs, hindlimbs and on the tail in randomized order. Differences between pre- and postdrug responding in the PS test were evaluated by non-parametric statistical tests. Morphine sulfate (MOR), chlorpromazine HCI (CPZ), sodium pentobarbital (PB), chlordiazepoxide HCI (CDP), naloxone HC1 (NAL) and artificial cerebrospinal fluid as a control (CSF) 55 were administered i.c. (unilaterally) into the PAG. Drugs were dissolved in CSF and injected, as was CSF control, in a 0.5/A volume administered over a 30 sec injection period. Lomax 43 has shown that 90 ~o of a 1 #1 injection of radioactive M O R is retained with a 1 m m envelope of the site of injection. Thus, these 0.5 #1 injections would be expected to diffuse no farther than I mm beyond the cannula tip. The doses of M O R administered were 5 and 17.5 nmoles (equivalent to 1.5 #g and 5 #g of MOR, as the base, respectively). Doses of the other drugs were made on a molar basis equivalent to and/or greater than morphine: CPZ 5, 17.5 and 175 nmoles; PB 5, 17.5 and 54 nmoles; C D P 5 and 17.5 nmoles, and N A L 17.5 nmoles. The pH of the drug solutions was determined for all drugs, but was found not to be a significant factor in the effects observed after i.c. injection. Approximate pH values, which differed slightly with different drug concentrations, were: M O R , 5.2; PB, 9.4; CPZ, 5.5: CDP, 3.3; and NAL, 4.0. Current pulses delivered to the P A G to produce analgesia consisted of 64 Hz 0.5 msec bipolar pulse pairs delivered continuously through stimulus isolation units from a Grass $88 stimulator. These stimulus parameters were found to be effective in producing changes in escape thresholds, but did not produce observable lesions on

287 cresyl violet stained 40/zm coronal brain sections. The PAG stimulus intensity was elevated gradually in 10 #A/30 sec steps and the animals were stimulated at the desired level for at least 5 min prior to the beginning of TG testing. Melzack and Melinkoff52 have repoited that SPA requires about 5 min to fully develop. Two stimulus levels, 40 and 120/~A, were routinely employed. Exceptions to this occurred when a large TG threshold increase occurred at 40/~A or when there was obvious excitation and/or motor impairment. In several cases it was decided not to continue raising TG stimulus levels because of the marked analgesic effect of 40/~A PAG stimulation on responding (see Fig. 6). Since these animals were also to be tested after i.c. drug injections into the PAG, high level stimulation of the trigeminal nerve was avoided in the TG test. No significant differences between the control TG thresholds and drug day control thresholds were found as a result of SPA. In all, 4 experiments were performed. During each experiment every animal received one dose of each of 4 drugs and control (CSF) according to a 5 × 5 latin square design77. Drug injections were always separated by at least a 3 day interval. Pretreatment control levels of responding were assessed immediately prior to drug administration and the results were expressed as the per cent of predrug control responding. All tests were applied to each animal with at least a 1 min delay between tests. The HP, TF and PS tests were administered in a randomized order prior to administration of the TG test. The TG test was administered last because it required a longer time to perform than the other tests. The latin squares for the TG, HP and TF tests were evaluated by analysis of variance to determine the effects of drug treatments as well as to assess variation due to the animals and/or the order of drug injection. In no case was there a systematic decrease in MOR's efficacy after previous injections of other drugs. Pooled data for all treatments were evaluated by the least significant difference procedure at P <: 0.0577 after a one-way analysis of variance (Figs. 3, 4 and 5). Following the experiments, all rats were sacrificed with an overdose of pentobarbital and transcardially perfused with saline followed by a 10 ~ formalin solution. After storage in 10~ formalin for 1 week, frozen 40 #m coronal brain sections were cut and stained with cresyl violet. Composite diagrams of serial sections were drawn to verify placement of the cannula/electrode assemblies; the assembly tract was approximately 0.5 mm in diameter (diagrammatically represented in Fig. 2). RESULTS Four experiments were performed and each will be treated separately since each was designed as a latin square and analyzed separately. Cannula/electrode assembly placements are represented in Figs. 1 and 2 to facilitate comparison of the effects of MOR and SPA at different sites within the PAG. Tables of the effects of MOR and SPA at individual PAG placements are included in the brief discussions of the individual experiments. Overall comparisons of i.c. drug injection effects from all 3 i.c. experiments are presented in Fig. 3 for TG test, Fig. 4 for the HP test and Fig. 5 for the TF test. Rats in which SPA was evaluated were tested only in the TG test; overall SPA effects are plotted in Fig. 6.

