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Paralemniscal reticular formation: Response of cells to a noxious stimulus
217
Brain Research, 267(1983) 217- 223 Elsevier Biomedical Press
Paralemniscal Reticular Formation:
R e s p o n s e o f C e l l s to a N o x i o u s S t i m u l u s
S. G. PATRICK HARDY*, HENRY J. HAIGLER** and GEORGE R. LEICHNETZ
Departments of (S. G.P. H.) Community Health and (H.J.H.) Pharmacologr', School of Medicine, Emory Universit), Atlanta, GA 30322 and (G.R. L.) Department of A natom)', Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298 (U.S.A.) (Accepted October 26th, 1982)
Key words: paralemniscal reticular formation - rat - nociception - analgesia - periaqueductal gray - horseradish peroxidase -microiontophoresis - norepinephrine
Extracellular single unit recordings were made in the paralemniscal reticular formation in adult male rats. A majority of the ceils studied were characterized as nociceptive because a noxious stimulus evoked a change (either an increase or decrease) in their spontaneous firing rates. Norepinephrine (NE) administered microiontophoretically usually mimicked the response to the noxious stimulus (foot pinch). After a neuron had been characterized with respect to its response to NE and the noxious stimulus, horseradish peroxidase (HRP) was iontophoretically ejected from the micropipette. Following iontophoresis of HRP into the paralemniscal reticular formation, retrograde and orthograde labeling was observed in the periaqueductal gray and the nucleus • raphe magnus. These data support a possible role of the paralemniscal reticular formation in an endogenous analgesic system. INTRODUCTION
It has been well documented that stimulusproduced analgesia can be elicited from sites located along the midline (raphe) of the brainstem ~.6.u.~3. Recently it was demonstrated that stimulus-produced analgesia can also be elicited from lateral brainstem sites (lateral reticular formation) in rabbits 9, cats 3 and primates 17. Furthermore, it has been demonstrated that certain regions of the lateral reticular formation are reciprocally connected with the spinal cord z~4.2~. Because these regions of the lateral reticular formation are located along the medial extent of the lateral lemniscus they will be referred to in this paper as the paralemniscal reticular formation (PRF). Because the PRF is reciprocally connected with the spinal cord, and is also a site from which stimulus-produced analgesia can be elicited, we concluded that the PRF may play a role in an endogenous ~inalgesic system. To examine this question further we per-
formed the following experiments to determine (a) if neurons in the PRF respond to a noxious stimulus, (b) if NE, a putative nociceptive neurotransmitter 8, would mimic the effect of the noxious stimulus and (c) what anatomical relationships, if any, exist between the PRF and areas that are believed to be part of an endogenous analgesic system. MATERIALS AND METHODS
A total of 29 Sprague-Dawley rats (Charles Rivers Laboratories, Wilmington, MA) weighing between 250 and 350 g were used in this study. All animals were anesthetized with chloral hydrate (400 mg/kg). Stereotaxic coordinates were initially derived from K6nig and Klippel ~j and Palkovits and Jacobowitz ~6 but were adjusted based on pilot experiments. Fourteen of these rats were used in electrophysiological experiments in which PRF neurons were studied initially and then HRP was ejected iontophoreti-
* Present address: Department of Anatomy, University of Mississippi, Schoolof Medicine, Jackson, MS 39216, U.S.A. ** Present address: Section Head, CNS Pharmacology, SEARLE Research and Development, SEARLE Pathway, Skokie, IL 60077, U.S.A. 0006-8993/83/0000- 0000/$03.00 © 1983 Elsevier Science Publishers
218 cally into that region. In the other 15 rats. HRP was injected into other areas (see below) using a Hamilton syringe or ejected from a micropipette. In the electrophysiological experiments, spontaneous and evoked firing rates of cells within the PRF were studied using a 7-barrel micropipette having an overall tip diameter of 5 7/~m. The recording barrel was filled with 2 M NaC1, the balance barrel was filled with 4 M NaCI, and two barrels were filled with 4 7% HRP (SigmaVI). A fifth barrel was filled with NE (0.2 M, pH 4.0) and the remaining two barrels were filled with various other drugs that were not involved in this study. The micropipette was lowered through the brain by a hydraulic microdrive (David Kopf Instruments). Action potentials, recorded extracellularly, were initially amplified by a single channel wide band electrometer (W-P Instruments). The output was displayed on a single beam oscilloscope (Tektronix D10) and was made audible by an audio amplifier (Grass AM 8). When the amplitudes of action potentials exceeded a certain preset threshold, pulses from a spike height discriminator (W-P Instruments) were recorded on a pen recorder (Gould Brush 220) to form analog records of the single unit firing. The pulses were also led into a microprocessor for the evaluation of single unit firing (Dilogic Corp., MESUF- 1). Initially each cell was categorized as nociceptive or non-nociceptive based on whether a noxious stimulus (NS; foot pinch) evoked a change in its firing rate. The NS used to evoke a response from the cells was pressure delivered to the left hind foot with an analgesy meter (Stoelting, no. 51302) using a method modified from Randall and Selitto ~9. This apparatus allows the same pressure to be delivered repeatedly to the foot via a small stylus; this pressure was increased from 90 to 540 lbs/in 2 without changing the position of the foot or touching the animal. Using this apparatus it has been determined that a pressure of 165 lbs/in 2 is sufficient to elicit pain-related behavior in the unanesthetized rat 7. Furthermore, it has been demonstrated that this same amount of pressure will produce desynch-
