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Pain pathways

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RESPONSES OF CAT LAMINA I SPINOTHALAMIC TRACT (STT) CELLS TO TONIC NOXIOUS MECHANICAL STIMULI.

D. Andrew & A. D. Craig, Atkinson Pain Research Laboratory, Division ofNeurosurgery, Barrow Neurological Inst, Phoenix, AZ 85013, USA.

Aim of Investigation: To determine the responses ofnociceptive lamina I STT cells to sustained mechanical stimulation. Psycho-physically, such stimuli evoke pain sensations that increase over time (Andrew & Greenspan, Soc. Neurosci. Abstr. 27: 396.11, 1997).

Methods: Adult cats were anesthetized with sodium pentobarbital (40 mg/kg, I.P.), and prepared for recording lamina I STT cells with receptive fields on the distal hindlimb. Six co-axial electrodes were positioned in the contralateral thalamus under electrophysi-ological control. Single, antidromically identified lamina I STT cells were classified as COLD cells if they responded to innocuous cooling; as HPC cells if they responded to noxious heat, pinch and noxious cold; or as NS cells if they responded only to pinch or to pinch and noxious heat. HPC and NS cells were further studied with mechanical stimuli (25, 50 & lOOg applied with a probe of tip area 0.1 mm2) of 2 minutes duration.

Results: Recordings were made from 14 lamina I STT cells (11 HPC, 3 NS) with glabrous skin receptive fields. Responses to tonic stimuli were varied, encompassing those that exhibited slowly-adapting responses to those that maintained their discharge. Response patterns were largely independent of whether a cell received C-fiber inputs or not.

Conclusions: Similar to peripheral nociceptors, lamina I STT cells show a range of response patterns to tonic mechanical stimuli. This variation in responses demands a detailed comparison of their stimulus-response functions with those previously described for the primary afferents and with the psychophysical data.

Acknowledgments: Supported by NIH (NS25616) and Barrow Neurological Foundation.

RESPONSES OF LAMINA I SPINOTHALAMIC NEURONS TO REPEATED BRIEF CONTACT HEAT STIMULI.

A.D. Craig and D. Andrew, Atkinson Pain Research Laboratory, Division ofNeurosurgery, Barrow Neurological Inst, Phoenix AZ, U.S.A.

Aim of Investigation: To test the hypothesis that lamina I STT cells manifest the temporal summation ("windup") observed psycho-physically in human pain sensation with repeated brief contact heat stimuli by Vierck et al. (J.Neurophysiol. 78:992, 1997).

Methods: Single, antidromically identified lumbosacral lamina I STT cells with receptive fields on the glabrous hindpaw were recorded in Nembutal- or Saffan-anesthetized cats and characterized with quantitative, natural stimuli as NS (nociceptive-specific), HPC (polymodal nociceptive), or COLD (thermoreceptive-specific) cells. Repeated brief contact heat stimuli were applied with a 4 cm2 Peltier element preheated to 45-58C with a constant contact time (0.7 sec) using intervals of 2, 3 or 5 sec.

Results: Four HPC and one NS lamina I STT neurons have been tested. All five cells showed accelerating responses to repeated stimuli at 2 and 3 sec intervals but weak or no summation at 5 sec intervals. Response magnitude was inversely related to interstimu-lus interval and directly related to stimulus strength. Strong suprathreshold stimuli were required, yet a single brief contact did not evoke a response. Increases in skin temperature could not explain these responses.

Conclusions: The observed responses match well the human pain responses to such stimuli reported by Vierck et al. (1997), who concluded by using alternating skin sites and dextromethorphan administration that central summation must be responsible. These observations indicate that lamina I STT neurons can explain the temporal "windup" of second pain.

Acknowledgments: Supported by NIH grant NS 25616 and the Barrow Neurological Foundation.

GLUTAMATE IMMUNOREACTIVITY IN RAT C1-C2 PROPRIOSPINAL NEURONS.

Kenneth E. Miller. Gabriel M. Garber*, Margaret J. Chandler*, Robert D. Foreman, Dept Cell Biology, Dept Physiology, Univ Oklahoma Health Sciences Center, Oklahoma City, OK 73190 USA

Aim of Investigation: Determine the neurochemical content of C1-C2 propriospinal neurons.

Methods: Retrograde tracing techniques were used in combination with immunohistochemistry for glutamate. Au-WGA-apoHRP was injected bilaterally into the lumbosacral dorsal horn in anesthetized rats. After 3-4 days survival, rats were anesthetized and perfused with fixative, 4% paraformaldehyde, 0.3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The C1-C2 spinal segments were removed and transverse sections processed for histochemistry. Retrograde labeling was localized with silver intensification of colloidal gold conjugated to WGA-apoHRP. Sections then were processed for glutamate with avidin-biotin-peroxidase immunohistochemistry.

Results: Retrogradely labeled neurons were located in: lateral spinal nucleus, nucleus proprius, central gray region, and ventral hom. The majority of the retrogradely labeled neuronal cell bodies in these regions were glutamate immunoreactive.

