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Effect on the nociceptive threshold and EEG activity in the rat of morphine injected into the medial thalamus and the periaqueductal gray
EFFECT ON THE NOCICEPTIVE THRESHOLD AND EEG ACTIVITY IN THE RAT OF MORPHINE INJECTED INTO THE MEDIAL THALAMUS AND THE PERIAQUEDUCTAL GRAY J. C. YEUNG. T. L. YAKSH and T. A. RUDY School
of Pharmacy.
University (Accepted
of Wisconsin. 27 Octohrr
Madison.
WI 53706. U.S.A
1977)
Summary-Rats were chronically implanted with EEG electrodes and. either microinjection cannulae aimed at the medial thalamus (MT) and periaqueductal gray (PAG). or a spinal catheter. Morphine sulphate injected intraperitoneally produced both analgesia. as measured by the tail flick and pinch-agitation tests, and episodes of high-voltage slow-wave activity (HVSA) in the EEG. Morphine injected into the MT produced HVSA but not analgesia. Conversely, morphine injected into PAG sites or into the lumbar subarachnoid space produced analgesia but did not alter EEG activity. These data suggest that the MT may mediate the EEG alterations produced by systemically administered morphine. They also indicate that analgesia (as measured by the tests employed) and HVSA are entirely independent actions of morphine mediated by different neuroanatomical substrates. The functional significance of the cortical HVSA produced by morphine and its mechanism of production are discussed.
In several animal species, it has been demonstrated that the systemic administration of morphine produces, not only analgesia, but also a concomitant synchronization of the EEG (Bradley, 1968; Domino, 1968; Killam, 1962; Valdman, 1967). Whereas the brain sites subserving morphine’s antinociceptive action have been extensively studied (Criswell, 1976; Jacquet and Lajtha, 1973. 1974; Pert and Yaksh, 1974, 1975; Sharpe, Garnett and Cicero, 1974; Tsou and Jang, 1965; Yaksh, Yeung and Rudy, 1976). the neuroanatomical substrate of the EEG synchronizing action of morphine remains largely undetermined. There exists excellent evidence that the periaqueductal gray (PAG), particularly its caudal aspect, is the major supraspinal structure mediating the analgetic action of narcotics (Jacquet and Lajtha, 1974; Sharpe et al., 1974; Yaksh et al., 1976). Although the possibility that the caudal PAG may also subserve morphine’s EEG synchronizing action has not been examined directly, Albus and Herz (1972) have reported that profound synchronization could be elicited in the rabbit by injection of morphine into the fourth ventricle after occlusion of the aqueduct at its rostra1 end. This finding and the extensive anatomical connections between the PAG and other brain regions which potently influence cortical activity, such as the bulbar reticular formation and the midline and intralaminar thalamic nuclei (Chi, 1970; Hamilton, 1973; Hamilton and Skultety, 1970; Ruda, 1975), suggest that the PAG may be involved in the genesis of narcoticinduced EEG synchronization. Key words: morphine. thalamus. periaqueductal
EEG.
antinociception.
Although Albus and Herz (1972) observed that morphine injected intraventricularly in such a manner as to limit its diffusion to the fourth ventricle and aqueduct could elicit typical synchronized cortical waves of high voltage and slow activity (HVSA). they found that morphine injections limited to the ventricular spaces rostra1 to the aqueductal mouth could also elicit HVSA. Thus, it would seem that there must exist a second substrate for the EEG synchronizing action of narcotics. It is well known that the medial thalamus (MT), an extension of the ascending reticular system, plays an important role in the control of ongoing cortical activity and the state of wakefulness (Krupp and Monnier, 1966; Moruzzi, 1972). Low-frequency electrical stimulation of certain MT nuclei in a variety of species has been associated with the evocation of synchronized HVSA cortical waves (Dempsey and Morison, 1942; Jasper, 1949; Morison and Dempsey. 1942; Starzl and Magoun, 1951). Destruction of the MT has led to a marked reduction in both the frequency and amplitude of the observed cortical activity, or to cortical activity that showed poorly organized recruiting responses and poorly organized spindles (Kennard, 1943; Knott, Ingram and Chiles, 1955). These observations, in conjunction with the recent finding that the MT exhibited stereospecific narcotic binding (Kuhar, Pert and Snyder, 1973; Pert, Kuhar and Snyder, 1975). suggested that the MT might be the rostra1 periventricular structure mediating the HVSA observed by Albus and Herz (1972). In the present study, therefore, the putative contributions of both PAG and MT to narcoticinduced HVSA were evaluated.
medial
gray.
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J.
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C. YEUNG, T.
L.
