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The periaqueductal gray: An examination of the distribution of opioid and non-opioid sites, their interaction, and the role of serotonin
Nichols, Deborah Sue, Ph.D.
The Ohio State University, 1987
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University Microfilms International rot PEki AuuEDUui an ukAi : AN EXACti NAT I UN ut THE UiSTRIBuiiuu ur
ueiuiD AND NuN-OPI01D SITES. THE Ik INTEkAo HuN ,
AND THE ROLE OF SEROTONIN
DISSERTATION
rresen teu in Par tia i t u t riiimen t or r ne kequirementa tut
the Decree Doctor of Pniiosopny in the uraauate
Scnoot of tne Ohio State University
By
ueoorah Sue Nicnois, B.S., M.Ea.
ihe Ohio State U n iversity
1967
u i sser t ation Oomfni t tee ; Approvea Dy b . n. i nor n
U.o . ner n t son
Aov I ' s d r
..) . i.. nr esriahan uepar t me tit at rsvtnoiuu1. To The Three Men
I Love,
L.T*, John, &> Mark ACKNOWLEDGEMENTS
I woula line to express my sincere appreciation to Dr. Beverly
Thorn for her support, enthusiasm, ana guidance throughout this project. A second cnank-you to Dr. Gary Berntson for his advice and
assitance tnroughout this project. Thanks also go to Drs. Bresnanan,
Bruno, and Stokes for their helpful comments and encouragement.
Gratitude is expressed to Orlando Mullins, for his technical assistance, Kathy Moreno, for her clerical assistance, ana Julia Wans
and Gary Griggs, for their research assistance. To my husband, L.T.,
I offer firs t my love and then my thanks for his support, love, ana
that gentle push when I needed it most (an additional thank-you tor
the Apple 1IC on whicn this has all been w ritten.) To my sons, Jonn and Mark, I thank you for those times you played quietly so i could work, your help with the rats, and your understanding when things got hectic*, I love you both. November, 8, 1955 Born - Dayton, Ohio
1978 ...... B.S. , The Ohio State University, Columbus, Ohio
1982 M.Ed., University of Toledo, To Iedo, Oh i o
1983-Present ...... Graduate Teaching Assistant The Ohio State University Columbus, Ohio
FIELDS OF STUDY
Major Field: Psychobiology
Studies in: Psychophysiology of Mental Retardation and Developmental D isabilities, Gary G. Berntson
Pain-medlating Systems, Beverly E. Thorn TABLE OF CONTENTS
ACKNOWLEDGEMENTS...... in
VITA...... iv
LIST OF TABLES...... vi
LI ST OF FIGURES...... vii
INTRODUCTION...... 1
METHODOLOGY...... 29
RESULTS...... 36
DISCUSSION...... S3
BIBLIOGRAPHY...... 64
v LIST OF TABLES
TABLE PAGE
1. Stimulation effects at dorsal ...... s ite s ...... 38
2. Stimulation effects at ventral sites ...... 39
3. Distribution of effects at anterior-posterior axes for each quadrant ...... 40
4. Results of regression on site specificity for mean difference scores ...... 44
5. Results of regression on site specificity for duration...... 44
6. Results of regression on site specificity for i ntensi ty ...... 44
vi LIST OF FIGURES
FIGURES PAGE
1. Stimulation effects plotted on rostral-caudal midbrain sections ...... 42
2. Effect of naloxone on stimu I at ion-produced analgesia plotted on rostral-caudal sections ...... 46
3. Effects of naloxone at dorsal and ventral sites ...... 47
4. Cross-tolerance effects at naloxone-reversible and naioxone-non-reversible sites ...... 49
5. Effect pf stimulation-produced analgesia, plotted on rostral-caudal sections ...... 51
6. Effect of methysergide at dorsal and ventral sites...... 52
vli INTRODUCTION
Stimulation of the midbrain periaqueductal gray (PAG) has been
reported to e lic it stimuI at ion-produced analgesia (SPA) in humans
(Hosobuchi, Adams, & Linchltz, 1977; Richardson & Akll, 1977; &
Richardson, 1962) and other animals (Reynolds, 1966; Mayer, Wolfe,
Akii, Carder, Uebesklnd, 1971; Balagura A Ralph, 1973). Reynolds
(1966) found this analgesia sufficient to allow an experimental
laparotomy to be done without need of any chemical anesthetic.
Although many areas within the centra) nervous system were found to
produce SPA in the rat (le . septal nuclei, dorsomedlal thalamic
nucleus, and ventral tegmentum) only stimulation of the mesencephalic
central gray matter and periventricular gray matter was found to
greatly reduce or totally abolish the responsiveness to all noxious
stimuli employed, including radiant heat applied to the tail. This
analgesia was reported to be equal to or greater than 10 mg/kg morphine on alI tests (Mayer & Liebesklnd, 1974). Furthermore, this
analgesic effect did not appear secondary to general sensory,
emotional, or motor mechanisms (Mayer et a l., 1971).
Neuroanatomical Pathways Mediating Pain;
Small fibers entering the dorsal root of the spinal cord are
responsible for the transmission of nociceptive (pain) information;
1 2
these fibers enter the medial aspect of the tract of Lissauer, sending collaterals to both the substantia gelatinosa and the marginal zone, as well as laminae IV and V (Kerr, 1975). Cells responding to nociceptive input are located In laminae I, I I , IV, V, & VI, with the substantia gelatinosa serving as an apparent relay station for the projection of peripheral fibers to these terminal zones (Fields &
Basbaum, 1978). Transmission of nociceptive information to higher
levels within the central nervous system is by way of multiple ascending pathways: the anterolateral (spinothalamic) system, the spinocervical system, the spinoreticular system, and a dorsal column system (Kerr, 1975; Fields & Basbaum, 1978).
The anterolateral system Is the classical pain pathway, lesions of which result In loss of pain contralateral and caudal to the lesion
(Spiller & Martin, 1921). This system originates from cells in laminae 1, IV, V, and VI (Fields & Basbaum, 1976), ascends within the anterolateral quadrant of the spinal cord, and terminates In the intralaminar, posterior, and ventrobasa! thalamus (Basbaum, Giesler. &
Nenetrey, 1977). The spinocervical system originates from laminae IV and V (Bryan, Coulter, & W illis, 1974), ascends in the dorsolateral funiculus (Fields & Basbaum, 1976), and crosses In the medulla together with the medial lemniscus (Kerr, 1975). The spinoreticular pathway is a major subcomponent of the anterolateral quadrant of the spinal cord (Fields & Basbaum, 1978) with cells originating from laminae 1, IV, and V (Fields, Clanton, & Anderson, 1977). It projects to the media) brainstem reticular formation, synapses, and then projects to the medial thalamus and hypothalamus (Fields & Basbaum, 3
1978). According to Fields & Basbaum (1978), this system has been
proposed to mediate the avers!ve-rootlvationai aspects of pain
perception. Although the dorsal cblumn-medlal lemnlscal system is not
associated with pain transmission, ceils within laminae IV and V that
respond to nociceptive input have been found to project through the
dorsal columns demonstrated to project to the thalamus and therefore may have a modulatory function in pain transmission (Fields & Basbaum, 1978). Fields and Basbaum (1978) also discuss three bulbospinal systems that display an Inhibitory function on nociceptive transmission; a ventral reticulospinal pathway, a monoamlnerglc raphe-spinal system, and a dorsal reticulospinal pathway. The ventral reticulospinal pathway arises from the dorsal nucleus re tic u la ris gigantocellularis and descends in the ventral spinal cord to terminate in laminae VII and V III. The monoaminergic system originates in the medullary raphe nuclei, and the dorsal reticulospinal system originates in the juxta- raphe medulla; both descend in the dorsolateral funiculus of the spinal cord with terminal fields In laminae I, I I , V, VI, and V II. Primary afferent volleys can be diminished by stimulation within the brainstem reticular formation or the pericentral, second somatic, and clngulate cortex (Kerr, 1975). It has been suggested that descending serotonergic neurons terminate on enkephalinergic neurons In the dorsal horn, which in turn may mediate presynapttc inhibition by way of opiate receptors on dorsal root afferent terminals (W illis , 1981). Furthermore, primary afferent depolarization and long latency dorsal root potentials have 4 been obtained following stimulation of the nucleus raphe roagnus, again suggesting a presynaptic action (Besson et a l., 1981). However, a postsynaptic site of action is also possible, since media) brain stem stimulation has been found to produce Inhibitory postsynaptic potentials in some dorsal horn neurons (Besson et a )., 1981). The PAG's antinociceptive action, therefore, may be produced by activation of one of these descending inhibitory systems with either a presyn- aptic or postsynaptic site of action. The interaction of ascending and descending pain-mediating systems with the PAG w ill be addressed in the subsequent discussion. Afferent and Efferent Projections for the PAG: The periaqueductal gray surrounds the cerebral acqueduct as it courses through the midbrain. Hamilton (1973a) described three nuclear regions within the PAG In the cat: the medial subnucleus, which surrounds the aqueduct and flares outward ventral Iy in a bell shape; the lateral subnucleus which constitutes the dorsolateral region; and the dorsal subnucleus which occupies the dorsal region above the medial subnucleus and between the two lateral subnuclei. These nuclear regions were found to differ in their projections. However, Ruda (1976) found that based on efferent projections of the PAG in the cat only two nuclear regions could be Identified (dorsal and la te ral). According to Hamilton (1973b>, the nucleus medial is was found to send projections to the ventral tespnentum, the nucleus dorsalis to the habenular nuclei, and the nucleus la te ralis to the tectum, tegpientum, periventricular nuclei of thalamus, dorsomedial and 5 ventrobasal thalamic nuclei, and posterior hypothalamus. Other researchers have found that long axons originating from all areas of the PAG project out of this region in all directions to interact with multiple areas of the forebratn and brainstem, encom passing and expanding those identified by Hamilton <1973b). Primary targets for ascending projections are the hypothalamus, thalamus, and other limbic system structures. Descending projections primarily terminate in the medullary tegmentum and the nucleus raphe magnus. Projections from the dorsal region of the PAG have been found to terminate within the periventricular nucleus of the thalamus, the periventricular gray matter of the third ventricle, and the dorsal hypothalamus (Chi, 1970), the parafasicularis, posterior and paraventricular hypothalamus, and the mesencephalic, pontine, and medullary reticular formation (Ruda, 1975), as welt as to midline and 1ntra1 aminar thalmic nuclei (Eberhart, Morrell, Krleger, & Pfaff, 1985). In addition, numerous projections from this dorsal region terminate in the nucleus raphe magnus (Fardin & Oliveras, 1982). The ventral region extends projections through the ventral tegroentum and medial forebrain bundle to the hypothalamus and preoptic area, caudoputamen, substantia innominate, s tria terminal is and amygdala. A second rostral projection is found in the diagonal band of Rroca with terminal fields in the medial septum, lateral septum, nucleus accumbens, olfactory tubercle, and frontal cortex (Eberhart et al, 1985). The rostral central gray also has projections to the thalamus, anterior hypothalamic nuclei, and the anterior periventric ular nuclei (Chi, 1970). Many of these projections originate in 6 catechol aminergic cell groups within the ventral PAG that contribute to the dorsal periventricular bundle (DPB), which projects rostrally and bifurcates into two