Conclusions: Physiological and anatomical studies from our laboratories have demonstrated a population of neurons in C1-C2; spinal segments with descending projections to lower segments (Miller et al., Brain Res. Bull. 47:43-47,'98; Zhang et al., Brain Res 709:337-42,'96). These neurons respond to a variety of stimuli and mediate inhibition ofthoracic & lumbar dorsal hom neurons activated by somatic & visceral afferent input. Results from the present study indicate that C1-C2; propriospinal neurons use glutamate as a neu-rotransmitter in caudal spinal segments.

Acknowledgments: Supported by NIH# HL-52986 (KEM, RDF).

SPIKE ACTIVITY OF SPINORETICULAR TRACT NEURONS EVOKED BY GLUTAMATE MICROIONTOPHORESIS OR PERIPHERAL STIMULATION DURING WAKEFULNESS AND SLEEP.

M.P. Kristensen. W. Pang*, N. Taepavarapruk*, S.A. McErlane*, and P.J. Soja, Faculty of Pharmaceutical Sciences, Univ of British Columbia, Vancouver, BC, V6T 1Z3, Canada.

Aim of Investigation: The amplitudes of peripherally evoked field potentials recorded within the spinoreticular tract (SRT) decline during active sleep (AS) relative to wakefulness (W; J. Neuro-physiol. 70:1708, 1993). Moreover, discharge rates of SRT cells decrease during AS (Soc. Neurosci. Abs. 23: 2341, 1997). The excitability of SRT cells may also be dependent on behavioral state.

The purpose of the current study was to evaluate this possibility by examining whether responses of individual SRT neurons to local glutamate application and peripheral nerve stimulation differ between W and AS.

Methods: Experiments were performed on 5 chronically instrumented, unanesthetized cats (J. Neurosci. Meths. 60:227, 1995). Spike activity of SRT neurons was recorded dunngjuxtacellular glutamate iontophoresis (0.5 M; pH = 8; -50 to -130 nA; 8 - 10 s epochs; 30 s intervals) as well as after low-intensity electrical stimuli (0.7 - 7 mA; 0.2 ms; 60 trials) applied to the ipsilateral quadriceps musculature and cutaneous tail or flank receptive fields.

Results: SRT neurons were antidromically identified (mean latency = 3.36 ms; mean threshold = 135 uA; n == 14). Glutamate-evoked discharge (mean of 5 - 11 consecutive trials; 7 cells) decreased from 14.2 8.3 Hz during W to 7.9 3.6 Hz dunng AS (p < 0.05; paired /-test). Responses did not vary significantly between quiet sleep and W. The average number of spikes elicited by fixed-amplitude peripheral stimuli decreased from W to AS in 9 of 13 tests (A " 13%;n= 13; 8 cells).

Conclusions: These data indicate that responses of individual SRT neurons tojuxtacellular applications of glutamate as well as to afferent stimulation are state-specific and decrease during AS. It is unclear to what extent pre- and postsynaptic mechanisms contribute to this excitability change across behavioral state. However, the present results suggest that SRT neurons may hyperpolarize, and that sensory transmission through the SRT may be impeded, dunng AS relative to W.

Acknowledgments: Supported by NIH (NS34716).

THE SPINO-PARABRACHIO-THALAMIC PATHWAY: EVIDENCE FOR AN INVOLVEMENT IN NOCICEPTION

Laurence Bourgeais*. Lenaic Monconduit*, Luis Villanueva and Jean-Francois Bernard, INSERM U 161, 2, rue d'Alesia, 75014 Paris, France.

Aim of Investigation: Anatomical studies in our laboratory have shown that the parabrachial internal lateral (PBil) subnucleus is the relay of a spino-parabrachial-thalamic pathway, in the rat. Indeed the PBil receives a dense projection from the spinal lamina V and projects densely to the paracentral thalamic (PC) nuclei.

Methods: The electrophysiological properties ofparabrachio-thal-amic neurons have been investigated using unitary extracellular recordings. Neurons were antidromically driven from the PC nucleus and located within and around the PBil subnucleus. The rats were anaesthetized with halothane (0.5%) in nitrous oxide and oxygen mixture (2/3-1/3).

Results: None of the PBil-thalamic neurons responded to innocuous stimuli. Nevertheless, they responded to mechanical, thermal and electrical noxious stimuli from receptive fields that included widespread cutaneous regions of the body. These neurons encoded the intensity of thermal stimuli in a noxious range. However, the response was maximum for temperature around 46-48C and clearly decreased for higher stimuli above 50C.

Conclusions: The present study demonstrated the existence of a spino-parabrachio-thalamic pathway that could be involved in at-tentional aspects related to nociception. Considering the projections of the PC nucleus to striatal and prefrontal cortex, this pathway could favor motivational behavior in response to noxious or strong events.

Acknowledgment: Supported by FINSERM and 1'Institut UPSA de la douleur.

LOCATION OF ASCENDING LAMINA I AND LAMINA V SPINOTHALAMIC TRACT AXONS IN THE MONKEY.