METHODS
Under sodium pentobarbital anesthesia (35 mg/kg. i.p.). each of 9 Holtzman male albino rats was prepared under stereotaxic control with bipolar EEG recording electrodes and an array of microinjection guide cannulae. The EEG electrodes were made of platinum tubing (27 g) and were implanted epidurally above the frontal cortex. The poles of the recording electrodes were 7 mm apart, and each pole rested on one side of the cortex. The microinjection guide cannulae (24 g T.W. stainless-steel) were aimed at the MT (AP: 3.8-3.2. L: l&0.0: HV: 0.54.0) and the PAG (AP: 1.2--0.8: L: 1.0-0.0; HV: 0.5-0.0). The stereotaxic co-ordinates were taken from the atlas of Pellegrino and Cushman (1967). In 3 rats. one cannula was implanted in the MT (in the midline) and two in the PAG. In the remaining 6 rats. two cannulae were implanted in the MT (bilaterally symmetrical, 0.5-1.0 mm off midline) and one was placed in the PAG. Following a 7-day recovery period, experiments were begun in which morphine was injected intracerebrally at sites in the PAG and MT and the effects on the nocicep&e threshold and EEG activity were observed. Each intracerebral injection consisted of 0.5 ~1 of either 0.9% saline or morphine sulphate dissolved in normal saline (10 pg/pl) delivered through a 29 g injector over a period of 20 sec. Initially, the injection was made 1.0 mm below the guide tip. When all experiments at this depth of penetration were completed. deeper sites (separated by 1 mm) were explored sequentially. Successive injections made at the same brain locus were separated by a period of at least 7 days. and each brain site was not injected more than twice with morphine. To facilitate brain mapping for morphine sensitivity in rats with bilateral MT cannulae. bilateral injections (IO pg/pl) were made. In some cases. when an effect on either the EEG or the nociceptive threshold was observed, unilateral injections were made on separate days and the effects re-evaluated. The effects of systemically injected morphine sulphate (10 and 30 mg/kg, i.p.) were examined in all animals. During the experimental sessions. each rat was loosely restrained in a hemi-cylindrical wire mesh cage with the tail protruding through a hole in one end. The tail-flick latency and the agitation response to skin pinches applied with forceps were checked before (0 min) and at 5. 20. 40 and 60 min following the intracerebral injection of morphine. In the pinchagitation test. forceps pinch was applied to the animal’s face and four limbs in a random fashion. The response was recorded as positive (presence of signs of agitation and/or withdrawal) or negative (absence of signs of agitation and withdrawal). In the tail flick test, the animal’s tail was laid in a groove perpendicular to a length of electrically heated nichrome wire located 1 cm below the tail. A cut-off time of 15 set was employed. After each assessment of the pinch agitation and tail-flick responses. a 2-min epoch of EEG
YAKSH and
T. A.
RUDY
activity was also taken, using a Grass EEG pre-amplifier (model 7P5B) and a Grass polygraph d.c. driver amplifier. Preliminary studies have shown that the EEG taken before the pain testing was similar to that taken after the testing. Whenever antinociception and/or a change in EEG (see below) was observed following morphine administration, the pharmacological specificity of the effect was ascertained by the intraperitoneal injection of naloxone hydrochloride (1 mg/kg). Naloxone was given 65 min following morphine administration, and measurements were again made 7 min after the naloxone injection. In a few experiments, 0.9% saline, instead of morphine or naloxone, was injected centrally (0.5 ~1 per site) or intraperitoneally (1 ml/kg) to rule out further a possible nonspecific effect of the procedures involved. To quantify the changes in EEG activity, the percentage of each 2-min epoch during which the rat displayed high voltage and slow activity (percentage HVSA) was measured. The HVSA was easily distinguishable and was defined for analysis purposes as those periods when the recorded amplitude exceeded twice that of the low-voltage fast activity (LVFA) observed before injection. At the end of the experimental series, each rat was sacrificed with an overdose of urethane and its brain perfused with 0.9% saline followed by 10% formalin. The brain was impregnated with gelatin as described by Wolf (1971) and was then sectioned on a freezing microtome. Sections were stained with cresyl fast violet. All brain injection sites were identified by microscopical examination and projected onto plates obtained from the atlas of Pellegrino and Cushman (1967). Three additional rats were implanted with bipolar frontal electrodes and subarachnoid spinal catheters with tip openings lying in the vicinity of the lumbar enlargement of the spinal cord as described by Yaksh and Rudy (1976). Following a ‘J-day recovery period, 5~11 of a solution of morphine sulphate dissolved in artificial CSF (IO pg/pl) was injected slowly over a period of 40sec. The same testing procedures employed in measuring the effects following intracerebra1 injections of morphine were employed in these studies. At the end of the experimental sessions. each animal was sacrificed with an overdose of urethane, and the location of the catheter tip opening in the spinal cord was verified by gross dissection. To compare the differences between means, Student’s t-test was employed. For the multiple comparisons of means observed at various times following treatment to that observed before treatment, a oneway analysis of variance was performed and the Newman-Keul’s test employed. The means are defined to be significantly different if P < 0.01 (2-tailed). RESULTS
During the experimental sessions, rats that received saline injections (intracerebral or systemic) generally
527
Morphine action on analgesia and EEG remained in a state of arousal associated with a desynchronized EEG characterized by 20-27 Hz LVFA waves. Intraperitoneai injection of morphine (10 or 3Omg/kg, i.p.f produced a dose-dependent behavioural antinociception and sedation associated with EEG slowing and synchronization. Episodes of HVSA (6-14 Hz) were observed to be interspersed among the LVFA waves. An example of morphine’s action (30 mg/kg, i.p.) on ongoing EEG and the noci-
ceptive threshold and its reversal by naloxone is illustrated in Figure I(a). Intracerebral injections of morphine into the MT in doses of 5 pg/OS ~1 unilaterally, or IO~g/~fl total if the injections were bilateral, evoked a marked slowing of the EEG with a concomitant increase in amplitude. Concurrently, the behaviour of the animal was observed to be mildly depressed and there was a marked decrement in the affective component of the
30 mg/kg i.p.
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Fig. 1. Examples of the effects of morphine administered intraperitoneally (a), into the medial fhalamus (b). into the p~riaqu~ductal gray (c) and intrathecalfy (d) on EEG activity. tail-flick latency and the pinch-agitation response. In all panels. upper two tracings represent EEG epochs recorded at slow and fast speeds, respectively (see time/voltage scales at bottom right). Solid bars indicate tail-flick latencies. Letters beside each bar denote the distribution of body surfaces rendered non-responsive to forceps pinch: 0 = no effect. B = hindpaws and forepaws rendered non-responsive. C = hindpaws. forepaws and face rendered non-responsive. Histological plates at right illustrate the intracerebral injection sites (dose = 5pg in 0.5~1 in both b and c). Abscissa indicates the times after injection when the measurements were taken: NAL denotes measurements made 7 min after the injection of naloxone (I mgikg i.p.). The nafoxone was injected 65 mm after morphine administration.