288 LO.7 2

14

1 mm ~ ,

Fig. 1. Diagrammatic parasagittal section of the rat brain indicating the location of the cannula/ electrode assemblies. Numbers represent experimental subjects and correspond to animal numbers contained in Tables I, II, III and IV and portrayed in Figs. 2 and 6. Abbreviations: PAG, periaqueductal gray matter; SC, superior colliculus; FR, fasciculus retroflexus; PC, posterior commissure; IC, inferior colliculus. Figure adapted from a cresyl violet/luxol blue parasagittal rat brain section. Animals 19-23, not shown, were outside of the PAG (dorsal and lateral).

Experiment 1 In this experiment, the effects of 5 nmole doses of MOR, CPZ, PB and C D P and 0.5/~1 of CSF were evaluated after i.c. injection into the P A G area. Testing in the various analgesiometric procedures was begun 10 min after i.c. administration of the drugs. Histological evaluation of the cannula/electrode assembly placements revealed that 3 assemblies were localized in the dorsolateral edge of the P A G (animals No. 2, 4 and 5), one passed through the P A G to a more ventral site adjacent to the P A G (animal No. 9), and one was localized dorsal to the P A G (animal No. 19). The analysis of variance of experiment 1 revealed only one significant F value for the drug treatments. Using Dunnett's test 77, a significant decrease in the H P response latency was found for PB when compared to CSF control. In the analysis of variance over all experimental groups, however, no significant PB effect was detected. The latin square analysis of the T F test revealed no significant drug effects. However, in the analyses of variance over all experimental groups, C D P was found to significantly elevate the T F latency (see Fig. 5). The effect of C D P in the TF test and PB in the H P test are isolated occurrences; no dose-response relationship was demonstrable for either drug in the overall study. Individual response values to MOR, PB and SPA for animals comprising experiment 1 are presented in Table I. The effects of CPZ and N A L were unremarkable; the effects of 5 nmole doses of all drugs are, however, presented in Figs. 3, 4 and 5 for the TG, H P and T F tests, respectively. In this first experiment, focal brain stimulation followed the i.c. drug treatments.

289 TABLE I Experiment 1 * (5 nmole drug dose) PB

Animal No.*** MOR

2 4 5 9 19

SPA**

TG

HP

TF

TG

HP

TF

TG

121 103 147 177 122

85 116 83 159 107

113 99 87 75 222

49 92 82 94 119

41 88 53 94 67

41 109 58 78 100

241 322+ 102 744 159

* Abbreviations: MOR, morphine; PB, pentobarbital; SPA, stimulation-produced analgesia; TG, trigeminal stimulation escape test; HP, hot plate test; and TF, tail flick test. Values within the body of the table are test results represented as ~o of pretreatment control. For additional information, see text. ** SPA evaluated at 120/~A, value denoted by + evaluated at 100/~A; see text for explanation. *** Animal numbers correspond to placements diagrammed in Figs. 1 and 2.

Marked changes in the T G threshold were apparent following P A G focal brain stimulation in this experiment (see Table I and Fig. 6). It is apparent from Table I that SPA is considerably more efficacious than 5 nmoles of M O R administered into virtually the same P A G site.