ronization of electroencephalogram activity in the anesthetized rat ~. The protocol for the delivery of the nociceptive stimulus was as follows. The left hind foot was trapped between the stylus and a pedestal by an applied pressure of 90 lbs/in2 for an equilibration period of 10 30 s. This allowed for any stimulation due to pressure or the change in joint position to be detected; even when such responses were present, which was infrequent, they disappeared rapidly. Without moving the foot, the pressure was increased to 540 lbs/in 2 in 14 s: this pressure was maintained for an additional 30 s. A computer was used to analyze the change in neuronal firing according to the method suggested by Hosford and Haigler 1°. Briefly, a oneway analysis of variance (ANOVA) was used to determine if the NS produced a significant effect (P <0.01) on spontaneously firing neurons. When there was no significant difference between the firing rates of two 10-s spontaneousfiring intervals the mean and variance of the last 10-s interval was stored in memory as the control period. Immediately after the onset of either the microiontophoresis of a drug or the noxious stimulus the computer accumulated the firing rate over the next 5, 10-s intervals (test period). The null hypothesis of no change in firing rate was rejected if there was a significant change in firing rate at the 0.01 level. All control periods were taken immediately prior to either noxious stimulus or drug administration. After recording from several cells (located within either the PRF or other brain sites) in each rat, the electrode tip was positioned in the PRF and horseradish peroxidase (4-7% in sterile saline) was ejected using an ejection current of 5 txA for 2 10 minutes. Following this ejection period the microelectrode was allowed to remain in position for a period of 5-10 min prior to its removal from the brain, thus decreasing the possibility of having HRP spread along the electrode track when the electrode was removed. Following a 24 h survival period the brains were removed, processed using a tetramethylbenzidine protocoP S and studied with both bright-field and dark-field microscopy. This re-
219 vealed the presence of labeled cell bodies and axons within the periaqueductal gray (PAG) and nucleus raphe magnus (NRM). To determine if the axonal labeling in these areas was orthograde or retrograde we administered HRP to these sites in a total of 15 additional rats. In 5 rats HRP (7-25% HRP in sterile saline) was ejected into the NRM using the iontophoretic procedure previously described. In 10 rats microinjections of HRP (0.01-0.02/~1; HRP in sterile saline) were made into the PAG, using a 5/~1 Hamilton syringe held in the microdrive apparatus. RESULTS Recordings were made from 48 separate single units located in the lateral reticular formation, inferior colliculus, ~:erebral Cortex and cerebellum. Thirty-three of these recordings were made in the PRF (P 0.5-2.0). In 30 of these PRF units the noxious stimulus changed the spontaneous neuronal firing rates. Therefore, these units were classified as nociceptive units. The remaining 3 units studied in this region were not affected by noxious stimulus. The rate of spon-
taneous firing was significantly increased in 16 units and significantly decreased in 14 units. In 18 of these 30 nociceptive units the response to noxious stimulus was mimicked by NE administered microiontophoretically (see Fig. 1). Of the 12 remaining nociceptive units in the PRF, 3 were not tested with NE, 8 responded to NE in a manner opposite to noxious stimulus mimicry, and 1 did not have a consistent response to NE. Control recordings (n = 6) were made of units in the inferior colliculus. Three of these units were classified as nociceptive based on the previously described criterion. In each of these nociceptive units the response to noxious stimulation was mimicked by NE. Additional control recordings (n = 9) were made in brain sites other than previously mentioned. Seven recording sites were located in the cerebral cortex or cerebellum and 2 were located in lateral reticular formation sites (P 2.8), caudal to the PRF. Of these 9 units only 1 unit (located in the cerebellum) was classified as nociceptive. Its spontaneous firing rate was increased in response to the noxious stimulus but this response was not mimicked by NE. Immediately following the single unit recordnociceptive,
response
•
nociceptive,
response
0
non-nociceptive
u ,%,i:,::. 0 P 0.5
P 1.0
P 1.5
,,\ ,:'):
is
mimicked
not
mimicked
with
NE
with
NE
o P 2.0
Fig. 1. Representativesinglesectionsillustrating the distributions of single unit recordingsites within the paralemniscalreticular formationand inferiorcotliculus.
220 TABLE I
Retrograde labeling sites following HRP ejections into the PRF Medulla
Lateral reticular nucleus Nucleus reticularis gigantocellularis Nucleus reticularis parvocellularis Facial nucleus * Nucleus raphe magnus * Nucleus reticularis magnocellularis
Pons
Nucleus reticularis pontis oralis Nucleus reticularis pontis caudalis
Midbrain
* Periaqueductal gray * Superior colliculus Pretectal nucleus Interstitial nucleus of Cajal Occulomotor complex
Diencephalon
Parafascicular nucleus Zona incerta Medial forebrain bundle Paraventricular nucleus
Other
Central amygdaloid nucleus Prefrontal cortex
yond the scope of this paper to consider all of the sites where labeling appeared, we will focus on
* Labeling patterns in these sites are discussed in this paper.
ings HRP was ejected into the PRF (P 0.5 P 2.0). This resulted in retrograde (see Table I) and orthograde labeling in numerous sites in the brainstem and diencephalon. Because it is be~
~
~
,
~
PLG 9
Fig. 2. Representative single sections illustrating the retrograde labeling in the periaqueductal gray (PAG) and nucleus raphe magnus (NRM) following a horseradish peroxidase (HRP) ejection into the paralemniscal reticular formation (PRF). Photomicrographs of labeling occurring in these sections can be seen in Figs. 3 and 4 for the PAG and NRM respectively. SC, superior colliculus: IC, inferior colliculus; VII, facial nucleus.
