hypothalamic influences on viscero-somatic

Journal of Physiology (1990), 424, pp. 427-444 427With 10 figuresPrinted in Great Britain

HYPOTHALAMIC INFLUENCES ON VISCERO-SOMATIC NEURONES INTHE LOWER THORACIC SPINAL CORD OF THE ANAESTHETIZED RAT

BY B. M. LUMBFrom the Department of Physiology, Medical School, University of Bristol, University

Walk, Bristol BS8 lTD

(Received 16 October 1989)

SUMMARY

1. Single unit electrical activity has been recorded from thirty-four viscero-somatic neurones in the dorsal horn of the lower thoracic spinal cord (T9-Ti 1) ofchloralose-anaesthetized rats. All neurones were driven by natural and/or electricalstimulation within their somatic receptive fields and gave excitatory responses toelectrical stimulation of the ipsilateral splanchnic nerve. Descending influences onthese neurones were tested by electrical and chemical (microinjections of DL-homocysteic acid) stimulation of sites in the rostral hypothalamus.

2. The electrical activity of most viscero-somatic neurones (64 %) was inhibited byelectrical stimulation at sites throughout the anterior hypothalamus-preopticregion. In any one cell, responses to stimulation of visceral and somatic afferent fibreswere inhibited to the same extent and any on-going activity was also depressed. Onlyone cell was driven by the conditioning stimulus and the electrical activity of theremaining cells (n = 7) was unaffected.

3. At certain hypothalamic sites the effects of electrical conditioning stimulationon the responses of viscero-somatic neurones were compared with those of localmicroinjection of DL-homocysteic acid. Electrical stimulation at all sites tested(n = 7) led to an inhibition of on-going and evoked neuronal activity. At twohypothalamic sites, both located in the ventral part of the preoptic area,microinjection of DL-homocysteic acid resulted in a complete abolition of theresponses to the test stimuli and in a cessation of any on-going activity.Microinjection of DL-homocysteic acid at the remaining five sites had no detectableinfluences on dorsal horn activity.

4. The results of this study include the first description of input propertiesiqfviscero-somatic neurones in the lower thoracic spinal cord of the rat. In addition,these results demonstrate that transmission of visceral and somatic informationthrough these neurones can be modulated by pathways that originate in the anteriorhypothalamus-preoptic region of the ventromedial forebrain.

INTRODUCTION

Stimulation of sites in the rostral ventromedial medulla (Ammons, Blair &Foreman, 1984; Chapman, Ammons & Foreman, 1985; Tattersall, Cervero & Lumb,1986) and in the midbrain periaqueductal gray (Sonoda, Ikenoue & Yokota, 1986;MS 8018

Chandler, Garrison, Brennan & Foreman, 1987) has been shown to modulate thespinal transmission of visceral afferent impulses. Similar inhibitory influences on thespinal transmission of somatic input are well documented (see Willis, 1982; Besson& Chaouch, 1987 for recent reviews). It is generally believed that this inhibitorymodulation participates in the antinociceptive effects of brain stimulation observedin behavioural experiments and which extends to pain of visceral origin (Giesler &Liebeskind, 1976).More rostral sites in the ventromedial forebrain, notably the anterior hypo-

thalamus-preoptic area (AH-POA), have also been implicated in antinociceptivemechanisms. For instance, microinjection of morphine into the AH-POA producesa profound analgesia (Pert & Yaksh, 1974) and conversely, electrolytic lesions placedin this region attenuate the analgesic effects of systemic morphine in rats (Pottoff,Valentino & Lal, 1979). Electrical stimulation in the AH-POA is reported toattenuate behavioural responses to noxious somatic stimuli in rats (Cunningham &Goldsmith, 1986) and monkeys (Oleson, Kirkpatrick & Goodman, 1980). Theavailable evidence suggests that these antinociceptive effects are mediated, at leastin part, by the activation of descending pathways which modulate nociceptivetransmission in the dorsal horn of the spinal cord (Carstens, Mackinnon & Guinan,1982) and in the trigeminal nucleus (Mokha, Goldsmith, Hellon & Puri, 1987).The results of a recent study from this laboratory indicate that the inhibitory

influences of AH-POA stimulation extend to visceral afferent input (Lumb &Cervero, 1989) as assessed by monitoring the motor output of a viscero-somatic reflex(reflex activity recorded from a spinal nerve (LI or L2) in response to electricalstimulation of visceral afferent fibres in the splanchnic nerve (SPLN)). This reflex(Downman, 1955) is thought to be the electrophysiological correlate of the sustainedmuscle contractions produced by noxious stimulation of abdominal viscera.The results of this previous study demonstrated a powerful inhibitory effect of