branches. The DPB is a rostrally projecting component of the dorsal longitudinal fasciculus of Schutz (Watson, Akil, Barchas, 1977). One branch of the DPB projects to the pretectal area, habenula, and paraventricular and parafascicular nuclei of the thalamus. The second branch projects to the dorsamedial hypothalamus (Watson et a l ., 1977). The PAG does not have a major projection to the spinal cord (Basbaum & Fields, 1978) but has been found to have numerous projections to brainstem areas Involved in pain mediation, including the spinal trigeminal, raphe magnus, gigantocelluIar pars alpha and paragigantocellular nuclei (Basbaum & Fields, 1978; Belts, Mullett, & Weiner, 1983). As can be seen from this description, the PAG has multiple pathways to the hypothalamic nuclei, thalamic nuclei, and medullary nuclei both in and adjacent to the reticular formation. These same areas, as well as others, have been found to send reciprocal projections back to the PAG. Projections to the PAG originate from the raphe nuclei (Brodal, Taber, & Walberg, 1960), especially the dorsal raphe (Conrad, Leonard, & Pfaff, 1974), the prefrontal cortex (Hardy, 1985), the mediobasal arcuate hypothalamus (Bloom, Battenberg, Rossler, Ling, & Guileman, 1978) as well as the superior colliculus, nucleus cuneiformis, externa) nucleus of the inferior colliculus, locus coeruleus, and parabrachlai nuclei (Mantyr, 1982). Although the anterolateral system has been found to send sparse projections to the PAG (Zemlan, Leonard, Kow, & Pfaff, 1978), nociceptive information may be transmitted to the PAG via direct 7 projections from the nucleus reticularis glgantocelIularis, nucleus reticularis paragigantoceI Iu 1 ar 1 s, and nucleus reticularis magno- celIularis (Mantyr, 1982), which have terminal fields from the spinoreticular pathway. These reticular formation nuclei also project to the raphe nuclei (Gallegher & Pert, 1978), which in turn project, as stated earlier, to the PAG. Therefore, the PAG receives input from multiple areas associated with pain mediation (the raphe nuclei, the medullary reticular nuclei, and the anterolateral system). Neurochemical Systems: The neurochemical nature of these systems projecting to and from the PAG is complex. In addition to the ventrally located catechol amine cells that contribute to the dorsal periventricular bundle, serotonergic cells have been Identified in the ventral PAG, extending laterally from mldllne (Yezierski, Bowker, Kevetter, Westlund, Coulter, 8. W illis, 1982). Furthermore, endogenous opioid peptides have also been identified in the PAG (Pert, Kuhar, & Snyder, 1976; Field & Basbaum, 1978; Watson, Akil, Khachaturian, Young, & Lewis, 1984) throughout Its interior. At present, three families of opioid peptides have been identi fied each deriving from cleavage of a protein precursor) pro-oplomeI- anocortin (POMC), prodynorphln, and proenkephalin (Hughes, 1984). From these precursors are derived at least five opioid peptides; from POMC, beta-endorphin; from prodynorphln, dynorphin and alpha- neoendorphln; and from proenkephalin, met-enkephal1n and leu- enkephalin (Berger, Akil, Watson, & Barchas, 1982). POMC is also the precursor for beta-1ipotropin and ACTH, both of which are synthesized with beta endorphin in the anterior lobe of the pituitary; beta- endorphin and beta-1ipotropin are also synthesized in the intermediate lobe of the pituitary 'Berger et a )., 1982). POMC neurons have been identified in the basal and tuberobasal hypothalamic region (Bloom et a l . t 1978) as well as the arcuate nucleus (Bloom et a l . t 1978; Watson et a l., 1984). Rostral POMC projections from these hypothalamic areas have been traced to the periventricular hypothalmus and preoptic sites, s tria terminal is, septum, nucleus accumbens and other forebrain structures (Watson et a l., 1984). Lateral projections terminate in the amygdala (Watson et al., 1984). A final periventricular projec tion extends to the colli cull, PAG, dorsal raphe, trigeminal nucleus, locus coeruleus and periventricular regions of the pons and medulla ( Watson et a l., 1984). These projectlonal areas are similar to those of the PAG and are also areas from which the PAG receives projections. Enkephalin-containing neuronal circuits have been identified in the medullary projections to the spinal cord and multiple areas of the limbic system, including amygdaloid efferents, hypohtalamlc-pituitary system, hypothalamus, entorhinaI-hippocampal system, cerebral cortex, olfactory nucleus and tubercle ( Watson et a I., 1984). Additional circuits have been identified in areas previously discussed in re l ation to pain mediation: PAG, nucleus raphe magnus, locus coeruleus, nucleus reticularis glgantocelIularis, nucleus reticularis paragigantocel)uIaris, dorsal horn, and spinal trigeminal nucleus (Watson et a l., 1984). DynorphIn-11ke imnunoreactivlty has been identified in the posterior pituitary and in the neurosecretory magnocellular nuclei of the hypothalamus; these cells also synthesize oxytocin and vasopressin (Watson, Akil, Flschll, Goldstein, Zimmerman, & Nil aver, 1981). Happing of dynorphln peptides loci often parallels enkephalin localization; dynorphin peptides have been identified in the caudate-putamen, hypothalamus, PAG, dorsal raphe, nucleus raphe magnus, nucIeus glgantoceIluI arts, nucleus paraglgantocelIularis, spinal trigeminal nucleus, and dorsal horn (Watson et a )., 1984). As is evident from the above description, endogenous oploid-contalnlng neurons are strongly associated with the hypothalamus, the limbic system, and pain-mediating stuctures. Opioid receptors have been identified throughout the central nervous system (Pert et a l., 1976; Field 8. Basbaum, 1978). The highest concentrations of opioid receptors are found in the midbrain (dorsal interpenduncuIar nuclei, PAG, andmldllne reticular formation), and striatum (Pert et a l., 1976). Intermediate levels of opioid receptors have been localized In areas of the hindbrain, specifically the locus coeruleus, spinal trigeminal nucleus, nucleus tractus solitarius, and the substantia gelatinosa. Again, these loci are areas associated with pain mediation. Multiple opioid receptors have also been identified (mu, delta, kappa, and epsilon) with varying a ffin ity for the existing opioid peptides (Hughes, 1984; Simon & H iller, 1984). Hu receptors have been described as ‘opiate- preferring' and delta receptors as ‘enkephalin-preferring" (Simon & H iller, 1984). This preference is demonstrated by the selectivity of opioid-receptor binding; the opioid peptides have been found to demonstrate some specificity in binding as follows: beta endorphins 10 binds equally at mu and delta receptors; leu- and met-enkephaltn bind at both delta and mu receptors but demonstrate a higher affin ity for delta receptors; dynorphin and beta-neoendorphin bind at kappa, mu, and delta receptors with decreasing a ffin ity . Epsilon receptors also demonstrate a high a ffin ity for beta-endorphin (Hughes, 1984). Behavioral Aspects of the PAG; As can be 3een from the preceeding discussion, the PAG has been found to have substantia] neuroanatomlcal and neurochemical ties with pain-mediating systems; however, activation of the PAG by morphine and focal brain stimulation has been found to e lic it multiple behaviors in addition to SPA. Stimulation of dorsal areas has been found to e lic it a hyperactivity syndrome characterized by “spontaneous and stimulus (light and loud noise) evoked running and bursts of motor activity which usually precludes analgesic testing" (Jenson & Yaksh, 1906, p .102). Other responses have also been detected by stimulation of the PAG including rotation, tremor, vocalizations, and gnawing (Fardin, 01iveras, & Besson, 1984a & b). In contrast, microinjections of etorphine into the PAG have been found to produce analgesia with an associated catatonia (Thorn-Gray, Levitt, H ill, 8. Ward, 1981). Pain Inhibltory Systems: The primary effect of morphine or focal brain stimulation on the PAG, however, is the production of analgesia. This ability of the periaqueductal gray to support SPA may be due to activation of an endorphinergic system, which is consistent with the multiple links 11 between the PAG and opioid peptide-containing neurons and the presence of multiple receptors and peptides within the PAG. Both mu and delta opioid receptors have been identified In the body of the PAG (Simon & H iller, 1984). Therefore, there is sufficient neuroanatomical and neurochemical evidence to link endorphinergic systems with the PAG. Furthermore, analgesic brain stimulation in humans has been associated with elevated enkephalin and beta-endorphin levels in third ventricu lar flu id (A kil, Richardson, Hughes,8. Barchas, 1978; A kil, Richardson, Barchus, & Li, 1979). Lewis and Gebhart (1977) have reported that analgesia is produced by micro-injections of morphine into the peri aqueductal central gray at loci adjacent to and with some overlap of those sites supporting SPA. Morphine had its greatest effect at medial sites whereas focal brain stimulation produced its greatest effect at the ventrolateral edge. This again suggests activation ot an endogenous opiate system within the PAG by both morphine and focal brain stimulation. In addition, s t1mu I ation-produced analgesia (SPA) from some PAG sites has been shown to be reversed by the morphine antagonist, naloxone (Adams, 1976; A kil, Mayer & Liebeskind, 1976; Hosobuchi et a l , 1977). Naloxone is a competitive antagonist at mu, delta, kappa, and epsilon receptor sites (Szekely & Ronai, 1982); however, it has a greater affinity for mu receptors and relatively poor affinity for delta and kappa receptors (Goldstein, 1964). Mu receptors are responsible for morphine Induced analgesia (Pasternak, Childers, & Snyder, 1980), and naloxone has been effective in reversing analgesia from both morphine microinjection into the PAG (Jensen & Yaksh, 1986) 12 and stimulation of the PAG (Adams, 1976, Akil et al. 1976, Hosobuchi et al 1977). However, naloxone at a dose of 2 mg/kg has been found to e lic it hyperalgesia in otherwise untreated rats (Berntson 8. Walker, 1977) but not in humans (Goldstein, 1904) unless accompanied by stress (Frid, Singer, Rana, 1979). Additional opioid characteristics of SPA from the PAG include that at subanalgesic levels it sums with subanalgesic doses ot morphine to produce effective analgesia (Samanin & Vaize11i , 1971), and it has been found to attenuate the morphine withdrawal syndrome (Beaulieu & Thorn,1986). Chronic stimulation of the PAG on its termination has also been found to result in some withdrawal behaviors similar to those of mild morphine withdrawal (Williams & Thorn, 1984). Furthermore, repeated stimulation of the PAG has been found to result in a diminution of effect, mirroring morphine tolerance (Mayer & Hayes, 1975). Examinations of this stimulation tolerance and morphine tolerance found that cross-tolerance could be produced between the two (Mayer 8. Hayes, 1975; Lewis 8. Gebhart, 1977). However, cross tolerance between morphine and SPA is uni-directional. Stimulation is ineffective in producing analgesia in a morphine-tolerant animal, but morphine will continue to produce analgesia in a stimulation-tolerant animal. This uni-directional effect, however, may be secondary to tne greater locus of action of morphine in comparison to the site specific action of focal brain stimulation (Lewis & Gebhart, 1977). According to Way 8, Rezvanl (1904), the phenomenon of opiate tolerance is characterized by three criteria; "1) decreased response