Fig. 3. Photomicrographs of filled cell bodies and axons in the periaqueductal gray (PAG) and deep layer of the superior colliculus (SC), following an ejection of HRP into the paralemniscal reticular formation. A: low power dark-field photomicrograph of the PAG and SC to show filled axons entering these regions. Note the dark, sparcely labeled gap separating the labeled PAG areas, pars dorsalis and pars lateralis. B: bright-field photomicrograph of A to show retrogradely labeled cell bodies in the PAG and SC. Arrows indicate individual labeled cells in the SC as well as a cluster of labeled cells in the PAG, pars dorsalis. The rectangle indicates a cluster of labeled cells in the PAG, pars lateralis. The dotted line represents the outer boundary of the PAG. Note that few labeled cells are present in thegap separating the retrogradely labeled PAG areas, pars dorsalis and pars lateralis. Aq, cerebral aqueduct. C: higher magnification of the area within the rectangle of g.
221 the labeling found in two areas presumed to be part of an endogenous analgesia system, the periaqueductal gray (PAG) and nucleus raphe magnus (NRM) (see Fig. 2). Following the H R P ejections in areas of the PRF where a nociceptive neuron was found, a consistent retrograde labeling pattern was observed in the region of the PAG and adjacent superior colliculus (see Fig. 3). In the superior colliculus (stratum p r o f u n d u m and stratum intermedium), primarily ipsilateral to the HRP ejection site, large (19 ~M) cell bodies were filled with HRP. This population of cells in the superior colliculus appeared to be contiguous with another population of smaller (12 ttM) labeled cells in the pars lateralis of the PAG. Small labeled cells in the PAG were also in the pars dorsalis. These two groups of labeled cells in the pars dorsalis and pars lateralis of the PAG were always separated by a gap where there were few, if any, labeled cells (see Fig. 3). Retrogradely filled cells bodies (approximately l(LI5 in any one section) were also found in the N R M and adjacent nucleus reticularis magnocellularis (see Fig. 4). Labeled cells were more numerous in the nucleus reticularis magnocellularis, but this group of cells appeared to be contiguous with labeling which extended into the NRM. In addition to labeled cell bodies, abundant axonal labeling was observed in the regions of the PAG and NRM, thereby indicating that some of these axons may have been orthogradely labeled. In order to examine this question, HRP was injected into the PAG or N R M in each of an additional 15 rats. In each case filled cell bodies were observed in the PRF following either the injection or ejection of HRP. These data support the hypothesis that some of the axonal labeling that had originally been seen in the PAG and NRM, after ejections of HRP into the PRF, resulted from orthograde labeling, and that bidirectional connections may exist between these structures. DISCUSSION
The results of this study provide additional
Fig. 4. Photomicrographs of filled cell bodies and axons in the nucleus raphe magnus (NRM) and adjacent nucleus reticularis magnocellularis (Rmc), following an ejection of HRP into the paralemniscal reticular formation. A: low power dark-field photomicrograph of the NRM and Rmc to show filled axons entering this region. P, corticospinal tract. B: bright-field photomicrograph of A to show retrogradely labeled cell bodies in the NRM and Rmc. Arrows indicate individual labeled cells in the NRM. The rectangle indicates a cluster of labeled cells in the Rmc. C: higher magnification of the area within the rectangle of B.
support that the PRF, a component of the lateral reticular formation, may be involved in an endogenous analgesic system. First, the majority (30/33) of spontaneously firing units in the PRF
222 are nociceptive, responding to a noxious stimulus with either an increase or decrease in neuronal firing. Secondly, the response to the noxious stimulus was frequently (60% of the time) mimicked by a putative nociceptive neurotransmitter, NE 8, administered microiontophoretically. The failure of NE to mimic the response to the nociceptive stimulus in all of the neurons in the PRF suggests that there are other nociceptive neurotransmitters in this region. In this regard, it should be noted that many of the PRF units in which NE mimicked the nociceptive response were interposed between the brachium conjunctivum and the lateral lemniscus (see Fig. 1). This specific region corresponds to the location of the catecholaminergic nucleus, A7, in which the cells are presumed to contain catecholamines~. Further evidence suggesting that the PRF is part of an endogenous analgesic system is the fact that reciprocal connections apparently exist between the PRF and two regions (PAG and adjacent superior colliculus: NRM and adjacent nucleus reticularis magnocellularis), the electrical stimulation of which produces profound analgesia tx'-~-"t~.-'°. In addition, the PRF is a principal contributor to the dorsolateral funiculus of the rat spinal cord 2.2t, the integrity of which is essential to central analgesic systemss. These data are in agreement with previous evidence that suggests the PRF is part of an endogenous analgesic system. For instance, the PRF is located within a region from which stimulusproduced analgesia may be elicited in various species~.~-~7. In addition, it has been demonstrated that the region corresponding to the PRF receives nociceptive input from the spinothalamic tract H and. in turn, contains numerous cells
REFERENCES 1 Basbaum, A. 1., Clanton, C. H. and Fields, H. L.. Opiate and stimulus-produced analgesia: functional anatomy of a medullospinal pathway, Proc. nat. Acad. Sci. U.S.A., 73 11976)4685 4688. 2 Basbaum. A. I. and Fields, H. L., The origin of descending pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation, J. comp. Neurol.. 187 (1979) 513-532. 3 Basbaum, A. l.,Marley, N. J. E., O'Keefe, J. and Clanton, C. H., Reversal of morphine and stimulus-produced
that project down the dorsolateral funiculus, along with projections from the NRM, to the spinal cord 2.2~. These projections may be part of a circuit which modulates the perception of, or response to, a noxious stimulus. The location of many of these PRF-spinal neurons corresponds precisely to the location of Dahlstr6m and Fuxe's catecholaminergic area AT --~. Furthermore, it has been demonstrated that the catecholamine, NE, is one of two neurotransmitters that mediates descending analgesia to the spinal cord :223. Accordingly, it is possible that A7 is a primary source for the NE involved in the descending analgesic system. Although the PRF may directly modulate the response to a noxious stimulus at a spinal cord level, it is also possible that it affects analgesic mechanisms through its projections to the PAG and NRM. One of several possibilities is that the PAG may influence the PRF which may in turn act upon the NRM. The existence of indirect projections from the PAG to the NRM has already been suggested by Pomeroy and Behbehani ~. Therefore, we conclude that the PRF may be a noradrenergic link in an endogenous analgesic system that modulates the response to a noxious stimulus either at a spinal or supraspinal level, ACKNOWLEDGEMENTS
We would like to thank Ms. Sandy Hosford and Ms. Joanne Botkin for their invaluable assistance. This project was supported in part by NIDA Grant 1-R01-DA-01344-06 to H.J.H. and NSF Grant BNS 7822971 to G.R.L.