J. C. YEUNG.T. L.
528
reaction to application of the forceps pinch (vocalization, defecation, urination and “fear”). The animal could still be easily aroused with non-noxious stimuli such as light, touch and loud clicking sounds. This effect of focally applied morphine in the MT on the EEG, however, was not accompanied by any degree of antinociception as measured by the tail flick and pinch-agitation tests. No discernible differences in the changes in ongoing EEG activity following systemic morphine or intracerebrally applied morphine into the MT were observed. An example of the effects obtained following the focal administration of mor50
r
0t
50
IO mg/kg
YAKSH
phine (5~g/OS~l) into a brain locus within the MT is shown in Figure I(b). The location of the brain locus is indicated in the corresponding coronal section. As with systemically applied morphine, naloxone (1 mg/kg. i.p.) completely antagonized the EEG synchronizing action of morphine within the MT. Further evidence that the synchronizing effect was not due to a non-specific action of the microinjection is suggested by the failure of microinjected saline (0.5 ~1 per site) into the MT to cause significant changes on the EEG. The mean + S.E.M. of the maximal changes in the percentage HVSA was 3.5 + 2.6 (n = 6) follow-
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Fig. 2. Summary of the differential effects on HVSA and tail-flick latency of morphine injected intraperitoneally and into the medial thalamus, periaqueductal gray and spinal subarchnoid space. First two panels: effect of injection of 10 and 30mg/kg (i.p.), respectively. Third and fourth panels: effect of unilateral and bilateral injection, respectively, of 5 pg into the medial thalamus (MT). Fifth panel: effect of injection of 5pg into the periaqueductal gray (PAG). Bottom panel: effect of intrathecal injection of 5Opg. Bar lengths indicate mean values and vertical lines, S.E.M. Asterisks denote values statistically significant at the 0.01 level. Solid bars represent the differences in the percentage of time spent in HVSA during 2-min EEG epochs recorded at various times after morphine injection as compared to a control (pre-injection) epoch. Hatched bars represent the change in tail-flick latency at each post-injection interval in comparison to pre-injection control value. Abscissa values indicate the times after morphine injection at which the data were recorded. The 72-min value represents data readings taken 7 min after injection of naloxone (1 mg/kg, i.p.) at the arrow. n = number of animals or number of injection sites included in the means (see text for details).
529
Morphine action on analgesia and EEG ing the microinjection of saline and was not significantly different from that observed before injection (P > 0.2). In contrast to the loci located in the MT, morphine injected into certain sites within the PAG resulted in antinociception but was without effect on the ongoing LVFA of the EEG. In Figure I(c) are shown the effects on the ongoing EEG activity. tail-flick latency and the pinch-agitation response of an intracerebral injection of morphine (5 /rg in 0.5 ~1) into a morphinesensitive site located within the PAG. The location of the microinjection site is indicated in the adjacent coronal section. Naloxone reversibility of the effects observed is also illustrated. The effects of an intrathecal injection of morphine (50 pig in 5 ~1) on tail-flick latency, the pinch-agitation
APt3.6
AP+ 1.0
response and ongoing EEG activity are shown in Figure I(d). The injection was effective in blocking the tail-Rick response and the pinch-agitation response of the four limbs but not the face. No change in the EEG. however. was discernible following such an injection. Figure 2 summarizes the time-effect relationships and pharmacological specificity of the EEG synchronizing and antinociceptive (tail-flick) actions of morphine administered intraperitoneally and into the MT, PAG and spinal subarachnoid space. The mean values illustrated for intraperitoneal and intrathecal injections are derived from all rats injected by these routes. The mean values for MT and PAG injections include only injections made into active sites (sites wherein microinjection clearly altered tail-flick
AP+3.4
AP-0.6
Fig. 3. Frontal sections of the rat brain (Pellegrino and Cushman atlas) indicating the approximate positions of morphine injection sites and the EEG and antinociception responses evoked. Top: Injection sites in or near the MT. Asterisks indicate sites where morphine injections elicited increase HVSA without affecting tail-flick latency. Open circles denote sites where morphine injection caused no change in either parameter. Bottom: Injection sites in or near the PAC. Solid circles indicate sites where morphine injections produced antinociception as measured by the tail-flick test and no increase in HVSA. Open circles denote sites where morphine injection caused no change in either parameter.
J.
530
C.
YEUNG.
T. L.
latency or EEG activity). As can be seen from the Figure. both the EEG and antinociceptive actions of morphine injected by all routes developed smoothly over time, became statistically significant at 2&40min after injection, and were completely reversed by naloxone. Naloxone (1 mg/kg, i.p.) alone was not found to affect the EEG and nociceptive threshold of the animal. The Figure also clearly indicates the selective actions of morphine on the EEG and tail-flick latency when injected into the MT and into the PAG or spinal subarachnoid space. respectively. The approximate anatomical locations of the microinjection sites yielding these selective actions on the EEG and tail-flick latency are presented in Figure 3. Brain sites studied but which did not mediate either of these two actions of morphine are also shown in this Figure. The number of inactive vs active sites in either the MT or the PAG are too few to delineate the borders of the responsive tissues. However, in the PAC. the distributions of active and inactive loci (as shown in Fig. 3) are in agreement with a previous study (Yaksh c’t al.. 1976) in which it was found that the PAG region of greatest morphine sensitivity was its ventral posterior aspect. The overlapping of positive and negative sites in the MT indicates that this structure, like the PAG. is not uniformly sensitive to locally injected morphine. DISCUSSION