E.-T. Zhang. A. Blomqvist, and A.D. Craig, Atkinson Pain Research Laboratory, Division ofNeurosurgery, Barrow Neurological Inst, Phoenix, AZ 85013 U.S.A., and Cell Biol. & Anat., Fac. Health Sci., Univ. Lmkoping, Sweden S-581 85.

Aim of Investigation: To test the hypothesis that spinothalamic lamina I and lamina V ascend in different locations in the spinal white matter.

Methods: Single or multiple iontophoretic injections of PHA-L or red (TRITC) dextran (10,000 MW, Molecular Probes) were made in lamina I and of green (FITC) dextran in lamina V in the lumbo-sacral or cervical cord, under physiological control in anesthetized cynomolgus monkeys. Oblique sections at Cl, C4 and T6 were examined after 4-6 weeks survival. In addition, we examined the C2 white matter of monkeys and humans for calbindin 28kD im-munoreactivity.

Results: Anterogradely labeled lamina I axons were found contra-laterally in the middle of the lateral funiculus, where a dense bundle of calbindin positive fibers is present in both monkey and human. Anterogradely labeled lamina V axons were seen more ventrally in the anterolateral funiculus.

Conclusions: Early studies of spinothalamic fibers in humans indicated two bundles, a lateral tract associated with 'pain and temperature', and an anterior tract associated with 'crude touch and movement'. Retrograde labeling studies tend to support this organization. Our observations confirm that lamina I axons ascend in the lateral bundle and that lamina V axons are located more ventrally. Because lamina 1 terminations in VMpo in primates and humans are calbindin-positive, these observations indicate that calbindin-positive lamina I axons ascend in the middle of the lateral funiculus and form the lateral spinothalamic tract, which is important for pain and temperature sensation in primates.

Acknowledgments: Supported by NIH grant NS 25616 and the Barrow Neurological Foundation.

ANALYSIS OF THE PROJECTION OF UNMYELINATED PRIMARY SENSORY NEURONES THROUGH THE DORSAL COLUMNS OF THE RAT SPINAL CORD.

Mark B. Plenderleith. Michelle B. Gerke, School of Life Science, Queensland Univ of Technology, Brisbane, Queensland, Australia, 4001.

Aim of Investigation: The dorsal columns are traditionally thought of as the major pathway through which large diameter myelinated primary sensory neurones, relaying information about non-noxious stimuli, project out of the spinal cord. However there is some evidence to suggest that unmyelinated primary sensory neurones also project out of the spinal cord through the dorsal columns. The aim of this study was to investigate this possibility using transgangli-onic transport of the plant lectin Bandeiraea simplicifolia I-isolectin B4 (BSI-B4) which is selectively taken up and transported by a subpopulation of unmyelinated primary sensory neurones.

Methods: Four adult, Wistar strain rats were deeply anaesthetised with Nembutal (60 mgKg"' i.p.) and 2u.lofa 4% solution of BSI-B4 conjugated to horseradish peroxidase was injected into the sciatic nerve on one or both sides. Following a survival period of 7 days these animals were reanaesthetised (Nembutal, 60 mgKg'1 i.p.) and perfused through the heart with 2.5% glutaraldehyde. The transported lectin was then visualised in vibratome sections of the L4 and T9 segments of the spinal cord using horseradish peroxidase histochemistry and these sections were then processed for electron microscopy.

Results: Transmission electron microscopic analysis revealed a large number of labelled axon terminals within the superficial dorsal horn of the L4 segments as well as labelled unmyelinatcd axons within Lissauer's tract. Analysis of the dorsal columns of the T9 segments revealed a large number ofunmyelinated axons, none of which appeared to contain the transported lectin observed in L4.

Conclusions: The results of this investigation suggest that the sub-population of unmyelinated primary sensory neurones which transport the lectin 881-84 do not project out of the spinal cord via the dorsal columns.

C-FOS INDUCTION IN THE RAT SPINAL CORD BY NOXIOUS AND INNOCUOUS STIMULATION FOLLOWING NEONATAL CAPSAICIN TREATMENT.

Carole Torsney. Jacqueta Meredith-Middleton and Maria Fitzgerald, Dept of Anatomy and Developmental Biology, Univ College London, Gower St. WC1E 6BT, UK.

Aim of the investigation: To assess functional consequences of central A fibre terminal reorganisation following capsaicin-induced destruction ofC fibres at birth.

Methods: 21 days after neonatal capsaicin treatment (100 mg/kg) rat pups received 10 minutes noxious or innocuous mechanical stimulation of the hindpaw. Animals were sacrificed 2 hours later and L4-L5 spinal cord transverse sections processed for c-fos im-munohistochemistry. The A fibre central termination pattern was mapped following sciatic nerve injection of the selective tracer, B-HRP.