analgesia by subtotal spinal cord lesions, Pain, 3 (1977) 43- 56. Carstens, E., Klumpp; D. and Zimmerman, D., Differential inhibitory effects of medial and lateral midbrain stimulation on spinal neuronal discharges to noxious skin heating in the cat, J. Neurophysiol., 43 (1980) 332 342. Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, A eta physioL scand., 62 Suppl. 232(1964) I 55.
223 6 Fields, H. L., Basbaum, A. I., Clanton, C. H. and Anderson, S. D., Nucleus raphe magnus inhibition of spinal cord dorsal horn neurons, Brain Research, 126 (1977) 441-453. 7 Haigler, H. J., Morphine: effects on serotonergic neurons in areas with a serotonergic input, Europ. J.Pharmacol., 51(1978)361 376. 8 Haigler. H. J. and Spring, D. D., Putative nociceptive neurotransmitter in the mesencephalic reticular formation, LifeSci., 29(1981)33-43. 9 Herz, A., Albus, K., Metys, J., Schubert, P. and Teschemacher, H., On the central sites for the antinociceptive action of morphine and fentanyl, Neuropharmacology, 9 (1970) 539- 551. 10 Hosford, D. A. and Haigler, H. J., Morphine and methionine-enkephalin: differential effects on spontaneous and evoked neuronal firing in the mesencephalic reticular formation of the rat, J. Pharmacol. exp. Ther., 213 (1980) 355-363. 11 K6nig, J. F. R. and Klippel, R. A., The Rat Braih: A stereotaxic Atlas of the Forebrain and Lower Parts of the Brainstem, Williams and Wilkins, Baltimore, 1963. 12 Mayer, D. J., Endogenous analgesia systems: neural and behavioral mechanisms. In J. J. Bonica (Eds.), Advances in Pain Research and Therap), Vol. 3, Raven Press, New York, 1979. 13 Mayer, D. J. and Price, D. D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379-404. 14 Mehler, W. R., Some neurological species differences - a posteriori,Ann. N, Y. A cad. Sci., 167 (1969) 424- 468. 15 Mesulam, M.-M., A tetramethylbenzidine method for light microscopic tracing of neural connections with horseradish peroxidase (HRP) neurochemistry. In Neu-
16
17 18
19 20 21
22
23
roanatomical Techniques, Short Course, Soc. for Neurosci., 1978, pp. 65-71. Palkovits, M. and Jacobowitz, D. M., Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. 1I. Hindbrain (mesencephalon, rhombencephalon),J, comp. Neurol., 157 (1974) 29-42. Pert, A. and Yaksh, T. L., Sites of morphine induced analgesia in the primate brain: relation to pain pathways, Brain Research, 80 (1974) 135- 140. Pomeroy, S. L. and Behbehani, M. M., Physiologic evidence for a projection from periaqueductal gray to nucleus raphe magnus in the rat, Brain Research., 176 (1979) 143- 147. Randall, L.. O. and Selitto, J. J., A method for measurement of analgesia on inflamed tissue, Arch. int. Pharmacodyn., I 11 (1957) 409-419. Soper, W. Y., Effects of analgesic midbrain stimulation on reflex withdrawal and thermal escape in the rat, J. comp. physiol. Psychol., 90 (1976) 91- 101. Watkins, L. R., Griffin, G., Leichnetz, G. R. and Mayer, D. J., Identification and somatotopic organization of nuclei projecting via the dorsolateral funiculus in rats: a retrograde tracing study using HRP slow-release gels, Brain Research, 223 ( 1981 ) 237- 255. Yaksh, T. L., Direct evidence that spinal serotonin and noradrenaline terminals mediate the spinal antinociceptive effects of morphine in the periaqueductal gray, Brain Research, 160(1979) 180- 185. Yaksh, T. L., Hammond, D. L. and Tyce, G. M., Functional aspects of bulbospinal monoaminergic projections in modulating processing of somatosensory information, Fed. Proc., 40 ( 1981 ) 2786- 2793.