Experiment 2

Cannula/electrode assembly placements in this second experiment were found to be localized at the lateral edge of the P A G in 3 cases (animals Nos. 7, I0 and 13) and passing through, but ventral to the P A G in one case (animal No. 12). Histological information was unobtainable from one animal (No. 24). Because of the marked effect of M O R in this rat, however, we felt confident that it represented a suitable placement for purposes of comparison of drug effects and it was retained in the statistical analysis. The order of i.c. drugs and SPA was reversed and focal brain stimulation preceded i.c. drug injections in this second experiment. Higher 17.5 nmole i.c. doses of MOR, CPZ, PB and C D P and 0.5/zl of CSF control were administered into the P A G after all animals had first been tested for SPA. Testing for drug effects was begun 10 min after drug injection. An analysis of variance of the latin squares for the TG, H P and T F tests revealed significant drug effects on all tests, but no significant variance due to animals or days of treatment. The significant drug effects were attributable only to M O R in all cases. Again, marked increases in the T G threshold were observed following P A G stimulation (see Table II and Fig. 6). Individual animal comparisons of test responses following M O R and SPA treatments are presented in Table II. Although significant increases in the T G threshold were apparent following both treatments, there is little apparent correlation between the magnitudes of effect of SPA and MOR-induced analgesia in the same animal. Overall drug comparisons are diagrammed in Figs. 3, 4 and 5.

290

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r i. osziii 23

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ilmmj Fig. 2. Diagrammatic coronal sections of the rat brain indicating the location of the cannula/electrode assemblies. Numbers represent experimental subjects and correspond to animal numbers contained in Tables I, II, III and IV and portrayed in Figs. 1 and 6. The stippled areas represent the approximate area and extent of a cannula/electrode track. (Figure adapted from KCSnigand Ktippe136.)

Experiment 3

In o r d e r to u n q u e s t i o n a b l y d e m o n s t r a t e the P A G as a morphine-specific site a n d to further estimate the m a g n i t u d e o f difference between M O R a n d the o t h e r drugs a d m i n i s t e r e d i.c., the effects o f a 17.5 nmole dose o f M O R were c o m p a r e d to a 175 n m o l e dose o f C P Z , a 54 n m o l e dose o f PB a n d a 17.5 n m o l e dose o f N A L a n d 0.5 #1 o f C S F control. I n this experiment, a 40 m i n d e l a y after i.c. d r u g a d m i n i s t r a t i o n was e m p l o y e d before testing to maximize d r u g diffusion a w a y f r o m the c a n n u l a a n d increase the l i k e l i h o o d o f detecting effects in the v a r i o u s analgesiometric tests following the e x t r a o r d i n a r i l y high doses o f C P Z , PB a n d C D P .

TABLE II Experiment 2* (17.5 nmole drug dose) Animal No.

7 10 12 13 24

MOR

SPA

TG

liP

TF

TG

116 206 186 334 350

285 230 284 153 209

143 57 265 123 303

>574** 113 712 1412 210

* See legend for Table I. For additional information, see text. SPA evaluated at 40/~A.

**

291 TRIGEMIN, dL 500

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THRESHOL O

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Mean t $ E

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100-

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CPZ

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PB

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CDP

NAL

Fig. 3. Results of i.e. drug injections into the periaqueductal gray on the trigeminal stimulation escape test. In this a n d the subsequent two figures, the following abbreviations apply: CSF, atificial cerebrospinal fluid control; M O R , m o r p h i n e ; CPZ, chlorpromazine; PB, pentobarbital; CDP, chlordiazepoxide; and NAL, naloxone. D a t a pooled from all experiments. * indicates significant difference from C S F control, P < 0.05; see text for additional information.

Of the 5 animals originally prepared for this experiment, only 3 cannula/ electrode placements were found to be localized within the PAG after histologic examination. Four additional animals were prepared and two of these were subsequently found to have assembly placements in the PAG. The resulting group of 5 animals (Nos. 1, 3, 6, 8 and 11) all had cannula/electrode assemblies localized in the TABLE

III

Experiment 3* Animal No.