Results: Loss of small diameter DRG cells in capsaicin treated animals was confirmed using IB4-binding and CGRP immunore-activity. Furthermore, A fibre terminals projected more dorsally, extending into 43% of lamina II compared to untreated littermates (Shortland et al, 1990). Lamina II shrinkage, estimated using Nissl-stained material, was small and not significant. Numbers of Fos immunostained nuclei in a defined region of lamina II were reduced in capsaicin treated rats (181.3) compared to vehicle controls (271.5) (67%, p<0.0001), following noxious stimulation. However, the response to innocuous stimulation remained unchanged with 70.3 Fos nuclei in capsaicin treated rats compared to 70.4 in vehicle controls.

Conclusion: Neonatal capsaicin treatment prevents A fibres withdrawing to deeper laminae postnatally suggesting competition between A and C fibre afferents. This exuberant A fibre termination did not lead to c-fos induction in the adult superficial dorsal hom by innocuous stimulation, as occurs in the neonate, implying that these superficial terminals in lamina II no longer function in the same way as in the neonate

Acknowledgments: Carole Torsney is supported by the Wellcome Trust.

STEREOLOGICAL QUANTIFICATION OF SPINAL NEURONS EXPRESSING C-FOS IN PIGS AFTER PERIOPERATIVE NOCICEPTION

Kirsten Lykkegaard*. Ove Svendsen, Royal Veterinary and Agricultural Univ, Dept of Pharmacology and Pathobiology, DK-1870 Copenhagen, Denmark

Aims of Investigation: To assess perioperative nociception quanti-tatively using unbiased objective stereological quantification of spinal neurons expressing c-fos in the dorsal hom.

Methods: Three groups of pigs (app. 25 kg) were kept anesthetized during the following procedures. Group one received a unilateral infiltration ofisotonic saline in the flank followed by a surgical incision, exposing the abdominal cavity. The wound was subsequently closed. Group two received an infiltration of local anaesthetic before the surgical procedures were performed. Group three only received an infiltration of local anaesthetic. Two hours after surgery the pigs were perfused with formalin. Five thoracic and five lumbar segments of the spinal cord were removed. To apply the stereologically fractionator method cryostat sections 100 u.m thick were cut at 12 millimeters intervals and immunohistochemi-cally stained for fos protein. A computerized program (C.A.S.T.-Grid, Olympus, Denmark) was used for the quantification of the total number of fos positive neurons.

Results: Preliminary results show that between one and three- hundred-thousand neurons in the ipsilaterally dorsal hom express c-fos. Blocking with local anaesthetic however virtually reduced the ipsilaterally c-fos expression to base-line level.

Conclusion: C-fos expression seem to correlate well with the expected afferent nociceptive transmission in the respective groups. To the knowledge of the authors, assessment of the total number of c-fos expressing neurons following a noxious stimulus has never been performed before. The method however, gives valuable quantitative information in an objective and unbiased manner.

EXPANSION OF C-FOS SOMATOTOPIC ORGANIZATION AFTER GRADED CO2 PULSE LASER STIMULATION.

Wei-Zen Sun. Bai-Chung Shyu, Jeng-Yung Shieh*, Depts ofAn-esthesiology, National Taiwan Univ, Taipei, Taiwan, Republic of China.

Aim of Investigation: In the present study, we measured the extent and distribution of c-fos expression in the superficial dorsal hom neurons (laminae 1/11) across the lumbar segments to examine the change ofsomatotopic organization after graded intensities of CO2- laser pulse stimulation.

Methods: The right hindpaw of male Sprague-Dawley rats, weighing 250-350 g, was irradiated with CO2 laser pulses at 2 min interval for 60 min using three levels of energy intensity, i.e., (1) subthreshold (0.5xTH) (2) threshold (TH) (3) suprathreshold (5xTH). The superficial lamina was divided into 10 compartments from media to lateral border (X-axis). The number ofFos-LI labeled neuron in each compartment (Z-axis) was then mapped along the lumbar spinal segments from L, to Le (Y-axis) and across X-axis using Contour Plot.

Results: At subthreshold level, repeated CO2 laser pulse stimulation evoked scarce c-fos expression exclusively at the medial third of the superficial laminar neurons near L4-L; segments. The increase in intensities led to greater number of Fos-LI labeled neurons and wider contour area across the X-Y plane.

Conclusions: We conclude that temporal summation of brief CO2- laser stimulation induces enlarged somatotopic presentation and enhanced neuronal activity at the superficial dorsal hom neurons depending on the pulse intensity. These results clearly provide anatomical evidence for central receptive field expansion after graded intensities of phasic pain stimulations.

Acknowledgments: Supported by Grant. NSC85-2331-B002-015.

ANTINOCICEPTION PRODUCED BY MICROINJECTING SUBSTANCE P NEAR THE A7 CATECHOLAMINE CELL GROUP MAY BE REDUCED BY STRUCTURAL CHANGES IN DENDRITES: ROLE OF INTRACELLULAR CALCIUM.

Byron A. Heidenreich. Herbert K. Proudfit, Dept of Pharmacology, Univ of Illinois at Chicago, Chicago, IL 60612, U.S.A.

Aim of Investigation: To determine the role ofintracellular calcium in the antinociceptive effect and morphological changes in noradrenergic dendntes produced by microinjecting substance P (SP) near the A7 catecholamine cell group.