The appearance of synchronized EEG slow waves following systemic morphine administration has been observed in a variety of species including rat (Cahen and Wikler. 1944; Colasanti and Khazan. 1973; Nakamura and Winters, 1973; Sawyer, Critchlow and Barraclough. 1955). rabbit (Gangloff and Monnier. 1955: Longo. 1962; Silvestrini and Longo. 1956). dog (Wikler, 1952; Wikler and Altschul. 1950) and man (Andrews, 1943; Gibbs and Maltby, 1943). Similar changes in the EEG activity have also been reported following intraventricular administration of morphine in rabbit (Albus and Herz. 1972). In the present experiment. it was shown for the first time that intracerebral administration of morphine into the medial thalamus also leads to the appearance of cortical HVSA waves. Injections of morphine into the PAG and into brain regions bordering on the MT and the PAG produced only a slight slowing of the EEG or had no effect. Since an extensive mapping of brain loci for sensitivity to morphine’s actions on the ongoing EEG has not been performed. it is not known if the MT is solely responsible for the production of HVSA waves following systemic morphine administration. However. that other brain regions may also be involved in the production of synchronized slow waves following the systemic administration of morphine is indicated by the finding of Albus and Herz (1972) that EEG slowing appeared following morphine adminis-
YAKSH
and T. A. RUDY
tration limited to the 4th ventricle and part of the aqueduct of the rabbit. Due to the nature of the experimental technique employed by Albus and Herz (1972). it was not possible to determine exactly the locus of morphine’s action except that the periventricular structures posterior to the third ventricle were involved. In addition, the observation by Teitelbaum, Catravas and McFarland (1974) that lesioning of the MT resulted in the re-appearance of EEG slowing in morphine-tolerant rats upon systemic morphine challenge, tends to suggest the existence of brain regions other than the MT that are capable of mediating this action of morphine. Some of the possible candidates in this regard are the pontobulbar reticular formation, the septum, the hippocampus and the amygdala. All of these areas exhibit stereospecific narcotic binding (Kuhar rt nl., 1973) and electrical stimulation of these structures elicits EEG synchronization (Kreindler and Steriade. 1964; Moruzzi, 1960; Parmeggiani. 1962; Rosvold and Delago, 1956). The mechanism by which morphine in the MT may exert its effect on the ongoing EEG is at present unclear. Both anatomically and electrophysiologically. the MT has been shown to be reciprocally connected with widespread areas of the cerebral cortex, the neighbouring thalamic nuclei. the hypothalamus and the striatum. The brain stem reticular formation also sends fibres which terminate within the intralaminar nuclei of the thalamus (Kupp and Monnier, 1966; Scheibel and Scheibel. 1967). Electrical stimulation of the MT at low frequencies in the cat resulted in recruitment and synchronization of the EEG. whereas stimulation at high frequencies led to cortical desynchronization (Dempsey and Morison, 1942). Electrical stimulation of the mesencephalic reticular formation led to LVFA (Moruzzi. 1960; Moruzzi and Magoun. 1949). and also counteracted the synchronizing action of low-frequency electrical stimulation of the MT (Jasper, Naquet and King, 1955; Moruzzi and Magoun. 1949). It was hence suggested by Moruzzi (1961) that a high activity-level of the ascending reticular activating system desynchronizes the thalamic neurones and diminishes thereby the synchronizing influence of the unspecific thalamic system. In accordance with this hypothesis. it was shown that mesencephalic transection of the ascending reticular formation resulted in increased spindle activity in the cortical EEG (Brookhart. Arduini, Mancia and Moruzzi. 1957: Monnier, Kalberer and Krupp. 1960). and that unilateral lesioning of the mesencephalic reticular formation resulted in ipsilateral appearance of cortical HVSA waves (Watson, Heilman, Miller and King, 1974). Pharmacological studies at the cellular level in which morphine was iontophoretically applied to various brain regions and spinal dorsal horn neurones have shown that the predominant, naloxone-reversible effect was a depression of cell firing rate (Bramwell and Bradley, 1974; Cavillo, Henry and Neuman, 1974: Frederickson and Norris. 1976; Haigler. 1976). Based on the above-mentioned observations, it is pos-
Morphine action on analgesia and EEG tulated that morphine in the MT may exert its EEG synchronizing action by blocking the active desynchronizing input from the mesencephalic reticular formation. Alternatively. morphine may exert a direct inhibitory action on MT neurones. This would decrease the firing rate of the MT units and, hence, increase the chance of phase-locking of these units, leading to the observed synchronized slowing of the EEG. The significance of morphine’s EEG synchronizing effect with respect to the behaviourally expressed effects of systemically administered morphine is not known. In the present study, however, it was observed that morphine injected into the PAG or spinal subarachnoid space produced strong antinociceptive effects without concomitant EEG slowing and that injections in the MT induced EEG slowing but not analgesia. Thus, EEG slowing is neither required for the expression of morphine’s antinociceptive action as measured by the tests employed in this study nor is it an obligatory consequence of the activation of the neural substrate mediating this type of analgesia. A mild sedation and a decrease in the affective components (vocalization, defecation, “fear” and avoidance) were observed following the intracerebral administration of morphine into the MT. In man, morphine is anecdotally reported to reduce the affective component of the reaction to nain. It remains oossible. therefore. that morphine’s ‘medial thalamic action on the EEG may be related to the affective rather than to the receptive component of narcotic-induced antinociception. REFERENCES
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Ruda, M. A. (1975). Autoradiographic study of the efferent projections of the midbrain central gray of the cat. Ph.D. Dissertation, University of Pennsylvania. Sawyer. C. H., Critchlow, B. V. and Barraclough, C. A. (1955). Mechanism of blockade of pituitary activation in the rat by morphine, atropine and barbiturates. Endocrinology
51: 345-354.
Scheibel. M. E. and Scheibel. A. B. (1967). Structural organization of nonspecific thalamic nuclei and their projection toward cortex. Brain Res. 6: 6C94. Sharpe, L. G.. Garnett. J. E. and Cicero, T. J. (1974). Analgesia and hyperreactivity produced by intracranial microinjections of morphine into the periaqueductal gray matter of the rat. Behav. Biol. II: 303-314. Silvestrini. B. and Longo, V. G. (1956). Selective activity of morphine on the EEG arousal reaction to painful stimuli. Experientia 12: 436437. Starzl. T. E. and Magoun, H. W. (1951). Organization of the diffuse thalamic projection system. J. Neurophysiol. 14: 1333146. Teitelbaum, H.. Catravas. G. N. and McFarland. W. L. (1974). Reversal of morphine tolerance after medial thalamic lesions in the rat. Science 185: 449-451. Tsou. K. and Jang, C. S. (1965). Studies on the site of analgesic action of morphine by intracerebral microinjection. Scientia Sinica 13: 1099-I 109. Valdman, A. V. (1967). Pharmacology of the brain. Progr. Brain
Res. 20: l-92.