AH-POA stimulation on the viscero-somatic reflex and, importantly, it located thecell bodies of origin of this descending inhibitory system in the AH-POA but couldnot give precise information on the site of action of the inhibitory effects. In view ofthis, the present study was undertaken to test whether activation of descendingpathways with their cell bodies located in AH-POA could inhibit the responses ofindividual dorsal horn neurones to stimulation of visceral afferent fibres. Preliminaryresults have been published in abstract form (Lumb, 1989).

METHODS

Experiments were carried out on twenty-three adult male rats with body weights between220 and 400g. In all animals anaesthesia was induced with halothane (3% in 100% 02) andthen maintained witha-chloralose (initial dose: 80-100 mg kg-'I.v.). Supplementary doses ofa-chloralose (30 mg kg-'I.v.) were given as required to maintain a level of anaesthesia so that no

precipitate changes in blood pressure were observed in response to minor noxious stimuli. Lack ofwithdrawal reflexes during recovery from paralysis was also used as an indicator of adequateanaesthesia. The animals were paralysed with pancuronium bromide and ventilated with a positivepressure pump. Arterial blood pressure, end-tidal CO2 and rectal temperature were monitored andmaintained within physiological limits.The lower thoracic spinal cord was exposed by a laminectomy from T9 to T12. The animals

were mounted in a rigid frame with the head positioned in a stereotaxic instrument. A pool was

428 B. M. LUMB

VISCERO-SOMATIC NEURONES

made with skin flaps over the exposed areas of the spinal cord and filled with a 4% agar. To gainaccess to the spinal cord with the recording electrodes, a 'window' was cut in the agar and was filledwith warm paraffin oil at 37 'C. Recording stability was improved by clamping the vertebralcolumn and by a bilateral pneumothorax.

Recording techniquesAt the start of each experiment, cord dorsum potentials were recorded in response to electrical

stimulation with intradermal needle electrodes of the skin covering the ipsilateral flank. Using thismethod it was possible to ascertain the dermatomes of the T9, T10 or T 1 segments. Extracellularsingle-unit recordings were then made in the dorsal horn of one of these segments using glassmicropipettes filled with 4 M-NaCl. Single units were isolated by their responses to intradermalstimulation of their somatic receptive fields. Amplified recordings were displayed on an oscilloscopeand analysed 'on-line' and 'off-line' using a microcomputer (Cervero, 1985).

Stimulation of afferent fibresSomatic afferent fibres were activated by natural stimulation of their receptive fields or by

electrical stimulation using intradermal electrodes. Natural stimulation included innocuous(brushing or stroking) and noxious (pinching and squeezing) procedures. Receptive field areas wereassessed and each neurone was ascribed to one of three categories depending on the size of itsreceptive field: small (approximately < 1 cm2), medium (1-3 cm2) and large (> 3 cm2).

Visceral afferent fibres were activated by supramaximal electrical stimulation (15-40 V, 0 5 ms,0.3 Hz or less) of the ipsilateral splanchnic nerve (SPLN). The greater splanchnic nerve wasdissected through a lateral incision as it emerged from the paravertebral muscle and prepared forelectrical stimulation by wrapping two Ag wire electrodes around it. The SPLN was then crushedperipherally and the area around the electrode was insulated using a dental impression material.