Methods: SP or the calcium ionophore A23187 was microinjected just dorsal to the A7 cell group in lightly anesthetized rats. Antino-ciception was assessed using the tail flick test and structural changes in tyrosine hydroxylase-immunoreactive (TH-ir) A7 neurons were assessed by light microscopy.

Results: Microinjection ofSP into the A7 cell group produced an-tinociception, but the antinociceptive effect of 32.5 pmol was less than that of 3.25 pmol. The 32.5 pmol dose ofSP produced dramatic structural changes in the small dendrites of TH-ir A7 neurons, which were transformed into a senes ofvaricosities connected by very thin segments, while lower doses produced few or no varicosities in TH-ir dendrites. A23187 (30 pmol) produced antinociception and marked varicosities in the dendrites ofA7 neurons.

Conclusions: These results suggest that SP-induced receptor inter-nalization and/or structural changes in the dendntes ofA7 neurons attenuates responsiveness to SP at high doses. Increases in intra-cellular calcium evoked by A23187 mimic the effects ofSP on nociception and dendritic morphology. These findings suggest that the structural changes produced by SP are mediated by increases in cytosolic calcium via the G-protein coupled inositol phosphate signal transduction pathway.

Acknowledgments: Supported by USPHS grant DA03980 from the National Institute on Drug Abuse.

SPINAL AFFERENTS TO THE VASODEPRESSOR REGION OF THE CAUDAL MIDLINE MEDULLA OF THE RAT.

Jason Potas*, Kevin Keay, Richard Bandler. Dept of Anatomy and Histology, Univ of Sydney and Centre for Anaesthesia and Pain Management Research, Royal North Shore Hospital, Sydney, NSW, Australia

Aim of Investigation: Activation of the caudal midline medulla (CMM) of the rat evokes hypotension and bradycardia. Inactivation of the CMM blocks hypotension and bradycardia evoked by deep somatic pain or blood loss. The aim of this study was to identify the populations of spinal neurons which project to the CMM.

Methods: The retrograde tracer, cholera-toxin subunit B (CTB), was deposited iontophoretically into the vasodepressor region of the CMM in rats anaesthetised with ketamine and xylazine. After a 7-10 day survival period, rats were re-anaesthetised, perfused and sections of medulla and spinal cord reacted for presence of CTB using standard immunohistochemical techniques.

Results: Afferents to the CMM arose from all spinal cord levels, with approximately 60% of labeled neurons located in the upper cervical spinal cord (C1-C4). CMM-projectmg neurons were distributed bilaterally in the deep dorsal hom (laminae IV and V), lamina X and the medial ventral hom (laminae VII and VIII). No projection to the CMM arose from the superficial dorsal hom.

Conclusions: Spinal afferents to the CMM arise exclusively from laminae which receive deep somatic and visceral, rather than cutaneous, primary afferents. Input to the CMM arising from the upper cervical spinal cord is of particular significance.

Acknowledgments: Supported by Australian NHMRC grant 970688.

AFFERENTS OF THE CAUDAL VENTROLATERAL MEDULLA: A RETROGRADE TRACING STUDY IN THE RAT

Angeles Cobos*^, Isaura Tavares. Armando Almeida, Deolinda Lima, Inst of Histology and Embryology, Faculty of Medicine, IBMC, Univ ofOporto, 4200 Porto, Portugal and Dept of Morphological Sciences, Univ of Santiago de Compostela, Spain.

Aim of Investigation: To determine the brain sources of afferent fibers to the lateral reticular formation of the caudal ventrolateral medulla (VLMiat), an area which produces intense antinociception and participates in hypertension-induced analgesia through inhibition of pain transmission at the spinal dorsal hom.

Methods: Male Wistar rats were injected in the left VLMiat, under halothane anaesthesia, with 0.3 |^1 of the retrograde tracer cholera toxin subunit B (CTb). Three days later the rats were reanesthetised with 35% chloral hydrate, i.p., and perfused with fixative through the ascending aorta. Coronal 40 ^m serial sections of the brain were serially obtained. One in every three sections was immuno-stained for CTb by the ABC method and counterstained with formol-thionin.

Results: Retrogradely labelled neurons were observed in areas involved in the production ofautonomic responses (nucleus of the tractus solitarius, parabrachial complex, A; noradrenergic cell region, periaqueductal gray and paraventricular nucleus), emotional motor reactions (central amygdalaloid and sublenticular nucleus, bed nucleus of the stria terminalis and lateral hypo-thalamus), somatic motor reactions (lateral reticular nucleus, inferior olive, dentate cerebellar nucleus, red nucleus and motor cortex) and visceral sensory (insular cortex) and motor (limbic cortex) responses.

Conclusions: According to the present data, activation of the VLMiat is intimately related with emotional, autonomic and motor events which suggests that its antinociceptive effects may be part of complex defense behaviours triggered in situations of physical threatning.

Acknowlegments: Supported by the Biotech project n B104-CT98-0076 of the European Commission.