to the same dose of drug; 2) Increased dose of drug to yield the same 13 response; and 3) lessened response to drug with continued exposure" (p.i09). Stimulation tolerance fulfills these criteria In that chronic stimulation results in a loss of effect and if done over a period of days, requires a gradual Increase in the current required to produce analgesia. Abstinence from stimulation results in a return to the effectiveness of original stimulation application (Lewis & Gebhart, 1977). Three cellular mechanisms have been proposed for the mediation of this effect: modulation of adenylate cyclase activity by opioids, changes in the composition of neural membranes, and opioid effects on calcium ions (Szekely & Ronai, 1962). Opiates as well as opioid peptides have been found to Inhibit basal and £ prostaglandin (PGE)- stimulated adenylate cyclase activity. Opiate receptors appear to De* coupled to adenylate cyclase with subsequent inhibition of adenylate cyclase activity upon opiold-receptor binding. Furthermore, opioid acfeninistration has been found to result in potassium channel opening with resultant inhibition of neurotranamitter release; this effect may be produced by the opioid inhibition of adenylate cyclase (North, 1984). This suggests a pre-synaptic site of action for opioids (H ill, Morris, 8. Pepper, 1984). Consequently, chronic acini n i strat i on of opioids results in hypertrophy of the adeylate cyclase system and in increased production of PGE (C o llier, 1984). Opiates and opioid peptides have also been found to inhibit calcium ion uptake into the synaptosomes, thereby decreasing the levels in the synaptosomal fraction. Inhibition of calcium binding leads to an elevation of tree calcium with a subsequent opening of potassium channels. Therefore, 14 this effect of opioids would also inhibit presynaptlc neurotransmitter release (North, 1984). Alternatively, inhibition of calcium binding results in an increase in free calcium as well as potassium, which produces hyperpolarization of the neuron (North, 1984). Concurrent with the development of tolerence, brain calcium ion levels are elevated (C o llier, 1984). Furthermore, C ollier <1984) reports that "chronic opiate acini n 1st rat ion has been reported to result in decreased phosphorylation in synaptic plasma membranes" (p.5). Dephosphorylation of a channel protein may also lead to channel opening; therefore, this mechanism may also be secondary to opioids known effect on potassium channels (H ill et a )., 1984). Additional effects of chronic opiate actninlstrat ion are: 1) inhibition of monoamines and acetylcholine, resulting in super- sensitivlty and multiplication of these respective receptors; 2) reduction of brain concentrations of endogenous opioids via negative feedback from opioid receptors; and 3) a decrease in the number of opioid receptors & Ronai , 1983). This down regulation of opioid receptors and decrease in endogenous opioid levels may also be responsible for the tolerance e ffe c t. Whether the c rite ria and possible mechanisms of opioid tolerance are the same as those for stimulation-tolerance is not known. One possible explanation for the loss of effect following chronic stimulation is tissue damage at the site of the electrode (Mayer & 15 Hayes, 1975); however, these authors report that the stimulation puise configuration u tilized to produce SPA has been found to be non- injurious and the stimulation duration was "uniikely.. .to result in significant tissue damage" (p. 94). Furthermore, damage at the electrode tip has not been identified in either of the cross-tolerance studies (Mayer & Hayes, 1975; Lewis & Gebhart, 1977). Additional explanations for the development of tolerance to stimulation-produced analgesia, aside from those associated with opiate analgesia, include neuronal fatigue ( Delgado, 1981), negative kindling (Delgado, 1981) and monoamine depletion ( Blanch 1, SIniseal chi, Verattl, & Beani, 1985). Neuronal fatigue results from chronic stimulation of brain structures and occurs at different rates in different central nervous system loci (Delagado, 1981). The rate of neuronal fatigue within pain-mediating systems has not been determined; therefore, the role of neuronal fatigue in the occurence of stimuI atlon-tolerance is not known. Negative kindling is also site specific, occurring in some brain regions and not in others (Delgado, 1981). Again, this effect has not been examined in relation to stimulation-tolerance and therefore remains a possible explanation for this effect. Blanchi et al. (1985) report that SPA tolerance is accompanied by monoamine depletion: this is in agreement with the findings of Hosobuchi (1978) who found that L-tryptophan adninistration reversed SPA tolerance. Both neuronal fatigue and neurotransmitter depletion would result in a diminished capacity of excitation within the activated neuronal pool and thus would account for the loss of effect produced by chronic stimulation. Consequently, these reports suggest that in the 16 observance of SPA tolerance, tissue damage should firs t be ruled out, and the remaining explanations of neuronal fatigue, negative kindling, and monoamine depletion should then be considered. These explanations are additional considerations to those underlined in association with morphine tolerance. In contrast to the studies cited that suggest an opioid PAG system, there is also substantial evidence for a non-opio id PAG system responsible for stimu I at ion-produced analgesia. Stimulation of some sites within the PAG results in analgesia that is not naloxone- reversible (Yaksh, Yeung, & Rudy, 1976i Cannon, Prieto, Lee, & Liebeskind, 1982), Also, nociceptive testing using a phasic t a il- flick test or a tonic formalin test found stimulation of the PAG to De effective in producing analgesia to both; however, naloxone blocked the analgesia to the ta i1 -flic k test but not the formalin test (Dennis, Choiniere, 8, Melzack, 1980, Thorn 8. Plotkin, 1986). This suggests the presence of two separate systems, one opioid in nature that mediates SPA to the t a iI- f lic k test and the other non-opioid in nature that mediates SPA to the formalin test. A neuroananatomical distinction has been suggested between these opioid and non-opioid PAG systems. Cannon et al <1962) reported that naloxone (0.01-10 mg/kg) reliably elevated SPA thresholds for ventral but not dorsal stimulation placements. In this investigation, a total of 18 midline sites were sampled, 12 ventral and 6 dorsal. However, 2 of these dorsal sites were also reported to show elevated SPA thresholds following naloxone injection although of lesser magnitude than ventral sites. One problem associated with this study is the ana 11 number of sites sampled, only 6 in the dorsal region, and 2 of those yielded dlscrepent results. A second problem is the dispro portionate demarcation of dorsal versus ventral regions; the authors designated sites as ventral if “just within or below the dorsal raphe" and sites as dorsal if “In the midline PAG, from its dorsal border to just above the dorsal raphe (p.317). Thi3 limits the ventral area to an extremely small portion of the PAG and expands what the authors considered as the dorsal area to nearly two-thirds the total area of the PAG. A more traditional demarcation of the dorsal/ventral PAG was proposed by Hamilton (1973) and used by other researchers (Fardin, Oliveras, & Besson, 1984a & b). This demarcation includes in the ventral PAG a medial nuclear region, surrounding the aqueduct and extending outward in a bell shape to the ventral boundary,a lateral nuclear region, and a dorsal nuclear region. This demarcation, however, would place all of the Cannon et a I . (1982) dorsal placements within the ventral area. Furthermore, the Hamilton (1973a) division includes a lateral nuclear area and thereby excludes the dorsolateral and ventrolateral regions from inclusion within the ventral and dorsal areas. Fardin et a l . (1984b) found that SPA could also be produced in the ventrolateral area and that this SPA was similar to that within the ventromedial region; this, therefore, necessitates further evaluation of the consistency of SPA from stimulation in the dorsai and ventral regions across their medial and lateral areas. An alternative demarcation would be to divide the PAG into equal halves, designated as dorsal and ventral. Nonetheless, the Cannon et a). (1982) findings propose that the PAG Is a region of two analgesic id systems, differing In locus and naloxone-reversibiIity. It should be noted, however, that Yaksh et al <1976) report an inability to reverse SPA from any area within the PAG by naloxone injection, and many of the sites tested were located within the area designated as ventral by Cannon et al . (1982). In addition, the Cannon et a l . <1962) findings suggest a role ror the dorsal raphe in SPA from ventral sites, since there was no dis tinction between sites within the dorsal raphe and those within the ventral PAG. The dorsal raphe is a midline nucleus located witnin the ventral portion of the PAG or at Its ventromedial boundary. As one of the raphe nuclei, it forms a continuous column of midline nuclei with in the brainstem reticular formation (Barr & Kiernan, 1963). The raphe nuclei are the primary source of brain serotonin and are the origin of an ascending serotonergic system (Anden, Dahlstrom, Fuxe, Larsson, Olson, 8. Urngerstedt, 1966) with terminations in many of the same areas as the PAG. This ascending serotonergic system projects through the ventral tegnentum into the medial forebrain bundle and therein projects to the preoptlc area of the hypothalamus, anterior amygdala, olfactory tubercle, septal nuclei, and via the cinguium bundle to the sublculum (Conrad et a l., 1974) and to the ventrolateral geniculate, cortical and basolateral amygdala (Haigler 8. Aghajaman. 