Stimulation in the ventromedial forebrainA small area of the frontal bone was removed to allow stereotaxic placement of stimulating

electrodes in ventromedial forebrain structures. Stimulating electrodes were triple-barrelled glassmicropipettes (overall tip diameter 20-30 um) suitable for electrical stimulation and pressureinjection of DL-homocysteic acid (DLH) or control solutions (R. M. McAllan & S. Woollard,personal communication).The electrode barrel used for electrical stimulation was filled with a mixture of Wood's metal and

indium. A second barrel contained DLH solution (pH 7 4, 0-2 M) saturated with Pontamine SkyBlue. This barrel was connected to a pressure injection pump which allowed small amounts(typically < 50 nl) to be ejected with 05 s pulses at pressures between 5 and 30 lbf in-2(35-205 kPa). The third barrel was also connected to the pressure injection pump and containeda solution of NaCl (pH 7-4, 0-2 M) for control injections.The stimulating electrode was mounted in a micro-manipulator and driven into the brain at a

point between 0-8 and 3-2 mm rostral to bregma and between 0 5 and 2-0 mm lateral to the mid-line. From an initial position, 1 or 2 mm above the ventral surface of the brain, the electrode couldbe advanced ventrally in order to test the effects of electrical stimulation at different sites on dorsalhorn neuronal activity. Stimulus parameters used for brain stimulation (10 ms trains of 0-2 mspulses at 300 Hz) were those shown in a previous study to attenuate reflex responses evoked bystimulation of the SPLN. In initial experiments an investigation was made of optimalconditioning-testing intervals and was found to be between 10 and 30 ms. At certain sites theeffects of electrical stimulation were compared with those of microinjection of DLH.

Histological methodsThe position of the recording electrode was marked by ionophoretic deposition of PSB in the last

track of the experiment. At least two marks were in this track over a distance of 500,Pm or more.Sites of electrical and chemical stimulation in the ventromedial forebrain and an indication of theextent of DLH injections were determined from the spread of Pontamine Sky Blue from thepressure injection barrel of the stimulating electrode.At the end of the experiment the brain and the segments of the spinal cord from which recordings

had been made were removed and fixed in 10 % formal saline. The stimulating and recording siteswere later recovered in 50 ,um transverse sections stained with Neutral Red.

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B. M. LUMB

RESULTS

Sample of neuronesExtracellular single-unit activity was recorded from fifty-five neurones in the

T9-T12 segments of the spinal dorsal horn. All of these cells were driven by naturaland/or electrical stimulation of their somatic receptive fields. Two classes ofneurones were distinguished on the basis of the effects of electrical stimulation of theipsilateral SPLN: (1) viscero-somatic neurones, i.e. those also showing an excitatoryresponse to SPLN stimulation (n = 34), and (2) somatic neurones, i.e. cells in whichsupramaximal stimulation of the SPLN failed to evoke neuronal activity (n = 21).Since the purpose of this study was to examine aspects of descending control ontransmission of visceral afferent impulses in the spinal cord, the analysis of resultswill be focused on those obtained from viscero-somatic neurones. However, whennecessary, comparisons will be drawn between certain properties of somatic andviscero-somatic neurones.

General properties of viscero-somatic neuronesBy definition electrical stimulation of the SPLN evoked action potentials in all

viscero-somatic neurones included in this study. An example of a viscero-somaticneurone, illustrating responses to electrical stimulation of its somatic receptive fieldand to electrical stimulation of the SPLN is given in Fig. 1.

In thirty-three cells the latencies of the responses to SPLN stimulation weremeasured and these ranged between 2 and 18 ms (6-3 ±4-1 ms; mean + S.D.) comparedwith 2-15 ms (4-7 + 2-8 ms; mean +S.D.) for responses to intradermal stimulation oftheir somatic receptive fields. All viscero-somatic neurones showed early responses tostimulation of somatic and visceral structures which were consistent with activationof primary afferent fibres conducting in the A fibre range. In addition, eight cells(24 %) showed a second, longer latency and higher threshold response to stimulationof visceral and somatic afferent fibres which was consistent with activation ofafferent C fibres. The neurone whose activity is shown in Fig. 1 is an example of onesuch cell. The differences in the latencies of the late responses of this cell tostimulation of somatic and visceral afferent fibres is most likely due to the differencesin peripheral conduction distances.The somatic receptive fields of all the neurones were located on the ipsilateral flank

although there were characteristic differences in the receptive field properties ofsomatic and viscero-somatic cells. Viscero-somatic units tended to have largersomatic receptive fields than those of somatic neurones and all but one were drivenby noxious stimulation of the skin. In contrast, 21 % of somatic cells were drivenexclusively by innocuous stimulation (Fig. 2).