Watson. R. T.. Heilman, K. M.. Miller, B. D. and King. F. A. (1974). Neglect after mesencephalic reticular formation lesions. Neurology 24: 294-298. Wikler. A. and Altschul. S. (1950). Effects of methadone and morphine on the electroencephalogram of the dog. J. Pharmac.
exp. Ther.
98: 437446.
Wilker, A. (1952). Pharmacologic dissociation of behaviour and EEG “sleep patterns” in dogs: Morphine, N-allylnormorphine and atropine. Proc. Sot. exp. Biol. Med. 79: 26lI265. Wolf. G. (1971). Elementary histology for neuropsychologists. In: Methods in Psychobiology (Myers. RI D.. Ed.). Vol. I. DD. 281-300. Academic Press, New York. Yaksh, T.‘L. and Rudy. T. A. (1976). Chronic catheterization of the spinal subarachnoid space. Physiol. Behav. 17: 1031-1036. Yaksh. T. L.. Yeung, J. C. and Rudy, T. A. (1976). Systematic examination in the rat of brain sites sensitive to the direct application of morphine: Observation of differential effects within the periaqueductal gray. Brain Rrs. 114: 83-103.
of Pharmacy.
University (Accepted
of Wisconsin. 27 Octohrr
Madison.
WI 53706. U.S.A
1977)
Summary-Rats were chronically implanted with EEG electrodes and. either microinjection cannulae aimed at the medial thalamus (MT) and periaqueductal gray (PAG). or a spinal catheter. Morphine sulphate injected intraperitoneally produced both analgesia. as measured by the tail flick and pinch-agitation tests, and episodes of high-voltage slow-wave activity (HVSA) in the EEG. Morphine injected into the MT produced HVSA but not analgesia. Conversely, morphine injected into PAG sites or into the lumbar subarachnoid space produced analgesia but did not alter EEG activity. These data suggest that the MT may mediate the EEG alterations produced by systemically administered morphine. They also indicate that analgesia (as measured by the tests employed) and HVSA are entirely independent actions of morphine mediated by different neuroanatomical substrates. The functional significance of the cortical HVSA produced by morphine and its mechanism of production are discussed.
In several animal species, it has been demonstrated that the systemic administration of morphine produces, not only analgesia, but also a concomitant synchronization of the EEG (Bradley, 1968; Domino, 1968; Killam, 1962; Valdman, 1967). Whereas the brain sites subserving morphine’s antinociceptive action have been extensively studied (Criswell, 1976; Jacquet and Lajtha, 1973. 1974; Pert and Yaksh, 1974, 1975; Sharpe, Garnett and Cicero, 1974; Tsou and Jang, 1965; Yaksh, Yeung and Rudy, 1976). the neuroanatomical substrate of the EEG synchronizing action of morphine remains largely undetermined. There exists excellent evidence that the periaqueductal gray (PAG), particularly its caudal aspect, is the major supraspinal structure mediating the analgetic action of narcotics (Jacquet and Lajtha, 1974; Sharpe et al., 1974; Yaksh et al., 1976). Although the possibility that the caudal PAG may also subserve morphine’s EEG synchronizing action has not been examined directly, Albus and Herz (1972) have reported that profound synchronization could be elicited in the rabbit by injection of morphine into the fourth ventricle after occlusion of the aqueduct at its rostra1 end. This finding and the extensive anatomical connections between the PAG and other brain regions which potently influence cortical activity, such as the bulbar reticular formation and the midline and intralaminar thalamic nuclei (Chi, 1970; Hamilton, 1973; Hamilton and Skultety, 1970; Ruda, 1975), suggest that the PAG may be involved in the genesis of narcoticinduced EEG synchronization. Key words: morphine. thalamus. periaqueductal
EEG.
antinociception.
Although Albus and Herz (1972) observed that morphine injected intraventricularly in such a manner as to limit its diffusion to the fourth ventricle and aqueduct could elicit typical synchronized cortical waves of high voltage and slow activity (HVSA). they found that morphine injections limited to the ventricular spaces rostra1 to the aqueductal mouth could also elicit HVSA. Thus, it would seem that there must exist a second substrate for the EEG synchronizing action of narcotics. It is well known that the medial thalamus (MT), an extension of the ascending reticular system, plays an important role in the control of ongoing cortical activity and the state of wakefulness (Krupp and Monnier, 1966; Moruzzi, 1972). Low-frequency electrical stimulation of certain MT nuclei in a variety of species has been associated with the evocation of synchronized HVSA cortical waves (Dempsey and Morison, 1942; Jasper, 1949; Morison and Dempsey. 1942; Starzl and Magoun, 1951). Destruction of the MT has led to a marked reduction in both the frequency and amplitude of the observed cortical activity, or to cortical activity that showed poorly organized recruiting responses and poorly organized spindles (Kennard, 1943; Knott, Ingram and Chiles, 1955). These observations, in conjunction with the recent finding that the MT exhibited stereospecific narcotic binding (Kuhar, Pert and Snyder, 1973; Pert, Kuhar and Snyder, 1975). suggested that the MT might be the rostra1 periventricular structure mediating the HVSA observed by Albus and Herz (1972). In the present study, therefore, the putative contributions of both PAG and MT to narcoticinduced HVSA were evaluated.
medial
gray.
525
J.
526
C. YEUNG, T.
L.