Brain Research, 267(1983) 217- 223 Elsevier Biomedical Press
Paralemniscal Reticular Formation:
R e s p o n s e o f C e l l s to a N o x i o u s S t i m u l u s
S. G. PATRICK HARDY*, HENRY J. HAIGLER** and GEORGE R. LEICHNETZ
Departments of (S. G.P. H.) Community Health and (H.J.H.) Pharmacologr', School of Medicine, Emory Universit), Atlanta, GA 30322 and (G.R. L.) Department of A natom)', Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298 (U.S.A.) (Accepted October 26th, 1982)
Key words: paralemniscal reticular formation - rat - nociception - analgesia - periaqueductal gray - horseradish peroxidase -microiontophoresis - norepinephrine
Extracellular single unit recordings were made in the paralemniscal reticular formation in adult male rats. A majority of the ceils studied were characterized as nociceptive because a noxious stimulus evoked a change (either an increase or decrease) in their spontaneous firing rates. Norepinephrine (NE) administered microiontophoretically usually mimicked the response to the noxious stimulus (foot pinch). After a neuron had been characterized with respect to its response to NE and the noxious stimulus, horseradish peroxidase (HRP) was iontophoretically ejected from the micropipette. Following iontophoresis of HRP into the paralemniscal reticular formation, retrograde and orthograde labeling was observed in the periaqueductal gray and the nucleus • raphe magnus. These data support a possible role of the paralemniscal reticular formation in an endogenous analgesic system. INTRODUCTION
It has been well documented that stimulusproduced analgesia can be elicited from sites located along the midline (raphe) of the brainstem ~.6.u.~3. Recently it was demonstrated that stimulus-produced analgesia can also be elicited from lateral brainstem sites (lateral reticular formation) in rabbits 9, cats 3 and primates 17. Furthermore, it has been demonstrated that certain regions of the lateral reticular formation are reciprocally connected with the spinal cord z~4.2~. Because these regions of the lateral reticular formation are located along the medial extent of the lateral lemniscus they will be referred to in this paper as the paralemniscal reticular formation (PRF). Because the PRF is reciprocally connected with the spinal cord, and is also a site from which stimulus-produced analgesia can be elicited, we concluded that the PRF may play a role in an endogenous ~inalgesic system. To examine this question further we per-
formed the following experiments to determine (a) if neurons in the PRF respond to a noxious stimulus, (b) if NE, a putative nociceptive neurotransmitter 8, would mimic the effect of the noxious stimulus and (c) what anatomical relationships, if any, exist between the PRF and areas that are believed to be part of an endogenous analgesic system. MATERIALS AND METHODS
A total of 29 Sprague-Dawley rats (Charles Rivers Laboratories, Wilmington, MA) weighing between 250 and 350 g were used in this study. All animals were anesthetized with chloral hydrate (400 mg/kg). Stereotaxic coordinates were initially derived from K6nig and Klippel ~j and Palkovits and Jacobowitz ~6 but were adjusted based on pilot experiments. Fourteen of these rats were used in electrophysiological experiments in which PRF neurons were studied initially and then HRP was ejected iontophoreti-
* Present address: Department of Anatomy, University of Mississippi, Schoolof Medicine, Jackson, MS 39216, U.S.A. ** Present address: Section Head, CNS Pharmacology, SEARLE Research and Development, SEARLE Pathway, Skokie, IL 60077, U.S.A. 0006-8993/83/0000- 0000/$03.00 © 1983 Elsevier Science Publishers
218 cally into that region. In the other 15 rats. HRP was injected into other areas (see below) using a Hamilton syringe or ejected from a micropipette. In the electrophysiological experiments, spontaneous and evoked firing rates of cells within the PRF were studied using a 7-barrel micropipette having an overall tip diameter of 5 7/~m. The recording barrel was filled with 2 M NaC1, the balance barrel was filled with 4 M NaCI, and two barrels were filled with 4 7% HRP (SigmaVI). A fifth barrel was filled with NE (0.2 M, pH 4.0) and the remaining two barrels were filled with various other drugs that were not involved in this study. The micropipette was lowered through the brain by a hydraulic microdrive (David Kopf Instruments). Action potentials, recorded extracellularly, were initially amplified by a single channel wide band electrometer (W-P Instruments). The output was displayed on a single beam oscilloscope (Tektronix D10) and was made audible by an audio amplifier (Grass AM 8). When the amplitudes of action potentials exceeded a certain preset threshold, pulses from a spike height discriminator (W-P Instruments) were recorded on a pen recorder (Gould Brush 220) to form analog records of the single unit firing. The pulses were also led into a microprocessor for the evaluation of single unit firing (Dilogic Corp., MESUF- 1). Initially each cell was categorized as nociceptive or non-nociceptive based on whether a noxious stimulus (NS; foot pinch) evoked a change in its firing rate. The NS used to evoke a response from the cells was pressure delivered to the left hind foot with an analgesy meter (Stoelting, no. 51302) using a method modified from Randall and Selitto ~9. This apparatus allows the same pressure to be delivered repeatedly to the foot via a small stylus; this pressure was increased from 90 to 540 lbs/in 2 without changing the position of the foot or touching the animal. Using this apparatus it has been determined that a pressure of 165 lbs/in 2 is sufficient to elicit pain-related behavior in the unanesthetized rat 7. Furthermore, it has been demonstrated that this same amount of pressure will produce desynch-