1

3 6 8 I1 20 21 22 23

MOR**

SPA

TG

HP

TG

126 286 362 301 222 95 206 183 833

179 29 64 357 100 125 230 51 400

--*** 93 >281 + 141 455 --*** 354 >255 + 78

* See legend for Table I. F o r additional information, see text. ** M O R dose, 17.5 nmoles, evaluated 40 min after administration. *** Electrode failure, n o data collected.

292 lateral-ventrolateral aspect of the PAG. The data from these rats were analyzed as a complete block, two-way analysis of variance; only drug treatments were found to have a statistically significant effect in the various tests. This effect was found to be due only to MOR. Focal brain stimulation in this third experiment preceded drug testing. Again, marked increases in the T G threshold were obtained (see Table 1II and Fig. 6). Focal brain stimulation data for two animals (Nos. 1 and 20) are missing due to an electrical short in the P A G electrode. The effects of M O R and SPA are compared in Table III. Again, both treatments were effective but there was little apparent correlation in the magnitude of the antinociceptive effect of either treatment within a single animal. Animals denoted 20-23 in Table IIl represent assembly placements not in the P A G region (see Fig. 2). In general, there is an increased effect of M O R on analgesiometric tests the closer the cannode to the PAG. The cannula/electrode assembly of animal 20 was well lateral to the P A G and M O R was ineffective in all tests. Animals 21 and 22 demonstrated some M O R effects in the T F and HP tests although their cannula/electrode placements were found to be at least 0.5 m m lateral to the PAG. Prominent SPA was also noted at these lateral placements for these two animals, suggesting that SPA may also be produced in the mesencephalon outside of the PAG. The largest effect of any i.c. M O R injection occurred in animal 23, where the cannula/ electrode assembly was found to be approximately 2 m m lateral to the PAG. In fact, a large lesion in this animal extended to within approximately 0.2 m m of the PAG. Whether the large M O R effect observed was due to another locus of action for M O R lateral to the P A G or diffusion of M O R into the P A G is not known. It is interesting to note that there was no SPA in this animal in the T G test. Animals numbered 20-23 were not included in the statistical evaluation of these experiments. Because the heat lamp intensity degraded over several months, control TF latencies in the third experiment were excessively long. A new calibration procedure was initiated which resulted in control TF latencies of approximately 3.5 sec and the T F as well as the HP and PS tests were re-evaluated in another series of animals (Table IV). The cannula/electrode placements for these animals, numbered 14-18, were

TABLE IV Experiment 3* Animal No.

14 15 16 17 18

MOR HP

TF

178 88 231 78 164

162 238 258 98 168

* See legend for Table I. For additional information, see text. ** MOR dose, 17.5 nmoles, evaluated 40 min after administration.

293 HOT P L A T E

PAW-LICK

LATENCY

Mean * SE '..4

200

~r

I-I 5 . 0 n a n o m o l e s I~! 17.5 n o n o m o l e s P-J as i n d i c a t e d

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II CSF

CPZ

MOR

PB

CDP

NAL

Fig. 4. Results of i.e. drug injections into the periaqueductal central gray on the hot plate test (see legend for Fig. 3). found to be slightly dorsal and caudal to earlier assembly placements of animals in experiment 3 (see Fig. 2). Animals 14-18 were not evaluated in the TG test. Significant effects due to drug treatments were found in both the TF and HP tests and were found to be related only to M a R . The data for the individual test values are presented in Table IV and comparisons to all other drug tests are presented in Figs. 3, 4 and 5. TAlL

FLICK

LATENCY

Mean t S E [] [] []

~J 200

5 . 0 nanomoles 17.5 nanomoles as indicated

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CSF

MOR

CPZ

PB

CDP

NAL

Fig. 5. Results of i.e. drug injections into the periaqueductal central gray on the tail flick test (see legend for Fig. 3).