BRAIN AFFERENTS TO THE MEDULLARY DORSAL RETICULAR NUCLEUS. A CTB RETROGRADE TRACING STUDY IN THE RAT.

Armando Almeida. Angeles Cobos'* and Deolinda Lima, Inst of Histology and Embryology, Faculty of Medicine and IBMC of the Univ ofOporto, 4200 Porto, Portugal and 'Dept. Morphological Sciences, Univ of Santiago de Compostela, Spain

Aim of Investigation: The dorsal reticular nucleus (DRt) was recently shown to facilitate nociception by enhancing nociceptive transmission at the spinal level through the activation of a DRt-lamina I reverberative circuit. In this study, brain nuclei capable of modulating the pain-facilitating role of the DRt were identified by the use of retrograde tracing techniques.

Methods: Cholera toxin subunit B (CTh) was injected iontophoretically in the left DRT of male Wistar rats, under halothane anaesthesia. Ten days later the rats were reanaesthetized with 35% chloral hydrate, i.p., and perfused with fixative. Serial 50 u.m coronal sections of the brain were immunostained for CTb and counter-stained with formol-thionin.

Results: Brain areas containing retrogradelly labeled neurons included subcortical areas involved in antinociception, defensive behaviour and/or autonomic control (amygdala, bed nucleus of the stria terminalis, paraventricular and lateral hypothalamic areas, periaqueductal grey matter, rostral ventromedial and caudal ventrolateral medulla, nucleus of the solitary tract, parabrachial nuclei, area prostrema), sensory and motor somatic and visceral cortices, extrapyramidal motor areas (red nucleus, inferior olive, parvocel-lular and lateral reticular nuclei, fastigial and dentate cerebellar nuclei), sensory relay nuclei (cuneate nucleus and lamina I of the spinal trigeminal nucleus) and contralateral DRt.

Conclusions: The pattern of afferent projections to the DRt suggests that DRt-mediated pain facilitation is modulated by the ho-meostatic autonomic system, the emotional motor system and the antinociceptive system.

Acknowledgments: Supported by Biotech project no BIO-CT98-0076 of the European Commission.

NEURONS IN THE VENTROLATERAL PERIAQUEDUCTAL GRAY AND CUNEIFORM NUCLEUS PROJECT TO DIFFERENT NORADRENERGIC NEURONS IN THE BRAINSTEM THAT ARE INVOLVED IN THE MODULATION OF NOCICEPTION.

D. Bajic and H.K. Proudfit, Dept. of Pharmacology, Univ of Illinois at Chicago, IL 60612, U.S.A.

Aim of Investigation: Stimulation of neurons in the ventrolateral periaqueductal gray (PAG) and cuneiform nucleus (CnF) produces antinociception that is mediated in part by spinally projecting noradrenergic neurons. These studies determined the location of noradrenergic neurons that receive projections from these two nuclei.

Methods: The anterograde tracer, biotinylated dextran amine (BDA) combined with tyrosine hydroxylase immunocytochemistry was used to determine the projections of neurons in the PAG or CnF to spinally projecting noradrenergic cells in the A7, A5, and A6 (Locus Coeruleus) cell groups that are involved in modulating nociception.

Results: The BDA deposit in both the PAG and CnF produced anterograde labeling that was most dense on the ipsilateral side. PAG neurons project to noradrenergic neurons located in the medial aspect of the A5, A6, and A7 cell groups. In contrast, CnF neurons project to noradrenergic cells located in the lateral aspect of the A5 and A7 cell groups.

Conclusions: These results suggest that the antinociception produced by stimulation of sites in the PAG and the CnF is mediated in part by different populations of noradrenergic neurons. These different populations may innervate different segments of the spinal cord or may modulate nociception by different mechanisms.

Acknowledgments: Supported by USPHS grant DA03980 from the National Institute on Drug Abuse.

THE ROLE OF VENTRO-LATERAL PAG-NRM EXCITATORY PATHWAY ON FORMALIN TEST.

Gila Behzadi. Famaz Nikbakht* Physiology Dept, Faculty of medecine, Shaheed Beheshti Med. Sci. Univ, Tehran, Iran

Aim of Investigation: It is suggested that neurons in the VL-PAG projecting to nucleus raphe magnus (NRM), utilize an excitatory amino acid (EAA) as neurotransmitter. This projection plays an important role in the descending pain modulation system. The goal of this study was to determine the role of this precise pathway in pain perception.

Methods: Eighteen male rats were received (uni & bilateral n=12) stereotaxical ibotenic acid (l^ig /0.1ug) lesion, a specific EAA neurotoxin, in VL-PAG. After one week recovery the nociception was evaluated during 60 minutes by formalin test (50ul, 10%). At the completion of experiments, animals were re-anesthetized and perfusion fixed with 10% formaldehyde/saline solution. Serial vi-bratom sections (80 microns) were Nissi stained and examined to determine the lesion location.

Results: A significant increase (15%) was observed in the first phase of formalin behavior. However, in the second phase in 15-20 minutes with a latency of 5 to 15 min, the lesion groups showed a significant diminution of nociception, (15.5% for uni and 20% for bilateral).