ronization of electroencephalogram activity in the anesthetized rat ~. The protocol for the delivery of the nociceptive stimulus was as follows. The left hind foot was trapped between the stylus and a pedestal by an applied pressure of 90 lbs/in2 for an equilibration period of 10 30 s. This allowed for any stimulation due to pressure or the change in joint position to be detected; even when such responses were present, which was infrequent, they disappeared rapidly. Without moving the foot, the pressure was increased to 540 lbs/in 2 in 14 s: this pressure was maintained for an additional 30 s. A computer was used to analyze the change in neuronal firing according to the method suggested by Hosford and Haigler 1°. Briefly, a oneway analysis of variance (ANOVA) was used to determine if the NS produced a significant effect (P <0.01) on spontaneously firing neurons. When there was no significant difference between the firing rates of two 10-s spontaneousfiring intervals the mean and variance of the last 10-s interval was stored in memory as the control period. Immediately after the onset of either the microiontophoresis of a drug or the noxious stimulus the computer accumulated the firing rate over the next 5, 10-s intervals (test period). The null hypothesis of no change in firing rate was rejected if there was a significant change in firing rate at the 0.01 level. All control periods were taken immediately prior to either noxious stimulus or drug administration. After recording from several cells (located within either the PRF or other brain sites) in each rat, the electrode tip was positioned in the PRF and horseradish peroxidase (4-7% in sterile saline) was ejected using an ejection current of 5 txA for 2 10 minutes. Following this ejection period the microelectrode was allowed to remain in position for a period of 5-10 min prior to its removal from the brain, thus decreasing the possibility of having HRP spread along the electrode track when the electrode was removed. Following a 24 h survival period the brains were removed, processed using a tetramethylbenzidine protocoP S and studied with both bright-field and dark-field microscopy. This re-
219 vealed the presence of labeled cell bodies and axons within the periaqueductal gray (PAG) and nucleus raphe magnus (NRM). To determine if the axonal labeling in these areas was orthograde or retrograde we administered HRP to these sites in a total of 15 additional rats. In 5 rats HRP (7-25% HRP in sterile saline) was ejected into the NRM using the iontophoretic procedure previously described. In 10 rats microinjections of HRP (0.01-0.02/~1; HRP in sterile saline) were made into the PAG, using a 5/~1 Hamilton syringe held in the microdrive apparatus. RESULTS Recordings were made from 48 separate single units located in the lateral reticular formation, inferior colliculus, ~:erebral Cortex and cerebellum. Thirty-three of these recordings were made in the PRF (P 0.5-2.0). In 30 of these PRF units the noxious stimulus changed the spontaneous neuronal firing rates. Therefore, these units were classified as nociceptive units. The remaining 3 units studied in this region were not affected by noxious stimulus. The rate of spon-
taneous firing was significantly increased in 16 units and significantly decreased in 14 units. In 18 of these 30 nociceptive units the response to noxious stimulus was mimicked by NE administered microiontophoretically (see Fig. 1). Of the 12 remaining nociceptive units in the PRF, 3 were not tested with NE, 8 responded to NE in a manner opposite to noxious stimulus mimicry, and 1 did not have a consistent response to NE. Control recordings (n = 6) were made of units in the inferior colliculus. Three of these units were classified as nociceptive based on the previously described criterion. In each of these nociceptive units the response to noxious stimulation was mimicked by NE. Additional control recordings (n = 9) were made in brain sites other than previously mentioned. Seven recording sites were located in the cerebral cortex or cerebellum and 2 were located in lateral reticular formation sites (P 2.8), caudal to the PRF. Of these 9 units only 1 unit (located in the cerebellum) was classified as nociceptive. Its spontaneous firing rate was increased in response to the noxious stimulus but this response was not mimicked by NE. Immediately following the single unit recordnociceptive,
response
•
nociceptive,
response
0
non-nociceptive
u ,%,i:,::. 0 P 0.5
P 1.0
P 1.5
,,\ ,:'):
is
mimicked
not
mimicked
with
NE
with
NE
o P 2.0
Fig. 1. Representativesinglesectionsillustrating the distributions of single unit recordingsites within the paralemniscalreticular formationand inferiorcotliculus.
220 TABLE I
Retrograde labeling sites following HRP ejections into the PRF Medulla
Lateral reticular nucleus Nucleus reticularis gigantocellularis Nucleus reticularis parvocellularis Facial nucleus * Nucleus raphe magnus * Nucleus reticularis magnocellularis
Pons
Nucleus reticularis pontis oralis Nucleus reticularis pontis caudalis
Midbrain
* Periaqueductal gray * Superior colliculus Pretectal nucleus Interstitial nucleus of Cajal Occulomotor complex
Diencephalon
Parafascicular nucleus Zona incerta Medial forebrain bundle Paraventricular nucleus
Other
Central amygdaloid nucleus Prefrontal cortex
yond the scope of this paper to consider all of the sites where labeling appeared, we will focus on
* Labeling patterns in these sites are discussed in this paper.
ings HRP was ejected into the PRF (P 0.5 P 2.0). This resulted in retrograde (see Table I) and orthograde labeling in numerous sites in the brainstem and diencephalon. Because it is be~
~
~
,
~
PLG 9
Fig. 2. Representative single sections illustrating the retrograde labeling in the periaqueductal gray (PAG) and nucleus raphe magnus (NRM) following a horseradish peroxidase (HRP) ejection into the paralemniscal reticular formation (PRF). Photomicrographs of labeling occurring in these sections can be seen in Figs. 3 and 4 for the PAG and NRM respectively. SC, superior colliculus: IC, inferior colliculus; VII, facial nucleus.