294

Experiment 4 Experiment 4 was conducted to evaluate the effects of MOR, CPZ, PB, C D P and CSF when administered by the subcutaneous route. This group of animals was implanted with a trigeminal stimulating electrode only; no cannula/electrode assembly was implanted in the PAG. The doses and times of testing after drug administration were: M OR, 5 mg/kg, tested at 40 min; CPZ, 5 mg/kg, tested at 40 min; PB, 10 mg/kg, tested at 30 rain; CDP, 5 nag/kg, tested at 40 min; and CSF, 0.7 ml, tested at 40 rain. These times and doses were selected on the basis of earlier work done in this laboratory. Only the results for the T G test are reported since no drug other than M O R produced any significant change in the HP, TE or PS tests. As can be seen in Fig. 7, MOR, CPZ and PB all produced significant elevations of the T G threshold. It should also be noted that 5 mg/kg of M O R given subcutaneously did not produce as great an increase in the T G threshold as did 17.5 nmoles of M O R administered i.c. directly into the P A G sites evaluated in these experiments (see Fig. 3). Regarding the PS test, it was performed on all MOR-treated animals. However, complete comparison with all other drugs was obtained only in the two groups of animals receiving high i.e. doses of the other drugs. Morphine consistently reduced responses on the PS test whereas no other drug did. Individual PS site data for the MOR-treated rats were also evaluated, but no significant differences between M O R ' s effect at the various body sites tested were found. We did note that the tail was 2000

1000

e7 k

5oo

.022

100

4'0 FOCAL

' BRAIN

8'0 STIM.,

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I :~0 uA

Fig. 6. Results of focal brain stimulation of the periaqueductal gray on the trigeminal stimulation escape test. Numbers represent experimental subjects and correspond to animal numbers contained in Tables I, II and III and Figs. 1 and 2. Animals 3, 5, 8, 10, 19 and 23 did not exhibit prominent stimulation produced analgesia and were omitted from this figure; see Tables I, II and III for actual values. See text for additional information.

295 TRI6EMINAL

ESCAPE

CSF

CPZ

THRESHOLD

300-

200

I00

MOR

PB

CDP

Fig. 7. Trigeminal stimulation escape thresholds following s.c. administration of CSF (artificial cerebrospinai fluid control), MOR (morphine), CPZ (chlorpromazine), PB (pentobarbital) and CDP

(chlordiazepoxide). Drug doses in mg/kg, as their bases, are indicated within the respective bars. Data represented as the mean ± S.E.; n = 5 animals/treatment. * indicates significant difference from CSF control, P < 0.05; see text for additional information.

consistently less analgesic than the other 4 body sites tested in this PS test following i.c. administration. This may relate to our testing procedure since the tail was also less affected after subcutaneous M O R administration. No somatotopy was apparent for the analgesic effects after unilateral i.c. morphine injection into the PAG. DISCUSSION

It has previously been demonstrated that both narcotics and centrally acting, non-narcotic, tranquilizers and depressants can elevate thresholds for response to various modes of trigeminal nerve stimulation. MitchelP ~ demonstrated that both morphine (2 and 4 mg/kg) and chlorpromazine were equipotent in elevating tooth pulp stimulation escape thresholds in cats, In the monkey, using gasserian ganglion stimulation, Weitzman and Ross aa found that morphine (0.5 and 2.5 mg/kg), chlorpromazine (0.3 mg/kg) and pentobarbital (5 mg/kg) would elevate their titration response threshold. In the present study, we have confirmed that morphine (5 mg/kg), chlorpromazine (5 mg/kg) and pentobarbital (10 mg/kg) all elevate the escape threshold in the T G test when administered by the subcutaneous route (Fig. 7). These data notwithstanding, we have herein demonstrated that only morphine when administered i.c. into the PAG region produces analgesic effects in the various tests employed, and that other, non-narcotic agents, injected into the identical site were ineffective. Only morphine significantly altered responding to all types of noxious stimuli tested, following either s.c. or i.c. routes of administration. Analgesia after i.c. morphine appears to be general in the sense that it is detect-