1977). In addition, a large proportion of these ascending projections terminate in the mesencephalon (Brodal et a l.. 1960). The dorsal raphe sends descending projections to the medullary nucleus raphe magnus (Fardin & 01iveras, 1982), dorsal te^nental nuclei, locus coeruleus, pontine reticular formation and caudal central gray tConrad 19 et a l., 1974), and to brainstem nuclei receiving nociceptive information from the spinal cord, the nucleus gigantoce1luIaris and nucleus reticularis pontis caudal is. The dorsal raphe receives afferent projections from the mesencephalic central gray (PAG), tegmentum, nucleus gigantocellularis, nucleus reticularis pontts caudaI is (Gallegher A. Pert, 1978), and locus coeruleus (Pierce, Foote, & Hobson, 1976). In addition, the caudal raphe nuclei, nucleus raphe magnus and nucleus raphe pontis, project to the spinal cord (Brodal et a l., 1960) with terminal fields in bilateral laminae I, II, V, VI, and VII of the spinal grey. The nucleus raphe magnus receives projections from the frontal cortex, zona inserts, dorsal and lateral PAG, dorsomedial nucleus of hypothalamus, nucleus parafasclcularis prerubralis, nucleus cuneiformis, deep superior colli cut us, dorsal column nuclei, and spinal trigeminal nuclei, as well as a smaller number of projections originating from the ventral and rostral periventricular gray (Carlton, Leichnetz, Young, & Mayer, 1983). As is obvious the raphe also has substantial links with both endorph- ergic and pain-mediating systems. The role of the raphe nuclei in SPA will be discussed in subsequent sections. The ventral and dorsal PAG have been reported to have d iffe r ential characteristics separate from their naloxone- reversibility. Cannon et al (1982) in a second experiment within that project report distinctions between ventral and dorsal sites on stimulation and duration parameters. Of 27 ventral sites tested, post-stimu1 at ion analgesia could not be demonstrated at 7 with the maximal current intensity (18 mi 11iamps) utilized in their experimental design; the current required to produce post-stimulation analgesia at ventral sites was much higher than the current required to produce post- stimuiation analgesia from dorsal sites. Ventral sites required almost double the current of dorsal sites. However, during stimulation current thresholds did not d iffe r between dorsal and ventral sites. These findings suggest that analgesia produced by ventral sites should have a shorter duration or require greater current intensity than that of dorsal sites when tested by a post- stimulation method. However, the seven ventral sites from which post-stimulation analgesia could not be elicited as described above were included in the analysis of dorsal-ventral stimulation d iffe r ences and may have artificially inflated the difference in post- stimulation thresholds between dorsal and ventral sites. These seven sites also appear to be significantly different from the other ventral sites. The remaining 20 ventral sites, from which post-stimu1 at ion thresholds were determined, did not appear to have current thresholds that were significantly different from the current thresholds of dorsal sites, suggesting that further evaluation of these parameters is necessary. However, there is additional evidence from other sources that ventral and dorsal differences exist within the PAG, Fardin, 01 iveras, 8. Besson (1984a 8. b) evaluated the a b ility of sites within the PAG to support SPA as well as the apparent side effects of such stimulation. They found that pure analgesia, that is analgesia without motor effects, could only be obtained from stimulation of the dorsomediat part of the dorsal raphe and the ventrolateral part of the PAG; therefore, according to these authors, analgesia cannot be 21 produced from the dorsal PAG without associated motor effects. These findings also suggest that medial - 1ateral differences exist within at least the ventral PAG. Lewis and Gebhart (1977) also report stimula tion to be most effective at lateral sites within the caudal PAG. These differences need to be further evaluated but suggest that the PAG is not equipotential for the production of SPA. Another area of interest has been the neuroanatomicaI pathway by which the PAG produces analgesia. Since SPA from both the dorsal and ventral PAG has been effective in abolishing the tai1-flick response to radiant heat, which is a spinal 1y-mediated reflex, this has resulted in a search for a descending system, terminating at the spinal cord, or a spinal system activated by a descending system. There is support for both. Early studies demonstrated that lesions of the dorsolateral funiculus (DLF) of the spinal cord abolished the analgesic effect of both PAG stimulation and systemic morphine (Basbaum, Clanton, & Fields, 1976; & Basbaum, Mar ley, O'Keefe & Clanton, 1977). Destruction of the DLF was also reported to disrupt front-paw shock induced analgesia, which is naloxone reversible, as wel i as hind-paw shock-induced analgesia, which is not naloxone reversible. Hind-paw shock-induced analgesia once in itia te d , however, produced an analgesia that persisted after the DLF was subsequently lesioned, suggesting that it activated an intraspinal system, which once activated continued to function without further descending input from the DLF non-opioid system. This was not true of the former system (Watkins & Mayer, 1982). However, there is no anatomical support for a direct projection from the PAG to the spinal cord (Besson, 01iveras, Chaouch, 8. Rivot, 1981), and therefore, an Indirect pathway may be responsible for PAG-originating analgesia. One possibility is a projection from the PAG to the nucleus raphe magnus and from the latter to the spinal cord. The PAG has been found to project to the nucleus raphe magnus in the medulla (Ruda, 197b; Gallegher 8, Pert, 1978; Fardin & Oliveras, 1982; Beitz et a l., 1983), which also receives projections from the dorsal raphe nucleus (Pierce et a l., 1976). Furthermore, the nucleus raphe magnus has numerous projections to the spinal cord, which descend within the dorsolateral funiculus (Basbaum et a l., 1976; Basbaum, Clanton, & Fields, 1978; Martin, Jordan, 8, W illis, 1978). Basbaum et al. (1976) found also that stimulation of the nucleus raphe magnus selectively inhibited dorsal horn nociceptive neurons. This finding In combination with those regarding projections from the PAG to the nucleus raphe magnus, suggest a role for that nucleus in SPA, possibly by activation of a descending pathway through the DLF from the raphe magnus to inhibit nociceptive neurons. In support of this concept, activation of the PAG by glutamate has been found to result in analgesia as well as an increase in the firing rate of neurons originating in the nucleus raphe magnus, both of which were reportedly naloxone-reversible; furthermore, lesions of the nucleus raphe magnus also abolished the analgesia produced by this glutamate- activation of the PAG (Behbehanl & Fields, 1979). Lesions of the nucleus raphe magnus have also been found to disrupt SPA from ventral but not dorsal PAG sites (Cannon, Prieto, & Liebesklnd, 1980, Prieto, Cannon, 8 Liebeskind, 1983). These sites, however, were not tested 23 for their naloxone re v e rs ib ility . In contrast, combined dorsal raphe and raphe magnus lesions were necessary to completely abolished morphine analgesia (Yaksh, Plant, & Rudy, 1977), thereby suggesting some differences in the neural substrates involved in SPA and morphine. Nonetheless, stimulation of the dorsal raphe was found to produce analgesia, which in turn was naloxone reversible (Swajkoski, Meyer, 8> Johnson, 1961) and at subanalgesic levels to sum with s u d - analgesic doses of morphine to produce effective analgesia (Samanin 8. V a lze lii, 1971). In a more recent study, Sandkuhler and Gebhart (1984) report that micro-injections of 1idocaine into the nucleus raphe magnus were ineffective in blocking SPA from the PAG, but blockage was obtained with injections into both the nucleus raphe magnus and the surrounding medullary reticular formation. Furthermore, this blockage was effective for both dorsal and ventral sites within the PAG, suggesting both areas operate through descending systems that pass through the medulla in the region of the nucleus raphe magnus. Therefore, both the dorsal raphe and the nucleus raphe magnus have been implicated in a descending pathway, originating in the PAG, descending in the DLF, and terminating in the dorsal horn: these findings also implicate serotonin in this descending pathway, since the raphe is the primary source of brain serotonin. Other studies have found that decreasing the amount of brain serotonin also inhibits analgesia from both morphine (Tenen, 1963; Dewey, Harris, Howes, 8. Nuite, 1970, & Vogt, 1974) and SPA (Akil & Mayer, 1972). Furthermore, the analgesia produced by micro-injections of morphine into the PAG or NRM is greatly diminished by clnanserln, a serotonin receptor blocker 24 (Dickenson, Oliveras, 8. Besson, 1979; Yaksh, Du Chateau, 8. Rudy, 1976). Lesions of the nucleus raphe magnus significantly reduce or aboii3h morphine analgesia (Yaksh et a l., 1977; Proudfit 8- Anderson, 1975), as do selective lesions of spinal serotonin terminals (Genovese, Zonta, & Mantegazza, 1973; Vogt, 1974). Correspondingly, morphine has been shown to increase 5-HT and tryptophan levels in the spinal cord of animals with chronic pain conditions induced by intra- dermaI injection of k ille d mycobacterium butyricum, which produces an a rth ritic -lik e syndrome (Wei 1 -Fugazza, Godefroy, Bineau, Thurotte, 8, Besson, 1984). Both PAG stimulation (Besson et a l , 1961) and 5-HT (Belcher, Ryal I , 8. Schaffner, 1978; Headley, Duggan, & Griersmith, 1978) decrease the firin g rate of dorsal horn nociceptive neurons. In addition, intravenous injections of the serotonin antagonist, meth- ysergide, has been found to inhibit the analgesia from PAG stimulation to pain produced by both thermal heat and C fiber stimulation (Foong, Herman, 8. Duggan, 1965). Methysergide is an ergot alkaloid derivative, which is a competitive serotonin receptor blocker (Stewart, Gerson, Sperk, Campbell, 8. Baldessarinl, 1978). Although methysergide is effective in blocking 5HT-2 receptors, which mediate excitation in the central nervous system and periphery, it is ineffective, as are all other serotonin antagonists in blocking alt serotonin receptors (Haigler K Aghajanian, 1977). Methysergide has been effective in blocking the protein synthesis disruption produced by 5-HT injection (Mollgard, Lundberg, Wlklund, Lachenmayer, & Baumgarten, 1978), prolactin-stimulating effects of serotonin receptor activation (Jacoby & Thomas, 1978), 5,6 DHT Induced pressor effect (Gothert 8, Klupp, 1978), and serotonin-induced head-weaving, forepaw treading, and hindlimb abduction but not the enhanced locomotor activity (Green & Heal, 1984). Of the serotonin antagonists tested, including mianserin, cinanserln, metergo 1ine, and cyproheptadine, methysergide was the only one that blocked all of these behaviors. However, there is some question as to the specificity of methysergide for serotonin receptors. Feldnan and Lebovitz (1972) found that methysergide also blocked dopamine-induced inhibition of glucose stimulated insulin release, suggesting that methysergide also blocks dopamine receptors in the periphery. As is evident from the effect of raphe lesions, serotonin depletion and methysergide-blockade, there is sufficient evidence to link the raphe nuclei and serotonin to analgesia originating from the PAG. However, Han and Xuan (1986) found micro-injection of cinan- serin Into the nucleus accumbens attenuated the analgesic effect of micro-inject ion of morphine Into the PAG and consequently suggest that PAG stimulation activates an ascending serotonergic pathway to the nucleus accumbens and thereby produces analgesia. This finding, although suggesting an alternative pathway, s t ill links serotonin to PAG produced analgesia. However, the role of serotonin may d iffer between the two PAG analgesia-producing systems (opioid and non-opioid). Additional research found that only SPA from sites in the ventral aspect of the PAG were affected by serotonin depletion frcm p-CPA injection, a serotonin synthesis Inhibitor (Akll & Mayer, 1972); however, this 26 finding was based on data from only two sites outside the ventral area and there was no attempt to distinguish the naloxone-reversibl1ity of the sites tested. This small number of sites necessitates further evaluation to determine the role of serotonin in SPA, and a deter mination of the consistency of this role in opioid and non-opioid SPA needs to be attempted. The findings of Thorn and Plotkin <1984) suggest that serotonin plays a roie in non-opioid as well as optoid SPA. These authors report that focal brain stimulation from the PAG produces analgesia to both a formalin and a ta i1 -flic k test. Naloxone failed to reverse SPA for the formalin test but reversed SPA for the tail-flick test; however, methysergide, the serotonin antagonist, reversed the SPA In the formalin test and attenuated the SPA in the t a i 1- f 1ick te s t. Problem Statement From this review there are several questions that remain unanswered. F