Fig. 3. Photomicrographs of filled cell bodies and axons in the periaqueductal gray (PAG) and deep layer of the superior colliculus (SC), following an ejection of HRP into the paralemniscal reticular formation. A: low power dark-field photomicrograph of the PAG and SC to show filled axons entering these regions. Note the dark, sparcely labeled gap separating the labeled PAG areas, pars dorsalis and pars lateralis. B: bright-field photomicrograph of A to show retrogradely labeled cell bodies in the PAG and SC. Arrows indicate individual labeled cells in the SC as well as a cluster of labeled cells in the PAG, pars dorsalis. The rectangle indicates a cluster of labeled cells in the PAG, pars lateralis. The dotted line represents the outer boundary of the PAG. Note that few labeled cells are present in thegap separating the retrogradely labeled PAG areas, pars dorsalis and pars lateralis. Aq, cerebral aqueduct. C: higher magnification of the area within the rectangle of g.
221 the labeling found in two areas presumed to be part of an endogenous analgesia system, the periaqueductal gray (PAG) and nucleus raphe magnus (NRM) (see Fig. 2). Following the H R P ejections in areas of the PRF where a nociceptive neuron was found, a consistent retrograde labeling pattern was observed in the region of the PAG and adjacent superior colliculus (see Fig. 3). In the superior colliculus (stratum p r o f u n d u m and stratum intermedium), primarily ipsilateral to the HRP ejection site, large (19 ~M) cell bodies were filled with HRP. This population of cells in the superior colliculus appeared to be contiguous with another population of smaller (12 ttM) labeled cells in the pars lateralis of the PAG. Small labeled cells in the PAG were also in the pars dorsalis. These two groups of labeled cells in the pars dorsalis and pars lateralis of the PAG were always separated by a gap where there were few, if any, labeled cells (see Fig. 3). Retrogradely filled cells bodies (approximately l(LI5 in any one section) were also found in the N R M and adjacent nucleus reticularis magnocellularis (see Fig. 4). Labeled cells were more numerous in the nucleus reticularis magnocellularis, but this group of cells appeared to be contiguous with labeling which extended into the NRM. In addition to labeled cell bodies, abundant axonal labeling was observed in the regions of the PAG and NRM, thereby indicating that some of these axons may have been orthogradely labeled. In order to examine this question, HRP was injected into the PAG or N R M in each of an additional 15 rats. In each case filled cell bodies were observed in the PRF following either the injection or ejection of HRP. These data support the hypothesis that some of the axonal labeling that had originally been seen in the PAG and NRM, after ejections of HRP into the PRF, resulted from orthograde labeling, and that bidirectional connections may exist between these structures. DISCUSSION
The results of this study provide additional
Fig. 4. Photomicrographs of filled cell bodies and axons in the nucleus raphe magnus (NRM) and adjacent nucleus reticularis magnocellularis (Rmc), following an ejection of HRP into the paralemniscal reticular formation. A: low power dark-field photomicrograph of the NRM and Rmc to show filled axons entering this region. P, corticospinal tract. B: bright-field photomicrograph of A to show retrogradely labeled cell bodies in the NRM and Rmc. Arrows indicate individual labeled cells in the NRM. The rectangle indicates a cluster of labeled cells in the Rmc. C: higher magnification of the area within the rectangle of B.
support that the PRF, a component of the lateral reticular formation, may be involved in an endogenous analgesic system. First, the majority (30/33) of spontaneously firing units in the PRF
222 are nociceptive, responding to a noxious stimulus with either an increase or decrease in neuronal firing. Secondly, the response to the noxious stimulus was frequently (60% of the time) mimicked by a putative nociceptive neurotransmitter, NE 8, administered microiontophoretically. The failure of NE to mimic the response to the nociceptive stimulus in all of the neurons in the PRF suggests that there are other nociceptive neurotransmitters in this region. In this regard, it should be noted that many of the PRF units in which NE mimicked the nociceptive response were interposed between the brachium conjunctivum and the lateral lemniscus (see Fig. 1). This specific region corresponds to the location of the catecholaminergic nucleus, A7, in which the cells are presumed to contain catecholamines~. Further evidence suggesting that the PRF is part of an endogenous analgesic system is the fact that reciprocal connections apparently exist between the PRF and two regions (PAG and adjacent superior colliculus: NRM and adjacent nucleus reticularis magnocellularis), the electrical stimulation of which produces profound analgesia tx'-~-"t~.-'°. In addition, the PRF is a principal contributor to the dorsolateral funiculus of the rat spinal cord 2.2t, the integrity of which is essential to central analgesic systemss. These data are in agreement with previous evidence that suggests the PRF is part of an endogenous analgesic system. For instance, the PRF is located within a region from which stimulusproduced analgesia may be elicited in various species~.~-~7. In addition, it has been demonstrated that the region corresponding to the PRF receives nociceptive input from the spinothalamic tract H and. in turn, contains numerous cells
REFERENCES 1 Basbaum, A. 1., Clanton, C. H. and Fields, H. L.. Opiate and stimulus-produced analgesia: functional anatomy of a medullospinal pathway, Proc. nat. Acad. Sci. U.S.A., 73 11976)4685 4688. 2 Basbaum. A. I. and Fields, H. L., The origin of descending pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation, J. comp. Neurol.. 187 (1979) 513-532. 3 Basbaum, A. l.,Marley, N. J. E., O'Keefe, J. and Clanton, C. H., Reversal of morphine and stimulus-produced
that project down the dorsolateral funiculus, along with projections from the NRM, to the spinal cord 2.2~. These projections may be part of a circuit which modulates the perception of, or response to, a noxious stimulus. The location of many of these PRF-spinal neurons corresponds precisely to the location of Dahlstr6m and Fuxe's catecholaminergic area AT --~. Furthermore, it has been demonstrated that the catecholamine, NE, is one of two neurotransmitters that mediates descending analgesia to the spinal cord :223. Accordingly, it is possible that A7 is a primary source for the NE involved in the descending analgesic system. Although the PRF may directly modulate the response to a noxious stimulus at a spinal cord level, it is also possible that it affects analgesic mechanisms through its projections to the PAG and NRM. One of several possibilities is that the PAG may influence the PRF which may in turn act upon the NRM. The existence of indirect projections from the PAG to the NRM has already been suggested by Pomeroy and Behbehani ~. Therefore, we conclude that the PRF may be a noradrenergic link in an endogenous analgesic system that modulates the response to a noxious stimulus either at a spinal or supraspinal level, ACKNOWLEDGEMENTS
We would like to thank Ms. Sandy Hosford and Ms. Joanne Botkin for their invaluable assistance. This project was supported in part by NIDA Grant 1-R01-DA-01344-06 to H.J.H. and NSF Grant BNS 7822971 to G.R.L.