METHODS
Under sodium pentobarbital anesthesia (35 mg/kg. i.p.). each of 9 Holtzman male albino rats was prepared under stereotaxic control with bipolar EEG recording electrodes and an array of microinjection guide cannulae. The EEG electrodes were made of platinum tubing (27 g) and were implanted epidurally above the frontal cortex. The poles of the recording electrodes were 7 mm apart, and each pole rested on one side of the cortex. The microinjection guide cannulae (24 g T.W. stainless-steel) were aimed at the MT (AP: 3.8-3.2. L: l&0.0: HV: 0.54.0) and the PAG (AP: 1.2--0.8: L: 1.0-0.0; HV: 0.5-0.0). The stereotaxic co-ordinates were taken from the atlas of Pellegrino and Cushman (1967). In 3 rats. one cannula was implanted in the MT (in the midline) and two in the PAG. In the remaining 6 rats. two cannulae were implanted in the MT (bilaterally symmetrical, 0.5-1.0 mm off midline) and one was placed in the PAG. Following a 7-day recovery period, experiments were begun in which morphine was injected intracerebrally at sites in the PAG and MT and the effects on the nocicep&e threshold and EEG activity were observed. Each intracerebral injection consisted of 0.5 ~1 of either 0.9% saline or morphine sulphate dissolved in normal saline (10 pg/pl) delivered through a 29 g injector over a period of 20 sec. Initially, the injection was made 1.0 mm below the guide tip. When all experiments at this depth of penetration were completed. deeper sites (separated by 1 mm) were explored sequentially. Successive injections made at the same brain locus were separated by a period of at least 7 days. and each brain site was not injected more than twice with morphine. To facilitate brain mapping for morphine sensitivity in rats with bilateral MT cannulae. bilateral injections (IO pg/pl) were made. In some cases. when an effect on either the EEG or the nociceptive threshold was observed, unilateral injections were made on separate days and the effects re-evaluated. The effects of systemically injected morphine sulphate (10 and 30 mg/kg, i.p.) were examined in all animals. During the experimental sessions. each rat was loosely restrained in a hemi-cylindrical wire mesh cage with the tail protruding through a hole in one end. The tail-flick latency and the agitation response to skin pinches applied with forceps were checked before (0 min) and at 5. 20. 40 and 60 min following the intracerebral injection of morphine. In the pinchagitation test. forceps pinch was applied to the animal’s face and four limbs in a random fashion. The response was recorded as positive (presence of signs of agitation and/or withdrawal) or negative (absence of signs of agitation and withdrawal). In the tail flick test, the animal’s tail was laid in a groove perpendicular to a length of electrically heated nichrome wire located 1 cm below the tail. A cut-off time of 15 set was employed. After each assessment of the pinch agitation and tail-flick responses. a 2-min epoch of EEG
YAKSH and
T. A.
RUDY
activity was also taken, using a Grass EEG pre-amplifier (model 7P5B) and a Grass polygraph d.c. driver amplifier. Preliminary studies have shown that the EEG taken before the pain testing was similar to that taken after the testing. Whenever antinociception and/or a change in EEG (see below) was observed following morphine administration, the pharmacological specificity of the effect was ascertained by the intraperitoneal injection of naloxone hydrochloride (1 mg/kg). Naloxone was given 65 min following morphine administration, and measurements were again made 7 min after the naloxone injection. In a few experiments, 0.9% saline, instead of morphine or naloxone, was injected centrally (0.5 ~1 per site) or intraperitoneally (1 ml/kg) to rule out further a possible nonspecific effect of the procedures involved. To quantify the changes in EEG activity, the percentage of each 2-min epoch during which the rat displayed high voltage and slow activity (percentage HVSA) was measured. The HVSA was easily distinguishable and was defined for analysis purposes as those periods when the recorded amplitude exceeded twice that of the low-voltage fast activity (LVFA) observed before injection. At the end of the experimental series, each rat was sacrificed with an overdose of urethane and its brain perfused with 0.9% saline followed by 10% formalin. The brain was impregnated with gelatin as described by Wolf (1971) and was then sectioned on a freezing microtome. Sections were stained with cresyl fast violet. All brain injection sites were identified by microscopical examination and projected onto plates obtained from the atlas of Pellegrino and Cushman (1967). Three additional rats were implanted with bipolar frontal electrodes and subarachnoid spinal catheters with tip openings lying in the vicinity of the lumbar enlargement of the spinal cord as described by Yaksh and Rudy (1976). Following a ‘J-day recovery period, 5~11 of a solution of morphine sulphate dissolved in artificial CSF (IO pg/pl) was injected slowly over a period of 40sec. The same testing procedures employed in measuring the effects following intracerebra1 injections of morphine were employed in these studies. At the end of the experimental sessions. each animal was sacrificed with an overdose of urethane, and the location of the catheter tip opening in the spinal cord was verified by gross dissection. To compare the differences between means, Student’s t-test was employed. For the multiple comparisons of means observed at various times following treatment to that observed before treatment, a oneway analysis of variance was performed and the Newman-Keul’s test employed. The means are defined to be significantly different if P < 0.01 (2-tailed). RESULTS
During the experimental sessions, rats that received saline injections (intracerebral or systemic) generally
527
Morphine action on analgesia and EEG remained in a state of arousal associated with a desynchronized EEG characterized by 20-27 Hz LVFA waves. Intraperitoneai injection of morphine (10 or 3Omg/kg, i.p.f produced a dose-dependent behavioural antinociception and sedation associated with EEG slowing and synchronization. Episodes of HVSA (6-14 Hz) were observed to be interspersed among the LVFA waves. An example of morphine’s action (30 mg/kg, i.p.) on ongoing EEG and the noci-
ceptive threshold and its reversal by naloxone is illustrated in Figure I(a). Intracerebral injections of morphine into the MT in doses of 5 pg/OS ~1 unilaterally, or IO~g/~fl total if the injections were bilateral, evoked a marked slowing of the EEG with a concomitant increase in amplitude. Concurrently, the behaviour of the animal was observed to be mildly depressed and there was a marked decrement in the affective component of the
30 mg/kg i.p.