irst, there has not been an extensive study, involving numerous electrode placements, to map opioid and non-opioid areas and to establish the potential of the PAG to produce SPA along its rostral-caudal extent. SPA has been examined primarily within the caudal region of the PAG (Yaksh et a l, 1977, Cannon et a l., 1982, Mayer 8, Liebeskind, 1974, & Fardin et al., 1984a & b); therefore, the effect of stimulation within the rostral PAG needs to be examined and compared to that of caudal sites. Secondly, if indeed opioid and non-oploid sites exist, the relationship of these two systems has not been explored. They appear to coexist within the PAG and to sim ilarly activate descending systems, which synapse within the raphe or medullary reticular formation and subsequently descend within the DLF of the spinal cord. Whether these systems act in isolation or are co activated has not been determined. There is some evidence for co activation in that stimulation of a single site within the PAG produces analgesia to both ta il-flic k (opioid) and formalin (non- -opioid) pain tests (Thorn & Plotkln, 1986); therefore, this stim ulation apparently activated both opioid and non-opioid neural systems. However, the directionality of this co-activation has not been determined. If co-activation does indeed occur, it could be either uni-directional (ventral to dorsal or dorsal to ventral) or bi-directional. Finally, the role of serotonin in opioid and non opioid analgesia remains ambiguous; there is evidence based on phasic and tonic pain tests to support its involvement in both , but there is also evidence based on a very small number of data points within what was designated as the dorsal PAG to suggest that it Is not involved in non-opioid PAG (Akil & Mayer, 1972). The present study attempts to address these Issues in the following manner: 1. To map the areas within the PAG which support stimu I at ion-produced analgesia on both a dorsal-ventral and a medial -1ateral axis. To determine the naloxone-reverslbi1ity of each site as an indication of its involvement in an opioid or a non-opioid system, and to evaluate the extent of the analgesic effect of PAG stimulation along an anterior-posterior, or rostral-caudal axis. 2. To examine the interaction between naloxone-reversible and non- reversible systems by attempting to produce cross-tolerance between naloxone-reversible and non-reverslble sites. To investigate the role of serotonin in naloxone-reversible and non-reversible SPA by examining the a b ility of reversible and non- reversible sites to produce analgesia, following serotonin receptor blockade. METHODOLOGY General Methods: Subjects: Alt subjects for the experiments described were experimentally naive Albino rats, 90-120 days of age and weighing 325-450 grams at the time of surgery. Animals were housed indiv- ually on a 12 hour light-dark cycle with ad libitum food and water. Surgery: Two monopolar stimulating electrodes, constructed of size 00 insect pins coated with epoxylyte except for .5 mm at the tip , were stereotaxical1y implanted into each ra t, using a Kopf stereotax. Electrodes were aimed at various loci within the midbrain peri aqueductal gray (see Experiment 1 methods). Coordinates were initially taken from the atlas of Pellegrino, Pellegrino, and Cushman (1979), and were adjusted according to this atlas following prelim inary histological examinations to allow more complete coverage of the PAG extent. Surgeries were performed while rats were under an anesthetic dose of sodium pentobarbltol (45 mg^kg) preceded by atropine sulfate to minimize congestion. Subjects were allowed 7 days recovery following surgery. Apparatus: A Grass S6C stimulator was used to deliver trains of biphasic rectangular-wave pulse pairs of 1-msec. duration at 50 Hz. Yeung et a l. (1977) reported that frequencies of 40-100 Hz were necessary to produce whole body antinociception at pulse durations of 29 30 less than 1 millisecond. Stimulation was applied at 2 minute intervals for a duration of 10 seconds. Intensity was Increased by 10 microampere increments at each stimulation until analgesia was apparent or until a maximum current of 300 microamps was reached. Nociceptive testing immediately followed the termination of stimula tion. Testing was terminated if observable motor activity or vocal izations were produced. During testing the animals were restrained using a cloth wrap. The rats were adapted to the wrapping procedure and electrode-stimulator attachment for one hour on the two days preceeding testing, which resulted In minimal escape behavior during formal testing. Nociceptive Testing: A standard t a iI- f lic k device, using a 500 watt incandescent projection bulb was used to deliver noxious radiant heat to the ta il, through a 1/8 inch aperture d rilled into the top of the encasement, over which the ra t's ta li was placed. A rheostat was connected to the bulb to control the heat intensity. The tall was blackened with a marking pen from 3 - 4 inches from its tip , and that area was placed over the heat source. Latency to remove the tall was recorded and the heat intensity adjusted to obtain four consecutive values with an average latency of 3.5-4.5 seconds. The operational criterion for analgesia was an elevation in tai1-flick latency of 30 percent over baseline. To prevent tissue damage, the heat source automatically shut off after 10 seconds. Drugs: The morphine antagonist, naloxone was used to determine the involvement of opioid systems in analgesia. Naloxone was chosen since it is a relatively potent opiate antagonist at mu receptors and 31 has been found to effectively reverse analgesia Induced by micro- injectlon of morphine into the PAG (Jensen & Yaksh, 1986) and anal gesia by stimulation of opioid sites within the PAG (Cannon et a l, 1982). Naloxone was dissolved in .9% saline solution and administered at a dose of 10 mg/kg and a volume of 1 ml/kg body weight. The serotonin antagonist, methysergide was used in Experiment 2. Methysergide was selected due to its effectiveness in blocking more serotonin-mediated responses than the other available antagonists (Halgler & Aghajanlan, 1977, Stewart et a), 1977, Moltgard et a l , 1977, Jacoby & Thomas, 1977, & Green & Heal, 1985), and because it has previously been reported to antagonize stimulation-produced analgesia (Thorn & Plotkin, 1984; & Foong et a l., 1985). Ideally, usage of multiple serotonin antagonists would have been beneficial; however, the limited number of sites tested precluded this alternative. Methy sergide was also dissolved in .9% saline solution and administered at a dose of 3 mg/kg and a volume of 1 ml/kg. Histology: Following testing, animals were euthanized with a 1 cc. Intraper1toneal injection of sodium pentobarbitol (65 mg/ml). Electrode placements were marked by passing .5 ml 11 lamps of anodal current through the electrode for 10 seconds. The animals were then intra-cardlalIy perfused with isotonic saline followed by 10\ formalin solution and then potassium ferrocyanide solution to stain the iron ions. Frozen sections of the brainstem were then cut at 40 microns, mounted, and stained with cresyl violet. Stereotaxic coordinates for each electrode placement was determined by direct projection of the brain sections onto atlas plates (Pellegrino et al., 1981). The 32 experimenter was blind to the behavioral results at this time. Data analysis: Multiple regression analyses were used to deter mine the role of site specificity in the production of analgesia, the current threshold required, and the duration of the analgesia. For these analyses atlas coordinates were used as predictors and mean base)Ine-stlmulatlon difference scores, duration and intensity were used as criteria (see results section for further descriptions). A discriminate function analysis was used to determine whether the membership into one of the non-effective site groups (no effect, motor effects with analgesia, and motor effects without analgesia) was site specific. Analyses of variance were conducted to assess the drug- slte interaction for both naloxone and methysergide, and a Newman- Keuls/ test was used for post hoc comparisons. Experimental Methods: Experiment 1: This firs t experiment was designed as a mapping study to investigate the opiate and non-opiate correlates of SPA within the PAG. Coordinates were chosen so as to divide the PAG Into dorsolateral, dorsamedla), ventrolateral, and ventromedial quadrants at three different anterior-posterior axes. For each quadrant at each of the three anterlor-posterlor axes, a minimum of 10 animals received an electrode aimed at the center of the quadrant. A total of 113 animals, each with two electrode implants, were tested, allowing a possible sampling of 226 sites. One week following surgery the animals began a series of tests, each separated by 48 hours. The left electrode site was tested for 33 SPA in the firs t test, and the right electrode in the second test. Thirteen animals with two positive sites underwent an extra test at the end of this second test to examine the interaction between opioid and non-oplold systems. In these animats, at the time of Implanta tion, one electrode was aimed at a dorsal site and the other at a ventral site (subsequent testing verified that these were non-opioid and opioid sites respectively). In this extra test, after the determination that the right site (Site 1) produced SPA, stimulation was switched to the left electrode (Site 2). Stimulation was adnin- 1 stored at this position every two minutes until tolerance was reached. Tolerance was defined as a return to baseline ta ll-flic k latency. Stimulation was then adnlnlstered at the original (right) site to determine if analgesia could s till be produced at that site. Four post-tolerance stimulations were given at Site 1 to determine If cross-tolerance was present. Following this test for cross-tolerance these animals were tested for naioxone-reversibl1ity as described below. Tests 3 and 4 established the naioxone-reversibl11ty of sites that produced analgesia in Tests 1 and 2. During these latter tests, baseline was established and then a preliminary test was done to assure that the site would s tilt produce analgesia. Following a single analgesia-producing stimulation, ta i1 -flic k latency was allowed to return to baseline levels; the Interval required for this return to baseline varied between animals, being dependent on the duration of the analgesia produced. Return to baseline was followed by a five minute interval. Naloxone was then injected subcutaneous!y and 10 34 minutes were allowed to elapse for drug action. Following this interval, stimulation and nociceptive testing were initiated at the pre-test current threshold; testing was terminated when four consecutive analgesic or non-analgesic values had been obtained. Current intensity was not increased during post-drug stimulation. Naloxone-Injected unstlmulated shams were not included in this study since naloxone injection had previously been determined to e lic it hyperalgesia in rats by this laboratory (Berntson & Walker, 1977). Experiment 2: This experiment examined the role of serotonin In opioid and non-optoid SPA. Sixteen animals from experiment 1 with only one positive site underwent a third testing session on that site. This testing session was done after the site had initially been tested for its ability to elicit analgesia and subsequently tested for naIoxone-reverslbi11ty. Similar to the test for naioxone- reversibl 1 1 ty , testing Included an In itia l baseline determination, a