296 able by several different types of analgesiometric procedures, including tooth pulp stimulation 21, noxious pinching29, 72, pin pricks 29 and shock titration procedures 62. In the case of the hot plate test, Sharpe et al. 72 found i.c. morphine to be effective while Jacquet and Lajtha 29 did not. In the experiments described herein, morphine's analgesic effect has been clearly demonstrated on all of the tests employed. However, little correlation between tests was apparent in the results observed in any given animal. Many animals were unresponsive ("analgesic") in one or two test procedures, but were not necessarily so in other tests. This may be an indication of a semi-somatotopic arrangement in the PAG as was demonstrated for noxious stimulus evoked potentials by Liebeskind and Mayer 41. Chlorpromazine's ability to inhibit escape responding to noxious stimulation may be the result of effects on several possible loci of action. Chlorpromazine is known to block many transmitter systems throughout the brain, dopamine prominent among them. Depression of dopaminergic function in the basal ganglia 33,v6 is known to be involved in many alterations of motor function, including catalepsy 13. Ungerstedt et al. a3 found that 10 #g of chlorpromazine injected into the caudate-putamen of the rat induced contralateral turning and asymmetric posture. Broekkamp and Van Rossum 7 have recently demonstrated that injections of haloperidol into the neostriaturn or nucleus accumbens inhibited self-stimulation in the ventral tegmentum. Morphine also inhibited self-stimulation when given via the ventricular system, but did not inhibit it when injected directly into the striatum. Morphine injected i.c. into the caudate, however, has been found to be ineffective in producing an analgesic effect in a shock titration procedure 6° or in the flinch-jump procedure 28. It has been reported 21,81 that the principle site of the analgesic effect of morphine after intraventricular injection is the area of the fourth ventricle or PAG. A medial subnucleus of the PAG has been shown to have efferent fibers passing to the ventral tegmenta[ area in the cat 19, suggesting that morphine's reported inhibition of self-stimulation in the ventral tegmentum could be mediated from the PAG. Chlorpromazine (5 #g) has also been demons'rated to inhibit escape and conditioned avoidance behavior when applied to the hypothalamus of rats, but not when injected into either the midbrain, medial or lateral septal area or amygdaloid complex 17. Morphine has been reported to produce an analgesic effect when injected directly into the hypothalamus2a,~z,v2, 82. Thus, in the hypothalamus, there appear to be at least two mechanisms for altering escape responding by drugs. Unlike the hypothalamus, the PAG appears to be one site where morphine alters specific behaviors required for response to noxious stimulation, and where other drugs do not alter these responses. Since other agents do not act directly at this site, it is probable that changes at this site relate to morphine's analgesic efficacy. Lesions of the ventral PAG have been reported to decrease escape responding in animals ls,4~. Similar PAG lesions have also reportedly failed to elevate shock titration thresholds 34,68,ss. In the present experiments, our highest dose of chlorpromazine (175 nmoles) most likely acted as a local anesthetic 71, but failed to produce an escape deficit in the T G test. This probably is due to the dorsolateral placement of the cannula/electrode assemblies relative to the sites investigated by Halpern TM. This