‘rl
110
lc L
Ic
‘rl ic 0 min
lo
lc
-L_- ,1. 20 min
60 min
10
Kc3
AP - 0.8
10 NAL
0. I mV L 5sec 3.1 mV I
I set
Fig. 1. Examples of the effects of morphine administered intraperitoneally (a), into the medial fhalamus (b). into the p~riaqu~ductal gray (c) and intrathecalfy (d) on EEG activity. tail-flick latency and the pinch-agitation response. In all panels. upper two tracings represent EEG epochs recorded at slow and fast speeds, respectively (see time/voltage scales at bottom right). Solid bars indicate tail-flick latencies. Letters beside each bar denote the distribution of body surfaces rendered non-responsive to forceps pinch: 0 = no effect. B = hindpaws and forepaws rendered non-responsive. C = hindpaws. forepaws and face rendered non-responsive. Histological plates at right illustrate the intracerebral injection sites (dose = 5pg in 0.5~1 in both b and c). Abscissa indicates the times after injection when the measurements were taken: NAL denotes measurements made 7 min after the injection of naloxone (I mgikg i.p.). The nafoxone was injected 65 mm after morphine administration.
J. C. YEUNG.T. L.
528
reaction to application of the forceps pinch (vocalization, defecation, urination and “fear”). The animal could still be easily aroused with non-noxious stimuli such as light, touch and loud clicking sounds. This effect of focally applied morphine in the MT on the EEG, however, was not accompanied by any degree of antinociception as measured by the tail flick and pinch-agitation tests. No discernible differences in the changes in ongoing EEG activity following systemic morphine or intracerebrally applied morphine into the MT were observed. An example of the effects obtained following the focal administration of mor50
r
0t
50
IO mg/kg
YAKSH
phine (5~g/OS~l) into a brain locus within the MT is shown in Figure I(b). The location of the brain locus is indicated in the corresponding coronal section. As with systemically applied morphine, naloxone (1 mg/kg. i.p.) completely antagonized the EEG synchronizing action of morphine within the MT. Further evidence that the synchronizing effect was not due to a non-specific action of the microinjection is suggested by the failure of microinjected saline (0.5 ~1 per site) into the MT to cause significant changes on the EEG. The mean + S.E.M. of the maximal changes in the percentage HVSA was 3.5 + 2.6 (n = 6) follow-
* I
nzf3
and T. A. RUDY
f
1’”
hid,,
% r MT
1'"
n= 14
5
loof-
MT
1
IO+g
I
5
I
I
40
20 Time,
I
60
^
I
72
IllI”
Fig. 2. Summary of the differential effects on HVSA and tail-flick latency of morphine injected intraperitoneally and into the medial thalamus, periaqueductal gray and spinal subarchnoid space. First two panels: effect of injection of 10 and 30mg/kg (i.p.), respectively. Third and fourth panels: effect of unilateral and bilateral injection, respectively, of 5 pg into the medial thalamus (MT). Fifth panel: effect of injection of 5pg into the periaqueductal gray (PAG). Bottom panel: effect of intrathecal injection of 5Opg. Bar lengths indicate mean values and vertical lines, S.E.M. Asterisks denote values statistically significant at the 0.01 level. Solid bars represent the differences in the percentage of time spent in HVSA during 2-min EEG epochs recorded at various times after morphine injection as compared to a control (pre-injection) epoch. Hatched bars represent the change in tail-flick latency at each post-injection interval in comparison to pre-injection control value. Abscissa values indicate the times after morphine injection at which the data were recorded. The 72-min value represents data readings taken 7 min after injection of naloxone (1 mg/kg, i.p.) at the arrow. n = number of animals or number of injection sites included in the means (see text for details).
529
Morphine action on analgesia and EEG ing the microinjection of saline and was not significantly different from that observed before injection (P > 0.2). In contrast to the loci located in the MT, morphine injected into certain sites within the PAG resulted in antinociception but was without effect on the ongoing LVFA of the EEG. In Figure I(c) are shown the effects on the ongoing EEG activity. tail-flick latency and the pinch-agitation response of an intracerebral injection of morphine (5 /rg in 0.5 ~1) into a morphinesensitive site located within the PAG. The location of the microinjection site is indicated in the adjacent coronal section. Naloxone reversibility of the effects observed is also illustrated. The effects of an intrathecal injection of morphine (50 pig in 5 ~1) on tail-flick latency, the pinch-agitation
APt3.6
AP+ 1.0
response and ongoing EEG activity are shown in Figure I(d). The injection was effective in blocking the tail-Rick response and the pinch-agitation response of the four limbs but not the face. No change in the EEG. however. was discernible following such an injection. Figure 2 summarizes the time-effect relationships and pharmacological specificity of the EEG synchronizing and antinociceptive (tail-flick) actions of morphine administered intraperitoneally and into the MT, PAG and spinal subarachnoid space. The mean values illustrated for intraperitoneal and intrathecal injections are derived from all rats injected by these routes. The mean values for MT and PAG injections include only injections made into active sites (sites wherein microinjection clearly altered tail-flick
AP+3.4
AP-0.6
Fig. 3. Frontal sections of the rat brain (Pellegrino and Cushman atlas) indicating the approximate positions of morphine injection sites and the EEG and antinociception responses evoked. Top: Injection sites in or near the MT. Asterisks indicate sites where morphine injections elicited increase HVSA without affecting tail-flick latency. Open circles denote sites where morphine injection caused no change in either parameter. Bottom: Injection sites in or near the PAC. Solid circles indicate sites where morphine injections produced antinociception as measured by the tail-flick test and no increase in HVSA. Open circles denote sites where morphine injection caused no change in either parameter.
J.
530
C.
YEUNG.