single analgesia producing stimulation and a return to baseline. This was followed by a five minute interval, after which methysergide was Injected subcutaneously followed by a 10 minute Interval to allow for drug onset. After this interval, stimulation and nociceptive test ing were Initiated until four consecutive analgesic or non-ana I agesic tai1-flick latencies were obtained at the Initial test threshold. Six animals that had not undergone electrode implantation were tested as shams to determine the effect of methysergide and the test ing procedure on ta iI-flic k latency. Procedures were identical to those described above, but no stimulation was delivered. Methysergide was injected at the same dosage as that received by the experimental 35 animals and sim ilarly was followed by a 10 minute interval to allow for drug onset latency. After this interval four tail-flick tests were conducted to determine post-inject ion latencies. RESULTS Experiment 1: One hundred and thirteen animals were included in this study, each having two monopolar electrodes, allowing for testing of two hundred and twenty-two sites within and around the PAG (four place ments were not tested due to faulty electrode connections). Of those sites tested, eighty-seven supported stlmu1 at ion-produced analgesia. Histological verification was possible for one hundred and ninety-nine total sites and seventy-eight of those supporting SPA. Damage to the brain during removal, preparation, or mounting resulted in the in ab ility to verify the remaining nineteen placements; data from these placements was not included in any analysis. Of the seventy-eight verified sites that supported SPA, f if t y - one were subsequently tested for naloxone-reversibi1it y , twenty-four of which were those that underwent prior cross-tolerance testing. Two of the sites Involved in the cross-tolerance test lost effectiveness before the test for naioxone-reversibl11ty was performed. Of the remaining naloxone-tested sites (those that were not part of the cross-tolerance test), sixteen were tested In Experiment 2 for methysergide-reversibi1ity . Loss of stimulation effectiveness, electrode breakage, and subject mortality accounted for the substan tial decrease in subject numbers across testing sessions (twenty-seven 36 37 between testa 1 and 2 and tests 3 and 4; and 9 between tests 3 and 4 and the methyserglde-reverslbiIity testing. The current threshold for producing SPA ranged from 10-100 micro- amps (only 1 site required greater than 60 microamps), with the exception of one site with an apparent faulty ground electrode connec tion, resulting in increased resistance and requiring 300 microamps of current. This site was excluded from the analysis of current intensity. Duration of analgesia ranged from less than the two minute interstimulus interval to 22 minutes per single stimulation (see Tables i,2,& 3). No correlation was found between duration of analgesia and the current threshold required (correlation = -.16;. Electrode placements were plotted along five anterior-posterior midbratn sections, taken from the atlas of P elllgrlno, Pelligrino, and Cushman (1979), and differing by .6mm (see Figure 1). Due to the large number of electrode placements to be mapped, the widespread nature of these placements, and the diversity of the mesencephalic structures, electrodes were plotted on five frontal sections rather than the three original stereotaxic placements described e arlier. Sections were chosen to be representative of the extent of the PAG and the surrounding neuroanatomical structures, to Include the oculomotor nucleus, the trigeminal nucleus, the dorsal raphe, and the locus coeruteus, and to demonstrate the changing spatial relationships of these nuclei to the PAG at different anterior-posterior midbrain sections. As each electrode placement was plotted, the anterior- posterior coordinate, to be used in further analyses, was determined from the midbrain section on which the electrode was plotted. Hedlal- TrtbLt i: Stimulation Effects at Dorsai Sites TIME TO CROSS 5 iit ha* SPA OUR. I NT X TWR N-R N-R TOLERANCE TOLERANCE */- (MIN.) MICROAMPS (SEC.) +/ - + /- (MIN.J PLACEMENT j * 4-6 50 7.8 _ + (2 300 7.9 j ♦ 6 30 8.2 4 ♦ 10 40 8.3 - +■ 0 *■ 2 60 7.8 6 2 10 8.7 + + ? ♦ (2 30 0.5 o * 2 20 8.2 * 9 (2 60 7.8 1 0 2 20 8.4 11 ♦ 6 10 0.2 +■ ■* 1. SPA - s im u la tio n produced analgesia ; OUR * duration of analgesia; I NT - in ten sity; a THB - mean threshold elevation in tai 1 -f 11 etc latency; N-R * naloxone reversiDie; H-R ■ methysergloe re v e rs ib le . iABIE 2: Stimulation Effects at Ventral Sites TIME TO CROSS SPA DUR. INT. X THR N-R M-R TOLERANCE TOLERANCE s ite No. ♦ - (MIN .) MICROAMPS ( SEC.) +/- +/ - (MIN./ PLACEMENT 1 13 t <2 50 7 .6 I 14 + <2 100 6 .8 + + 115 + 8 40 7 .3 116 + 2 10 6 .2 117 + 2 40 6 .6 lie + 2 30 6 .6 U9 + 6 40 7.0 120 ♦ <2 60 6 .2 + 121 + <2 50 8 .5 122 + <2 40 8 .2 + 12j + (2 20 5 .7 124 ♦ 12 20 5.9 125 + 6 10 7.4 126 +■ 4 20 6 .6 - 127 + 2 10 7 .3 128 ♦ <2 40 6.1 + 129 + 10 30 5 .9 130 + <2 20 6 .8 + + 131 + 2 10 6.0 + 132 + 2 30 6.0 + ♦ 133 + 2 20 7 .9 + 134 <2 10 7.5 - 135 + <2 10 8 .2 + 26 3 3 136 *■ 6 10 7.0 + 24 34 137 * <2 10 6 .8 + 26 35 138 + 2 40 7.4 +■ 24 3 6 139 + 2 30 7.4 + 140 + 2 30 5 .7 22 39 141 + 2 10 7.0 + 30 40 142 + <2 40 7.3 + 143 + 2 20 5 .7 + 1 4 4 + 4 30 7.7 + + SPA * stimulation produced analgesia; OUR > duration of analgesia; INI » intensity; X THR - mean threshold elevation In tai1-flick latency; N-R » naloxone reversioie; M-R - methysergide reversioie. un TABLE 3; Distribution of effects at each anterior-posterior axis for each quadrant. A-P QUAD + SITES -SITES X DUB* X I NT* X THE* NB ME COOB. (MIN.) MICBOAMPS (SEC.) + - + - -4.4 DM 11 9 4.9 18.2 6.7 4 5 4 0 DL 1 4 <2.0 60.0 7.8 0 0 0 0 VH 9 8 4.2 21.1 6.7 5 2 2 0 VL 1 4 2.0 10.0 7.3 0 0 0 0 -5.0 DM 11 17 2.9 19. 1 5.5 2 5 3 0 DL 1 2 6.0 30.0 8.2 1 0 0 0 VM 2 8 3.0 35.0 7.0 I 0 0 0 VL 0 2 ------ -5.6 DM 11 7 4.0 27.3 7.0 1 4 0 0 DL 1 6 6.0 10-0 8.2 1 0 1 0 VM 17 11 3.1 33.5 6. 7 10 1 3 0 VL 0 4 ------ -6.2 DM 6 10 3.0 13.3 7.8 2 2 2 0 DL 3 4 5.0 16.6 8.0 2 1 0 0 VM 2 11 2.0 40.0 7.8 2 0 0 0 VL I 1 2.0 10.0 6.2 0 0 0 0 -6.8 DM 1 4 2.0 40.0 5.8 0 0 0 0 DL 0 4 ------VM 0 4 ------VI 0 1 ------ A-P COOB “ distance from bregma (taken from the atlas of Pellegrino, Pe1legr ino, Cushman, 1981) ; QUAD 3 quadrant (DM * dornos•edial. DL ■ dorsolateral , VM - ventromedial, VL * ventrolateral); + SITES “ no. of sites producing analgesia; - SITES * no. of sites not producing analgesia; X DUE ■ s u n duration of analgesia; X INT. * aean intensity; X THE * mean threshold of elevation in tail-flick latency; NB - naloxone reversible; HE * methysergide reversible. * determined for analgesia-producing sites only 41 lateral and dorsal-ventral coordinates were based on atlas coordinates (Pellegrino et a l., 1981) Sites that did not produce analgesia In the absence of motor effects fell into one of three categories: no effect, motor effects with analgesia, and motor effects without analgesia. Forty-nine sites were tested up to 300 microamps without producing analgesia or any motor effect, thirty-one produced analgesia associated with motor effects, and forty-one proAiced motor effects without the associated analgesia. These sites were included in the first regression analysis (described below) but were not Included in the multiple regression analyses that examined intensity- and duration-slte specificity. As can be seen from the plots in Figure 1, sites supporting SPA are located throughout the interior of the PAG front its most anterior extent to the point at which the dorsal raphe is no longer present. Only one of the placements located in the most posterior aspect of the PAG supported SPA, but this site did not maintain its effectiveness to allow naloxone testing. A multiple regression analysis was conducted to determine the contribution of electrode placement to the production of stimulation-produced analgesia, utilizing the coordinates as des cribed above as predictors and the stimulation-produced change in tail-flick latency as the criterion. Mean base 1ine-stImu1 ation difference scores were determined for each site by subtracting a summed baseline mean from the summed post-stimulation latencies and dividing by the number of latencies; this score along with the designated coordinates was used in the multiple regression analysis. Electrode placement was found to be nonsignificant in determining B - o * ANALGESIA >2 ° ANALGESIA 1 2 NO EFFECT Figure 1: Stimulation effects plotted on rostral-caudal midbrain sections (A - E corresponding to -4.4, -5.0, -5.6, -6.2, & -6.3 from bregman). Analgesia > 2 = analgesia of greater than 2 minute duration; Analgesia < 2 = analgesia of less than or equal to 2 minute duration. 43 whether or not a gite would produce SPA or the degree of analgesia produced, as Indicated by the relative increase in ta ll-flic k latency (see Table 3). A second multiple regression analysis yielded a non significant effect for electrode placement in the determination of the intensity threshold required to produce analgesia (see Table 4). A final multiple regression analysis yielded a nonsiginificant effect for electrode placement on the duration of the analgesia produced (see Tab Ie 5). To determine if electrode placement was predictive of inclusion into one of the non-pure-analgeslc groups (no effect, analgesia with motor effects, or analgesia without motor effects), a discriminant function analysis was conducted, utilizing mean baseline-stimuI at ion difference scores and stereotaxic coordinates as the predictor variables for group membership. Mean difference scores, medial- lateral placement, and dorsal-ventral placement did not contribute to group discrimination; however, anterior-posterior electrode placement was predictive of group membership. Sites in the most rostral aspect of the PAG were more likely to produce motor effects accompanied by analgesia. Sites in the middle PAG sections were most likely to produce motor effects without analgesia, and sites in the caudal PAG were more likely to produce null effects for both motor effect and ana Igesia. Of the initial eighty-seven positive sites, fifty-seven were tested with naloxone to determine re v e rs ib ility . Of this fifty-seven fifty-one were histologically verified. The electrode placements of these naloxone-tested sites were plotted on four of the anterior- 44 TAbLt 4 : Results ot regression on site specificity for mean difference scores. varlaDie Squared Partial Squared Correlat ion Semi-part 1 a 1 Corre1 at ion Ant. -Post. .0006 .0005 Med.-Lat. .0294 .0291 Dor.-Vent. .0008 .0007 R = .0346 F(3,195> * 2.777 P = .041 ResuIts of regression on site specificity for duration. VarlaDle Squared Partial Squared Correlat Ion Semi-partial Corre i at ion Ant. -Post. .0210 .0176 Med.-Lat. .0000 .0000 Dor. -Vent. .0171 .0168 R — .0346 Ft3,74) « .885 p * — TAoLt. 61 Results ot regression on site specificity for intensity. Var i aDle Squared Partial Squared Correlat Ion Semi-partiai Correlation Ant. -Post. .0008 .0008 Med.-Lat. .0009 .0009 Dor.