297 finding emphasizes that the analgesia induced by morphine in the PAG is not likely due to a non-selective depression of this region, nor is it likely to be due simply to changes in escape performance since alterations in escape behavior and titration thresholds appear to have different mechanisms. Pentobarbital's effect on evoked potentials41,7s as well as on behavioral responses (ref. 86 and this report) is most likely due to a general depression of large groups of neurons in the CNS and not due to actions on the PAG alone 16. Morphine, in contrast, appears to be acting selectively in the PAG. Straw and Mitchell7s reported that both morphine and pentobarbital (at doses as low as 2.5 mg/kg) depressed tooth pulp stimulation evoked potentials recorded in the PAG. On the basis of their results, they suggested that the PAG was unlikely to be involved in morphine's analgesic effect because the non-narcotic pentobarbital produced a similar effect on evoked potentials in the PAG. Evoked potential studies must be evaluated somewhat critically, however, since they constitute the summed activity of a multitude of neuronal sinks and sources. Bradley and Dray 6 reported that iontophoretic application of morphine onto mesencephalic neurons produced both stimulation and depression of neuronal firing whereas Henry20 reported a depression of the discharge rate as well as spike amplitude of PAG neurons. Since many workersla,42,~7,~s,66 (and this report) have demonstrated an analgesia after stimulation of the PAG, depression of the overall neuronal firing in the PAG may be obscuring behaviorally significant stimulation of some of these neurons. Houser and Par625 reported that chlordiazepoxide produced analgesic effects in rats when tested in a grid shock spacial preference test. We had hoped to investigate whether this effect was due to a direct action on the PAG. However, we were unable to reproduce their results with s.c. doses of 5 or 10 mg/kg of chlordiazepoxide, up to 5 times higher than their reported EDs0. It may be that chlordiazepoxide is less effective in elevating a trigeminal stimulation escape threshold than a grid shock escape threshold. We were also unable to demonstrate a consistent effect of chlordiazepoxide when administered i.c. in any of the other analgesiometric tests we employed. Naloxone, a specific narcotic antagonist, was administered i.c. to determine if it alone would have any effect in the various tests. We found no such effects when doses equivalent to morphine were employed. Reversal of both the hyper- and hyporeactivity produced by morphine administered i.c. into the PAG by naloxone also administered i.c. into the PAG has been reported 29. We have found that naloxone administered systemically is also capable of antagonizing the analgesic effect of morphine administered i.c. into the PAG (unpublished observations). Marked elevations in the trigeminal escape threshold in these experiments were produced by electrical stimulation of the PAG. The precise placement of our electrodes is complementary to the work of other investigators2,a,42,47-49,57,66 in that the majority of our electrode placements were at the rostral level of the superior colliculus. Balagura and Ralph 8 have reported decreased responsiveness to pin jabs at a similar location and other brain stem areas located adjacent to the PAG. At this site, we were able to demonstrate profound increases in the trigeminal escape threshold. These increases are comparable in magnitude to the 3-10 × increases in the jaw jerk threshold re-

298 ported by Oliveras et al. 5s subsequent to stimulation of the dorsal raphe area in cats. Liebeskind and Mayer 41 reported that noxious stimulation applied to the face of rats produced the most prominent gross evoked potentials in the rostral PAG; thus, stimulation in this area may be especially effective in altering responding to trigeminaJ stimulation. In general, we observed that the effects of focal brain stimulation (120 #A) were larger than the effects of 17.5 nmole i.c. morphine on the TG test. Comparisons between the magnitude of morphine-induced and focal brain stimulation-induced analgesia revealed that both treatments were more effective at ventrolateral PAG placements. This is in agreement with previous work48, 7z. There was an absence of similarity, however, between the magnitude of effect produced by morphine and focal brain stimulation at the same site. Experiments recently completed suggest that the differences in efficacy between morphine and focal brain stimulation applied at coincident PAG sites may relate to the action of these treatments on anatomically different elements within the PAG (in preparation). In the experiments reported herein, multiple i.c. drug injections were employed to evaluate the specificity of morphine's analgesic effect in the PAG. While neural damage certainly occurs after cannula implantation and multiple i.c. drug injections, no significant decrease in morphine's effect was observed in these experiments as a result of this damage. Jacquet and Lajtha 29,3° have reported tolerance to the effects of i.c. morphine after multiple i.c. injections. The present study would substantiate their assertion that the tolerance they see is due to receptor or neural tolerance and not merely to glial scarring at the cannula tip that might prevent morphine's access to critical tissues. While this study, as well as others, has shown that analgesia can be produced after injection of morphine into the PAG, there is considerable evidence suggesting that the PAG is not the only central locus of morphines' action. Localization studies of stereospecific opiate binding sites have found such binding to be widespread. While binding sites are discrete with respect to various structures, they are found in many areas, including the cortex, limbic structures, caudate nucleus and spinal cord 2z,39,6°. Injection of narcotics into these brain sites has resulted in electrophysiologic evidence of narcotic-induced alterations in function. Iontophoretic application of morphine on cortical neurons depresses neuronal activity acutely but not in tolerant animals v°. Changes occur in the EEG after injection of morphine into the amygdala, and unilateral tolerance reportedly developed to this effect9. Local application of extremely small doses of morphine (2.5 ng) was found to inhibit nociceptive evoked electrical activity in the caudate nucleus, dorsal medial thalamic area, and PAG region 4. Direct actions of narcotics on spinal cord sensory transmission and numerous spinal reflexes have also been demonstrated aS,46,69,sv. If opiate binding sites are so widely distributed and apparently functional, why can analgesia be produced only when narcotics are injected into restricted midtine periventricular-PAG-fourth ventricle sites? A possible reason for this PAG specificity may not relate to the specificity of actions of narcotics, but to the nodal character of the PAG for processing afferent and efferent information required for responding to noxious stimuli 12,50,56,75.