analgesia by subtotal spinal cord lesions, Pain, 3 (1977) 43- 56. Carstens, E., Klumpp; D. and Zimmerman, D., Differential inhibitory effects of medial and lateral midbrain stimulation on spinal neuronal discharges to noxious skin heating in the cat, J. Neurophysiol., 43 (1980) 332 342. Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, A eta physioL scand., 62 Suppl. 232(1964) I 55.
223 6 Fields, H. L., Basbaum, A. I., Clanton, C. H. and Anderson, S. D., Nucleus raphe magnus inhibition of spinal cord dorsal horn neurons, Brain Research, 126 (1977) 441-453. 7 Haigler, H. J., Morphine: effects on serotonergic neurons in areas with a serotonergic input, Europ. J.Pharmacol., 51(1978)361 376. 8 Haigler. H. J. and Spring, D. D., Putative nociceptive neurotransmitter in the mesencephalic reticular formation, LifeSci., 29(1981)33-43. 9 Herz, A., Albus, K., Metys, J., Schubert, P. and Teschemacher, H., On the central sites for the antinociceptive action of morphine and fentanyl, Neuropharmacology, 9 (1970) 539- 551. 10 Hosford, D. A. and Haigler, H. J., Morphine and methionine-enkephalin: differential effects on spontaneous and evoked neuronal firing in the mesencephalic reticular formation of the rat, J. Pharmacol. exp. Ther., 213 (1980) 355-363. 11 K6nig, J. F. R. and Klippel, R. A., The Rat Braih: A stereotaxic Atlas of the Forebrain and Lower Parts of the Brainstem, Williams and Wilkins, Baltimore, 1963. 12 Mayer, D. J., Endogenous analgesia systems: neural and behavioral mechanisms. In J. J. Bonica (Eds.), Advances in Pain Research and Therap), Vol. 3, Raven Press, New York, 1979. 13 Mayer, D. J. and Price, D. D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379-404. 14 Mehler, W. R., Some neurological species differences - a posteriori,Ann. N, Y. A cad. Sci., 167 (1969) 424- 468. 15 Mesulam, M.-M., A tetramethylbenzidine method for light microscopic tracing of neural connections with horseradish peroxidase (HRP) neurochemistry. In Neu-
16
17 18
19 20 21
22
23
roanatomical Techniques, Short Course, Soc. for Neurosci., 1978, pp. 65-71. Palkovits, M. and Jacobowitz, D. M., Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. 1I. Hindbrain (mesencephalon, rhombencephalon),J, comp. Neurol., 157 (1974) 29-42. Pert, A. and Yaksh, T. L., Sites of morphine induced analgesia in the primate brain: relation to pain pathways, Brain Research, 80 (1974) 135- 140. Pomeroy, S. L. and Behbehani, M. M., Physiologic evidence for a projection from periaqueductal gray to nucleus raphe magnus in the rat, Brain Research., 176 (1979) 143- 147. Randall, L.. O. and Selitto, J. J., A method for measurement of analgesia on inflamed tissue, Arch. int. Pharmacodyn., I 11 (1957) 409-419. Soper, W. Y., Effects of analgesic midbrain stimulation on reflex withdrawal and thermal escape in the rat, J. comp. physiol. Psychol., 90 (1976) 91- 101. Watkins, L. R., Griffin, G., Leichnetz, G. R. and Mayer, D. J., Identification and somatotopic organization of nuclei projecting via the dorsolateral funiculus in rats: a retrograde tracing study using HRP slow-release gels, Brain Research, 223 ( 1981 ) 237- 255. Yaksh, T. L., Direct evidence that spinal serotonin and noradrenaline terminals mediate the spinal antinociceptive effects of morphine in the periaqueductal gray, Brain Research, 160(1979) 180- 185. Yaksh, T. L., Hammond, D. L. and Tyce, G. M., Functional aspects of bulbospinal monoaminergic projections in modulating processing of somatosensory information, Fed. Proc., 40 ( 1981 ) 2786- 2793.