-Vent. .0007 .0007 R = .0266 F O ,74) - .675 45 posterior axes previously described (see Figure 2); the most caudal midbrain section was not included since none of the naloxone-tested SPA-positlve sites were located in that region of the PAG. Mean difference scores were calculated for the pre-test analgesic latencies and the four post-injection stimulation latencies. Twenty-seven of the sites tested were completely or partially reversed by naloxone. Complete reversal of SPA was defined as a post-naloxone mean- difference score of less than one; partial reversal was defined as a 30 percent decrease in the post-naloxone mean-difference score as compared to the pre-naloxone score. The remaining twenty-four sites failed to demonstrate naloxone-reversibi1it y . To examine the effect of electrode placement on naloxone-reversibi11ty , an ANOVA (1 between x 1 within) was used with dorsal versus ventral electrode placement the between factor and pre- and post-inject ion mean differences the within factors. This yielded a significant effect for site, F (1,49) =10.941, p<.002, treatment, F (1,49) * 39.636, p<.001, and a site x treatment interaction, F (1,49) =11.114, p=.002. A Newraan-Keuls' post hoc test found this interaction to be due to the effect of naloxone at ventral sites, yielding a significant difference between the ventral post-inject ion group and the other three groups 3), resulting in increased SPA thresholds for ventral sites. Only thirteen animals were found to have two SPA-supporting electrode sites and potential cross-tolerance was measured in these 46 B ■ REVERSED ° NON - REVERSED Figure 2: Effect of naloxone on stimulation-nroduced analgesia plotted on rostral-caudal midbrain sections (A - D corresponding to -4.4, -5.0, -6.2, 6 -6.8 from bregma). gur 3 Efect f aooe t sal n ventral t , s ite s l a r t n e v and l a rs o d at naloxone of t c ffe E 3: re u ig F DIFFERENCE SCORES (SEC.) OSL VENTRAL DORSAL R NALOXONE PRE OT NALOXONE POST IE/ TEST / SITE 4 7 animals. The number of stimulations required to reach tolerance (logs of analgesic effect with stimulation applied every two minutes), ranged tram 4 to 15 with the exception of one animal that remained analgesic for 57 stimulations. The mean time to tolerance was 29.7 minutes if this exceptional value Is included and 26.8 minutes if this value is excluded. Four analgesic pre-tolerance latencies and four stimulation post-tolerance latencies were obtained for Site 1 (tolerance was in itia lly developed at Site 2). Mean base Iine st imulation difference scores were determined for these pre-tolerance and post-tolerance latencies. Cross-tolerance was observed at all sites as indicated by diminished post-tolerance mean difference scores. Only two sites failed to return completely to baseline levels (a mean difference score of less than one). At the time of testing, the opioid nature of the sites was not known but was later determined by naloxone testing. Naloxone testing determined that prior cross-tolerance had been produced from a non-naloxone-reversible site to a naloxone-reversible site (NNR-NR) in six animals, and from a naloxone-reversible site to a non-naloxone-reverslble site (NR-NNR) in 8 animals. One animal was tested for cross-tolerance in both direc tions with one week between testing sessions. An ANOVA (1 between x l within) with the direction of the development of tolerance (NNR-NR and NR-NNR) the between factor and treatment (pre- versus post-tolerance mean difference scores) the within factor, produced a significant effect for treatment, F (1,12) = 174.899, pC.OQl (see figure 4). gur ; rs t er nal r si le ib rs e v -re e n o x lo a n t a s t c e f f e e c n ra le to Cross 4; re u ig F mean difference SCORES ISEC.) NRN) n non- oxone-ever bl N.NR si . s e it s (NR.-NNR) le ib rs e v -re e n o x lo a -n n o n and (NNR-NR) NNR-NR R TOLERANCE PRE OT TOLERANCE POST TEST NR-NNR 49 50 Experiment 2: Sixteen animals, included in Experiment 1 , maintained an active SPA-supporting electrode site to allow a third testing session. Forty-eight hours after naloxone-reversibi11ty testing, these animals were tested for the effect of methysergide on SPA. Of the sites tested 7 were naloxone-reversible and 10 were non-naloxone-reversible. These sites are plotted along the four anterior-posterior axes, pre viously described, in Figure 5. One of the naloxone-reversible sites was not within the PAG, being located outside the most dorsal aspect of the PAG; however, this site was included In the dorsal group in the analysis of this data. A second site, within the dorsal group, was also located outside the dorsal aspect of the PAG; however, this site was non-naloxone-reverslble. Mean difference scores were determined for the pre-injection analgesic latency and the post-inject ion stimulation latencies, as described earlier. All but one site exhib ited a diminished mean difference score following methysergide injection. The site that failed to demonstrate methyserglde- reversibi1ity was the naloxone-reversible site outside the PAG. An ANOVA <1 between x 1 within) with site (dorsal-ventral) as the between factor and treatment effect (pre- and post-lnjection mean difference scores) as the within factor yielded a significant effect for treat ment, F (1,15) * 23.493, pc.001, without a slglnificant interaction effect, indicating that methysergide was effective in reversing the analgesia from both ventral and dorsal sites (Figure 6). AT test of the methysergide injected shams' pre- and post-injectIon mean t a ii- flick latencies, yielded a nonsignificant difference. 51 ■ REVERSED D NONREVERSED Figure 5: Effect of methysergide on stimulation-produced analgesia plotted on rostral-caudal midbrain sections (A - D corresponding to -4.4, -5.0, -5.6, -6.2, -6.8 from bregma) . F ig u re 6: 6: re u ig F DIFFERENCE SCORES (SEC.l Effect o f m e th y s e rg id e a t d o rs a l and v e n t r a l l a r t n e v and l a rs o d t a e id rg e s y th e m f o DORSAL OT METHYSERGIDE POST MUKYSERGIDE PRE TE/ T S E /T E IT S VENTRAL sites, 52 DISCUSSION The present study has provided the most extensive mapping ot the periaqueductal gray for SPA. The results Indicate that, with the ex ception of its most posterior aspect, the PAG appears to be relatively equipotential in its ability to support stimulation-produced anal gesia. The findings of Experiment 1 demonstrate that stimulation within the interior of the PAG is not dependent on the site of the electrode placement; all areas within the PAG along its rostral-caudal axis and varying in dorsal-ventral and medial-lateral orientation were equally likely to support SPA. Earlier reports suggested that SPA was more easily produced in the caudal PAG (Yeung et a l., 1977, Cannon et al,1982, 8. Fardin et a l., 1984a 4 b), that ventral PAG sites were more effective in e lic itin g SPA than were dorsal PAG sites (Fardin et a l., 1984b, Lewis & Gebhart), and that lateral sites were more effective than medial sites (Yeung et a l., 1977, Lewis 4 Gebhart, 1977). These reports, however, were based on relatively small numbers of tested sites. Stimulation was applied using bipolar electrodes in all of the aforementioned studies, which contrasts with the use of monopolar electrodes in the present study, and these earlier studies utilized much higher stimulation Intensities than the present study. These differences in methodology may account for the descrepant results, relating to site specificity. 53 Intensity of the threshold current to produce SPA, the degree of analgesia produced (as interpreted by the average increase in ta il- flick latency), and the duration of the analgesia produced were also found to be independent of electrode placement. Testing throughout this project was conducted post-stimulation, which was reported by Yeung et at. (1977) to be an inappropriate testing method because they were unable to produce post-stimulation analgesia in the absence ot motor effects. However, in this later study, post-stimulation analgesia was only obtained, according to the authors, at high current intensities, and they suggested that this may have been due to the electrode configuration (bipolar rather than monopolar) and stimula tion parameters used. Cannon et a l . (1982) reported that post stimulation analgesia was more easily elicited from dorsal regions than ventral regions. The latter authors suggested therefore that this method was an ineffective approach for evaluating SPA from ventral sites. However, in that study, the ventral group contained seven anomalous sites from which post-stimulation could not be produced. The remaining twenty ventral sites produced post- stimulation analgesia that was apparently Indistinguishable from that of the dorsal group; therefore, this ventral-dorsal distinction may have been a r tific ia lly inflated by the inclusion of these seven anomalous points in the analysis of post-stimulation effects. In the present study no such differential effectiveness was found between dorsal and ventral sites. The duration of the analgesia produced by stimulation in the present study ranged from less than the two minute interstimulation Interval to twenty-two minutes; however, duration was found to be independent of electrode placement, and therefore sites producing short duration analgesia were not located in the outer boundaries ot the PAG or at some distance from an obvious focal point. This suggests that there is no central nuclear region within the PAG that is activated by stimulation whereby all points central to that locus produce long duration analgesia and distal points produce short duration analgesia. It should be noted that in assessing analgesic duration, the intensity required to produce that analgesia is a confounding variable In that higher intensities might be expected to produce longer durations. However, not only was there no correlation between duration and Intensity in the present study but the three longest analgesic durations (18, 18, and 22 minutes from a single stimulation! were produced at the lowest possible stimulation intensity used in this study, which was 10 microamperes. That electrode placement is independent of the production of pure analgesia is in contrast to the Fardin et el <1984b> report that pure analgesia (analgesia without associated motor effects) could only be proceed from ventral sites at two locations: one within the dorsal raphe and the other in the ventrolateral PAG. As is evident from Experiment 1 not only was pure analgesia not confined to the ventral PAG in two small areas, but multiple sites throughout the PAG, both ventral and dorsal, supported analgesia without any observable motor effects. There is some tendency, as Is apparent from Figure 1, for ventral placements that produce SPA to be located within or adjacent to the dorsal raphe, suggesting a role for this nucleus in this 56 analgesic effect. The two pure analgesic sites of the Fardin et al. (1984b) study were also located within and lateral to the dorsal raphe, which is in agreement with the present findings. In this earlier study, the ventrolateral site described was immediately adjacent to the dorsal raphe nucleus as it appears Just laceral to midllne in the caudal extent of the PAG and not at the ventrolateral boundary of the PAG. Furthermore, Fardin et al (1984a 8. b> report that motor effects were produced more frequently in the dorsal PAG than in the ventral PAG; however, these authors continued to increase current intensity beyond that which produced analgesia to took for motor effects. This was not done in the present study. Morgan, Franklin, & Abbott (1986) found that stimulation of any area of the PAG w ill produce analgesia but that this effect is always accompanied by motor effects; they subsequently hypothesized that all SPA originating from the PAG is secondary to stress effects. In the present study, motor effects were e lic ite d twice as frequently in the absence of analgesia, often at the upper lim its of the current intensities employed, than in the presence of analgesia, suggesting