299 The mechanisms for morphine-induced and focal brain stimulation-produced analgesia in the P A G are unknown. These treatments certainly influence descending systems which can then alter responding to a variety of noxious stimuli 2,57,69. Serotonergic and dopaminergic systems appear to facilitate both SPA and morphine-induced analgesia while norepinephrine may be antagonistic T M . The involvement of the serotonergic raphe system in morphine analgesia is controversial, some investigators implicating serotonin and the raphe system 1,6a and others not 5,8,44. Adler et al. 1 recently reported that the changes in analgesia they find following lesions of the median raphe are not related to corresponding reductions in forebrain serotonin content. Thus, either regional changes in forebrain serotonin are critical or changes in serotonin in other parts of the brain are important65, 84. In the PAG, numerous other putative transmitter substances are also known to exist. These include acetylcholine (refs. 10, 59), glutamate and gamma-aminobutyric acid78, 74, epinephrine 2a and substance p24. Several laboratories have recently found endogenous compounds which act in a fashion similar to narcotic agonists on the guinea pig ilium preparation or mouse vas deferens 26,79,s°. Brain stimulation has been hypothesized to release such a morphine-like substance onto the opiate "receptor". Morphine itself has been hypothesized to act as an exogenous transmitter-like substance 38. Further evidence that both SPA and morphine may act at the same receptor site is provided by the repolted crosstolerance between the two 47. An extensive report of careful examination of the congruence and possible interaction of SPA and morphine efficacious P A G sites is forthcoming. ACKNOWLEDGEMENTS The authors gratefully acknowledge the contributions to the successful completion of these experiments made by Mr. K. Schaefer and Mrs. L. Miller. These data were presented, in part, at the 59th Annual Meeting, Federation of American Societies for Experimental Biology, Atlantic City, N.J., April, 1975. Supported by N I H Grants G M 22026 and NS 12114 to G.F.G. REFERENCES 1 Adler, M., Kostowski, W., Recchia, M. and Samanin, R., Anatomical specificity as the critical determination between raphe and morphine analgesia, Europ. J. Pharmacol., 32 (1975) 39-44. 2 Akil, H. and Liebeskind, J., Monoaminergic mechanisms of stimulation produced analgesia, Brain Research, 94 (1975) 279-297. 3 Balagura, S. and Ralph, T., The analgesic effect of electrical stimulation of the diencephalon and mesencephalon, Brain Research, 60 (1973) 369-379. 4 Bennett, C. T., Bevan, T. and Gall, K., Changes in nociceptive evoked activity of the caudate nuclei following local application of morphine, Neurosci. Abstr., 1 (1975) 281. 5 Blasig, J., Reinhold, K. and Herz, A., Effect of 6-OH dopamine, 5, 6-dihydroxytryptamine and raphe lesions on the antinociceptive actions of morphine in rats, Psychopharmacologia (Berl.), 31 (1973) 111-119. 6 Bradley, P. B. and Dray, A., Morphine and neurotransmitter substances: microiontophoretic study in the rat brain stem, Brit. J. PharmacoL, 50 (1974) 47-55. 7 Broekkamp, C. L. E. and Van Rossum, J. M., The effect of microinjections of morphine and haloperidol into the neostriatum and the nucleus accumbens on self-stimulation behavior, Arch, int. Pharmacodyn., 217 (1957) 110-117.

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