Conclusions: The chemical lesion in VL-PAG produced a hyperal-gesic first phase (acute pain), suggesting that the EAA containing neurons in VL-PAG do contribute to a descending influence on this kind of pain. The fact that there is a nociceptive attenuation in second phase indicates that, this inflammatory phase is going under an active inhibitory process, following selective VL-PAG lesion.

ASCENDING INTRANUCLEAR CONNECTIONS FROM THE CAUDAL PART TO THE ORAL PART OF THE SPINAL TRIGEMINAL NUCLEUS IN THE RAT: AN ANATOMICAL STUDY.

D.L. Voisin*. S. Domejean-Orliaguet*, M. Chalus*, R. Dallel and A. Woda (SPON : E. Tomb), Faculte de Chirurgie Dentaire, 63000 Clermont-Ferrand, France

Aim of Investigation: To investigate the existence of ascending intranuclear trigeminal pathways from the caudal part of the spinal trigeminal nucleus (Sp5C) to the oral part (Sp50) in the rat, and to study the distribution of the cells of origin of these connections.

Methods: The retrograde fluorescent tracer tetramethylrhodamine conjugated to dextran was injected lontophoretically into Sp50 of the anaesthetized rat. Two days later, rats were perfused with 4% paraformaldehyde, their brain removed and sectioned.

Results: Cell bodies labelled retrogradely from Sp50 were observed in laminae IV and V of Sp5C consistently. Labelled neurons were also found in lamina III, and to a much lesser degree, in lamina I. A topographic distribution was observed : neurons in the dorsal part of the Sp5C project to the Sp50 dorsally, while those in the ventral part of the Sp5C project ventrally.

Conclusions: These data provide an anatomical support for the indirect activation of Sp50 convergent neurons by nociceptive stimuli (Dallel et al., J. Neurosci., 1998, 18, 3529-36), via deeper laminae neurons of the Sp5C. The latter are thought to receive C-fiber inputs either directly via dendrites extending into the substan-tia gelatinosa, or indirectly via substantia gelatinosa neurons.

SPINAL SOURCES OF NOXIOUS VISCERAL AND NOXIOUS DEEP SOMATIC AFFERENT DRIVE ONTO THE VENTROLATERAL PERIAQUEDUCTAL GRAY OF THE RAT.

Colin Clement*, Kevin Keay, Brent Gordon. Katherine Podzc-benko*, Richard Bandler, Dept of Anatomy and Histology, Univ of Sydney and Centre for Anaesthesia and Pain Management Research, Royal North Shore Hospital, Sydney, NSW, Australia

Aim of Investigation: Pain of deep somatic or visceral origin selectively activates the ventrolateral periaqueductal gray region (vIPAG) of the midbrain. Spinal afferents to the vIPAG arise predominantly from the superficial and deep dorsal horn, lamina X and nucleus of the dorsolateral funiculus (nDLF), with approximately 50% ofvlPAG-projecting neurons found within the upper cervical spinal cord. The aim of this study was to identify the populations of spinal neurons which both project to vIPAG and arc activated by deep pain.

Methods: Following prior injection of retrograde tracer into the vIPAG, halothane-anaesthetised rats were subjected to noxious stimulation of deep somatic (hindlimb) or visceral (cardiac and peritoneal) structures. The immunohistochemical detection ofFos protein was used to determine the location ofretrogradely-labcled (vIPAG-projecting) spinal neurons activated by the deep noxious manipulations

Results: Ventrolateral PAG-projecting spinal neurons activated by visceral (cardiac, peritoneal) pain were restricted to the superficial and deep dorsal hom, lamina X and nDLF of the thoracic spinal cord. In contrast, vIPAG-projecting spinal neurons activated by deep somatic (hindlimb) pain were located in the nDLF of both the upper cervical and lumbosacral cord.

Conclusions: The findings suggest that pain arising from deep structures (somatic vs. visceral) influences activity within the ventrolateral column of the PAG via distinct pathways arising from different populations of spinal neurons.

Acknowledgments: Supported by Australian NHMRC grant 970688.

THE VMPO NUCLEUS OF THE HUMAN THALA-MUS: CYTOARCHITECTONIC AND IMMUNOCYTO-CHEMICAL CHARACTERISTICS

Blomqvist. E.-T. Zhang, and A.D. (Bud) Craig. Dept. Cell Biology, Fac. Health Sciences, Linkoping Univ, Sweden, and Div. Neurosurgery, Barrow Neurological Inst, Phoenix, AZ 85013.

Aim of Investigation: To describe the cyto- and chemoarchitec-tonic relationships of the human VMpo nucleus, the main termination site for nociceptive and thermoreceptive-specific lamina I spinothalamic tract neurons in the primate brain. Methods: Adjacent sections from 11 human thalami, cut in different planes, were stained with thionin and for calbindin-, substance P- and CGRP-immunoreactivity.