that motor effects alone are not indicative of sufficient stress to produce analgesia. Furthermore, motor effects accompanied by anal gesia were usually produced at relatively low current intensities. Experiment 1 also addressed the site specificity of the reduc tion in SPA produced by naloxone injection. Naloxone was found to have a d ifferential effect at ventral and dorsal sites. It completely abolished (reversal) or diminished (partial reversal) the analgesia produced from stimulation of ventral sites and had relatively no 5 7 effect on the analgesia produced from stimulation of dorsal sites. This contradicts the findings of Yaksh et al (1976) and supports the findings of Cannon et al (1982). The Yaksh et a l. (1976) stimulation was applied throu^iout the duration of the testing procedures, which ranged from 45 seconds to 2 minutes; this extremely long stimulation duration may account for the ineffectiveness of naloxone in reversing SPA in that study. The Cannon et a l. study confined its examination to midline points, however, and the present study found this dorsal- ventral differentiation to persist within more lateral aspects of the PAG. These findings strongly support the hypothesis that two analgesic systems co-exist within the PAG, one naloxone-reversible and the other non-naloxone reversible. It should be noted, however, that although ventral sites tended to be naloxone-reversible and dorsal sites non-naloxone-reversible, there was some overlap between these two regions. Furthermore, as was true with ventral sites in genera), naloxone-reversible sites were found primarily in the area adjacent to or within the dorsal raphe, with no distinction between those within and those adjacent to this nucleus. This further suggests a role for the dorsal raphe in the production of SPA at least from ventral sites. There is, therefore, considerable indirect evidence from Experiment 1 to suggest a role for the dorsal raphe in SPA. First, SPA was e lic ite d only from PAG regions along the anterior-posterior axis in which the dorsal raphe is present; SPA was never found in the more caudal PAG in the vicin ity of the locus coeruleus. Secondly, ventral sites, and more specifically naloxone-reversible sites, tended 56 to surround the dorsal raphe nucleus. The findings of Experiment 2 further suggest a role for the raphe nuclei, or at least for sero tonin, in the phenomenon of stimulation-produced analgesia from all sites within the PAG. Analgesia from both naloxone-reversible and non-reversible sites was reversed by the serotonin antagonist, methysergide, with no significant difference between this drug's effect at sites differin g in naloxone response. This is in support of the finding of Thorn and Plotkin (1986) that methysergide reversed analgesia to both a tail-fllck test and a formalin test even though naloxone only reversed analgesia to the ta il-flic k test. There are at least two possible explanations for this reversal of both naloxone-reversed and non-reversed analgesia by methysergide. First, stimulation of the PAG may activate two separate systems, one naloxone-reversible and the other non-naloxone-reversible , which exist as parallel systems without interaction, and that utilize serotonin at same point within each system. This explanation is unlikely since cross-tolerance developed between these two systems, thereby indicating some level of Interaction. However, it is possible that this interaction occurs rostral to the u tiliza tio n of serotonin and that different serotonergic neuronal pools are activated by PAG stimulation. An alternative explanation is that these two systems are not completely distinct en titles but at some level share a common neuronal pool, which utilizes serotonin. This latter explanation will be further addressed subsequently. Experiment 1 demonstrates that chronic stimulation of any point within the PAG results in a loss of the analgesic effect of 59 stimulation. This loss of effect is not only present at the site of original stimulation but also at a point distant to the original site, as wag evident from the cross-tolerance data. This was true despite the fact that the two sites of stimulation differed In their response to naloxone. The loss of effect at an individual electrode site has been termed tolerance by others (Mayer 8. Hayes, 1974, Lewis 8. GeDhart, 1977, Thorn-Gray, Johnson, 8. Ashbrook, 1982) and may be due to the same mechanians discussed In the introduction for morphine tolerance (hypertrophy of the adenylate cyclase system, alterations of the neural membranes, or changes in the calcium Ion concentration). The rapid development of tolerance to stimulation, however, makes these explanations suspect. Alternative explanations for this loss of effect from chronic stimulation are tissue damage at the electrode site (Mayer 8. Hayes, 1975), neuronal fatigue (Delgado, 1980), negative kindling (Delgado, 1980), and monoamine depletion (Bianchi, Sinisea I chi, Verattl, & Beanl, 1986), as discussed in the intro duction. In the present study, tissue damage is not a plausible explanation for this loss of effect in these animals since a sub sequent test was done on all but two electrode implants with continued production of analgesia possible at the original or nearly original threshold levels. However, a substantial number of sites (36) throughout this project demonstrated a loss of effect between testing sessions, which may have resulted from tissue damage secondary to stimulation at the preceeding testing session. Kindling is seen primarily with repeated stimulation over a number of consecutive days 6 0 (Delgado, 1981), which was not the procedure used to produce tolerance. In the present study, this loss of effect occurred within a single testing session. However, neuronal fatigue and neuro- transmitter depletion remain plausible explanations for this toss of effect following chronic stimulation. These explanations w ill be discussed in more detail in the subsequent discussion of cross- tolerance . The loss of effect not only at the original site of stimulation but at a site distant to the original has been called cross-tolerance (Thorn-Gray, Johnson, & Ashbrook, 1982). In the present study, cross-tolerance was seen between natxone-reversible and non-reversible sites, suggesting a bi-directional interaction between reversed and non-reversed systems. Aside front the mechanisms already discussed in relation to opioid tolerance, possible explanations for the cross- tolerance effect are current spread from the firs t electrode site to the second and the above mentioned phenomena of neuronal fatigue and neurotransmitter depletion. Current spread is an unlikely explanation for this loss of effect in the present experiment. Ranck (1973) reports that for a monopolar electrode the current spreads radially outward from the electrode with the current density diminishing as the square of the distance from the electrode tip. Bagshaw and Evans (1976) report that for a current of 10 microamps, current spread would be expected for a .15 millimeter radius, and for a current of 100 mlcroamps, expected spread would be over a radius of .51 millimeters. In the present study, all of the electrode pairs were separated by a millimeter or more (actual range 61 was from .97nro to 2.5ram after variations in dorsal-ventral, medial- lateral, and anterior-posterior directions were taken into account;, which would make activation of the second site by current spread from the initial site implausible at the current intensities used in the present cross-tolerance testing <10 to 40 microamps). Therefore, this loss of effect, if not due to traditional oploid-tolerance mechanisms, is probably due to neuronal fatigue or neurotransmitter depletion. The production of neuronal fatigue or neurotransmitter depletion, occurring simuttaneously in naioxone-reversiIbe and naloxone-non- reversible systems, can be explained by two mechanisms: co-activation of both systems by stimulation of either or the sharing of a common neuronal substrate. It is possible to hypothesize several mechanisms by which co activation coutd take place. F irs t, direct projections from the naloxone-reversible system in the ventral PAG to the non-naloxone reversible system in the dorsal PAG and vice versa would allow for this bi-directional co-activation. Alternatively, direct projections from each region to a second structure (eg.nucleus raphe magnus or reticular formation) with reciprocal projections back to the PAG could result in an indirect co-activation from stimulation of one region. This stimulation would therefore activate this second structure, thereby activating the alternate system via the reciprocal projections. Contrastingly, stimulation most likely activates both presynaptic and postsynaptic neurons simultaneously. As a result, stimulation could activate a conxnon pre-synaptic projection from a centrally located mechanism, (eg. hypothalamus) with projections to 62 both naloxone-reversible and non-reversible regions and thereby produce co-activat Ion. It Is also possible to hypothesize several sites at which naloxone-reversibie and naloxone-non-reversible PAG projections might share a cannon neuronal pool. Both dorsal and ventral regions of the PAG have rostral projections to the hypothalamus, thalamus, and other forebraln structures (Chi, 1970; Ruda, 1975; Eberhart et a l., 1985;& Watson et a l., 1977). Although this would be an indirect route for subsequent spinal projections, a cannon rostral neuronal pool is possible. Furthermore, a PAG rostra) projection has been linked to the analgesic action of morphine micro-inject ion into the PAG (Han & Xuan, 1986). However, there are also numerous cannon projection field s of both the dorsal and ventral PAG within the brainstem, specifically the nucleus raphe magnus (Fardin & Oilveras, 1962; Beitz et a l., 1983; & Basbaum & Fields, 1978), and the medullary nucleus re tic u la ris paraglgantoceilularis and nucleus retic u la ris giganto- c e llu la rls (Ruda, 1975; Beitz et a l ., 1983; & Basbaum & Fields, 1978). It is possible, therefore, that a cannon neuronal substrate could be activated in the brainstem by PAG-orlginating projections. If neurotransmitter depletion Is the mechanism by which stimuI ation-tolerance and cross-tolerance effects are produced within this cannon neural poo), serotonin may be the neurotransmitter involv ed. Stimu)ation-tolerance has been reversed by the aCkninistrati on of 5-hydroxytryptophan in the cat (Besson et a l ., 1981) and L-tryptophan in humans (Hosobuchl, 1978), suggesting that chronic stimulation may result in serotonin depletion. The findings of Experiment 2 63 demonstrate common usage of serotonin by both naloxone-reversibie and naloxone-non-reversible systems. Further research is necessary to determine the mechanisms involved in stimulation-tolerance and cross- tolerance and the site at which serotonin is utilized within the two analgesic systems of the PAG. Additional research Is also needed to determine if stimulation tolerance is accompanied by the same physio logical responses that accompany opioid tolerance. From the present study, several conclusions can be drawn regard ing stimulation ot the periaqueductal gray to produce analgesia. First, stimulation of multiple sites from Its rostral to caudal boundaries supports stimulation-produced analgesia in regions adjacent to the dorsal raphe. This analgesia is similar in duration and degree and demonstrates minimal site specificity. 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