T. L.
latency or EEG activity). As can be seen from the Figure. both the EEG and antinociceptive actions of morphine injected by all routes developed smoothly over time, became statistically significant at 2&40min after injection, and were completely reversed by naloxone. Naloxone (1 mg/kg, i.p.) alone was not found to affect the EEG and nociceptive threshold of the animal. The Figure also clearly indicates the selective actions of morphine on the EEG and tail-flick latency when injected into the MT and into the PAG or spinal subarachnoid space. respectively. The approximate anatomical locations of the microinjection sites yielding these selective actions on the EEG and tail-flick latency are presented in Figure 3. Brain sites studied but which did not mediate either of these two actions of morphine are also shown in this Figure. The number of inactive vs active sites in either the MT or the PAG are too few to delineate the borders of the responsive tissues. However, in the PAC. the distributions of active and inactive loci (as shown in Fig. 3) are in agreement with a previous study (Yaksh c’t al.. 1976) in which it was found that the PAG region of greatest morphine sensitivity was its ventral posterior aspect. The overlapping of positive and negative sites in the MT indicates that this structure, like the PAG. is not uniformly sensitive to locally injected morphine. DISCUSSION
The appearance of synchronized EEG slow waves following systemic morphine administration has been observed in a variety of species including rat (Cahen and Wikler. 1944; Colasanti and Khazan. 1973; Nakamura and Winters, 1973; Sawyer, Critchlow and Barraclough. 1955). rabbit (Gangloff and Monnier. 1955: Longo. 1962; Silvestrini and Longo. 1956). dog (Wikler, 1952; Wikler and Altschul. 1950) and man (Andrews, 1943; Gibbs and Maltby, 1943). Similar changes in the EEG activity have also been reported following intraventricular administration of morphine in rabbit (Albus and Herz. 1972). In the present experiment. it was shown for the first time that intracerebral administration of morphine into the medial thalamus also leads to the appearance of cortical HVSA waves. Injections of morphine into the PAG and into brain regions bordering on the MT and the PAG produced only a slight slowing of the EEG or had no effect. Since an extensive mapping of brain loci for sensitivity to morphine’s actions on the ongoing EEG has not been performed. it is not known if the MT is solely responsible for the production of HVSA waves following systemic morphine administration. However. that other brain regions may also be involved in the production of synchronized slow waves following the systemic administration of morphine is indicated by the finding of Albus and Herz (1972) that EEG slowing appeared following morphine adminis-
YAKSH
and T. A. RUDY
tration limited to the 4th ventricle and part of the aqueduct of the rabbit. Due to the nature of the experimental technique employed by Albus and Herz (1972). it was not possible to determine exactly the locus of morphine’s action except that the periventricular structures posterior to the third ventricle were involved. In addition, the observation by Teitelbaum, Catravas and McFarland (1974) that lesioning of the MT resulted in the re-appearance of EEG slowing in morphine-tolerant rats upon systemic morphine challenge, tends to suggest the existence of brain regions other than the MT that are capable of mediating this action of morphine. Some of the possible candidates in this regard are the pontobulbar reticular formation, the septum, the hippocampus and the amygdala. All of these areas exhibit stereospecific narcotic binding (Kuhar rt nl., 1973) and electrical stimulation of these structures elicits EEG synchronization (Kreindler and Steriade. 1964; Moruzzi, 1960; Parmeggiani. 1962; Rosvold and Delago, 1956). The mechanism by which morphine in the MT may exert its effect on the ongoing EEG is at present unclear. Both anatomically and electrophysiologically. the MT has been shown to be reciprocally connected with widespread areas of the cerebral cortex, the neighbouring thalamic nuclei. the hypothalamus and the striatum. The brain stem reticular formation also sends fibres which terminate within the intralaminar nuclei of the thalamus (Kupp and Monnier, 1966; Scheibel and Scheibel. 1967). Electrical stimulation of the MT at low frequencies in the cat resulted in recruitment and synchronization of the EEG. whereas stimulation at high frequencies led to cortical desynchronization (Dempsey and Morison, 1942). Electrical stimulation of the mesencephalic reticular formation led to LVFA (Moruzzi. 1960; Moruzzi and Magoun. 1949). and also counteracted the synchronizing action of low-frequency electrical stimulation of the MT (Jasper, Naquet and King, 1955; Moruzzi and Magoun. 1949). It was hence suggested by Moruzzi (1961) that a high activity-level of the ascending reticular activating system desynchronizes the thalamic neurones and diminishes thereby the synchronizing influence of the unspecific thalamic system. In accordance with this hypothesis. it was shown that mesencephalic transection of the ascending reticular formation resulted in increased spindle activity in the cortical EEG (Brookhart. Arduini, Mancia and Moruzzi. 1957: Monnier, Kalberer and Krupp. 1960). and that unilateral lesioning of the mesencephalic reticular formation resulted in ipsilateral appearance of cortical HVSA waves (Watson, Heilman, Miller and King, 1974). Pharmacological studies at the cellular level in which morphine was iontophoretically applied to various brain regions and spinal dorsal horn neurones have shown that the predominant, naloxone-reversible effect was a depression of cell firing rate (Bramwell and Bradley, 1974; Cavillo, Henry and Neuman, 1974: Frederickson and Norris. 1976; Haigler. 1976). Based on the above-mentioned observations, it is pos-
Morphine action on analgesia and EEG tulated that morphine in the MT may exert its EEG synchronizing action by blocking the active desynchronizing input from the mesencephalic reticular formation. Alternatively. morphine may exert a direct inhibitory action on MT neurones. This would decrease the firing rate of the MT units and, hence, increase the chance of phase-locking of these units, leading to the observed synchronized slowing of the EEG. The significance of morphine’s EEG synchronizing effect with respect to the behaviourally expressed effects of systemically administered morphine is not known. In the present study, however, it was observed that morphine injected into the PAG or spinal subarachnoid space produced strong antinociceptive effects without concomitant EEG slowing and that injections in the MT induced EEG slowing but not analgesia. Thus, EEG slowing is neither required for the expression of morphine’s antinociceptive action as measured by the tests employed in this study nor is it an obligatory consequence of the activation of the neural substrate mediating this type of analgesia. A mild sedation and a decrease in the affective components (vocalization, defecation, “fear” and avoidance) were observed following the intracerebral administration of morphine into the MT. In man, morphine is anecdotally reported to reduce the affective component of the reaction to nain. It remains oossible. therefore. that morphine’s ‘medial thalamic action on the EEG may be related to the affective rather than to the receptive component of narcotic-induced antinociception. REFERENCES
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