Results: The human VMpo is a cytoarchitectonically distinct nucleus, located posteromedial to the VPL/VPM, ventral to the anterior pulvinar and centre median, lateral to the limitans and dorsal to the medial geniculate and suprageniculate nuclei. Calbindin-ir fibers, representing spinothalamic tract input, were seen entering VMpo from the spinal lemniscus, and they formed patches of dense terminal-like staining that coincided with clusters of VMpo neurons. A few of these clusters also displayed terminal-like substance P-labeling. Small bursts of CGRP fibers, probably originating from the parabrachial nucleus, were found intercalated between the calbindin labeled clusters, but there was little or no overlap between the two markers. CGRP-1R was also present in a calbindin- negative area separating VMpo from VPL/VPM.

Conclusions: The location of the VMpo fits with clinical descriptions of the thalamic area from which pain, temperature and visceral sensations can be evoked. The immunohistochemical data suggest that these sensations may be represented by areas stained for distinct chemical messengers.

Acknowledgement: Supported by SMRC 7879 and NS 25616.

IS THE CERBELLUM INVOLVED IN PAIN?

Carl Y. Saab. AshrafA. Makki*, Michael J. Quasi*, Jingna Wei*, Elie D. Al-Chaer, William D. Willis, Univ of Texas Medical Branch, Dept of Anatomy and Neurosciences, Galveston, TX, 77555, USA

Aim of Investigation: To study the effect of peripheral capsaicin injection on the brain activity of rats, using functional magnetic resonance imaging (fMRI) and electrophysiology (EP).

Methods: Experiments were conducted on 4 male Sprague Dawley rats under anesthesia. For fMRI, serial images were taken before and after 0.1 ml of 0.3% capsaicin was delivered to the gastrocne-mius muscle. Twenty four hours later, we recorded extracellular responses of neurons in the cerebellar vermis, with receptive fields at the ankle, following a similar stimulation paradigm using the same rats.

Results: fMRJ revealed regional increases in cerebellar blood volume around the fourth ventricle, crus two cerebelli, the tenth cerebellar lobule, and the vermis. Other regions included the entor-hinal, perirhinal, and sensory motor cortices, thalamic nuclei, and areas around the cerebral aqueduct. EP showed -150 % increase in firing rate of presumed Purkinje cells that lasted for -400 sec, and then decreased to baseline levels gradually, over a period of at least 800 sec.

Conclusion: Increase in blood volume correlated well with an increase in neuronal activity of cells in the cerebellar vermis. The time frame of this activation and the areas involved are also in agreement with previous reports, describing the effect of peripheral capsaicin injection on brain activity. However, few studies have emphasized the role of the cerebellum in pain. This study suggests that the cerebellum is indeed involved in the processing of nociceptive information.

Acknowledgments: Supported by NIH Grants NS 09743 and NS 11255.

ARE THERE CEREBRAL SEP GENERATOR(S) COMMON ACROSS DIVERSIFIED NOXIOUS LASER, PELTIER-HEAT, ULTRA-SONIC, AND GALVANIC (NERVE, SKIN, MUSCLE, GUT) STIMULATION?

Andrew CN Chen. Lars Arendt-Nielsen, HBMCI Laboratory, SMI, Aalborg Univ., DK.-9220, Aalborg, Denmark

Aim: To investigate the cerebral generator(s) ofsomatosensory evoked potentials (SEPs) common among different noxious stimulation, so as to understand the convergent pain processing in the brain.

Methods: Noxious vs. innocuous stimulation by various modalities was studied separately in male subjects (Laser, n=10; Peltier-heat, n=13; ultrasonic, n= 12; galvanic skin/muscle, n=20, gut, n=13), with recording of 31 ch. EEC (bandpass: 0.1-100 Hz, sampling rate: 256 Hz, linked-ears reference). Prominent late SEPs were subjected to the analyses of intensity effects. These late components were then individually studied of its generator(s) using the source localization routines of the ASA program (ANT, Netherlands) to isolate the parameters related to nociceptive processing.

Results: Late SEPs were displayed in each of the study. They differed largely in peak latencies and amplitudes. Yet, they all shared a similar topographic sequence. The major late activation isolated was N160 (^57) and P285 (72) at vertex, spreading bilaterally. Using inverse-solutions for rotating dipole, the ASA yielded mostly a similar result within each of the studies, particularly for the Cz/P285. These generators exhibited a mean of 95.6% (3,0) correlation of the measured scalp topography and amounted to 90,9% (5,3) in goodness-of-fit. However, there was no singular generator unequivocally common to each of all studies. The "average" generator location calculated was at the PAN coordinates: -8,4 (7,4) x(mm), 1.2 (8,4) y(mm), 0,3 (12,8) z(mm).

Conclusion: This report can not support a specific cerebral generator common across diversified somatic stimulation. For the late vertex potentials, various generators are distributed as the mid-line deep dipoles in different modalities. They are clustered within 10 mm (s.d.) among each others.

Acknowledgement: Supported by the Danish National Research Foundation.

9th WORLD CONGRESS ON PAIN, 1999, Vienna, Austria, p.169 - 174

   

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