Figure 3 illustrates the location of the recording sites of somatic and viscero-somatic neurones. In agreement with previous reports in the cat, somatic neuroneswere distributed in all dorsal horn laminae. In contrast, viscero-somatic neuroneswere found predominantly in the superficial and deep dorsal horn (i.e. laminae I, IVand V) and none was recorded in laminae II and III.

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A C

~~~~~~7 JF

ASkin

_ I

500 Am

B,

-

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40c

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50 ms

Touch/pinch A

SPLNFig. 1. Viscero-somatic neurone in the thoracic spinal cord. A, location ofthe recording site ofthe neurone in lamina V. B, this cell was excited by noxious (pinch) and innocuous(touch/brush) stimulation of its somatic receptive field (shaded area). C, superimposedoscilloscope sweeps illustrating the response of the cell to electrical stimulation of itssomatic receptive field (upper trace) and to electrical stimulation of the SPLN (lowertrace) with single shocks at supramaximal intensity. Each trace represents the responseto five consecutive stimulus presentations and the arrows mark the time of application ofthe stimuli.

80 Somatic neurones 80 Viscero-somatic neuronesn= 19 n=31

*60 60 -

5

240- 40

OD0

56-20 -20-

0 0SMVL SMVL SMVL SMVL SMVL SMVL SMVL SMVL Receptive field size

1 2 3 Deep 1 2 3 Deep Receptive field class21.0 42.1 26.4 10.5 3.2 90.4 3.2 3.2 Percentage in each class

Fig. 2. Somatic receptive field properties of somatic and viscero-somatic neurones.Receptive field sizes: S, small; M, medium, L, large. Receptive field class: 1, respond toinnocuous stimulation of their somatic receptive fields; 2, respond to noxious andinnocuous stimulation; 3, respond exclusively to noxious stimulation; Deep, respond todeep probing and squeezing of subcutaneous tissue including muscle.

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B. M. LUMB

Somatic Viscero-somatic

500gAm 500gm

Fig. 3. Locations of the recording sites of somatic and viscero-somatic neurones. Thelocations are plotted on a standard transverse section of the T1O segment.

Effects of electrical stimulation of ventromedial forebrain sitesTwenty-two viscero-somatic neurones were tested with conditioning stimulation

of ventromedial forebrain sites in, and adjacent to, AH-POA. Neurones were dividedinto three categories according to the effects of this stimulation on their responses tostimulation of visceral and somatic afferents: (i) cells whose responses to visceral andsomatic stimulation and any on-going activity were reduced by the conditioningstimuli (n = 14), (ii) one cell which exhibited an initial excitatory drive to brainstimulation which was followed by a period of reduced responsiveness to the teststimuli, and (iii) cells whose evoked and on-going activities were unaffected by theconditioning stimuli (n= 7).

Cells inhibited from the ventromedial forebrainThis group (64%) represents the most common effect of stimulation in AH-POA

on the activities of thoracic viscero-somatic neurones. An example of the inhibitoryeffects of electrical stimulation at a site in the anterior hypothalamus is illustratedin Fig. 4. Using short (10 ins) trains of conditioning stimuli the threshold currentintensities necessary to reduce the neuronal responses to the test stimuli averaged95 1tA. Inhibitory effects on dorsal horn neurones were often evoked at relatively lowcurrent intensities (10-20 ,utA). Therefore, threshold intensities greater than ten timesthese values (i.e. > 200 ,tA) were considered to be indicative of stimulus spreadbeyond the immediate electrode location. Stimulation sites requiring these highcurrent intensities were regarded as negative locations.The example shown in Fig. 5 illustrates several features of the inhibitory effects of

AH-POA stimulation. First, the magnitude of the inhibitions is directly related to

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the intensity of the conditioning stimuli. For example, at a conditioning stimulusintensity of 50 ,sA both the early and late components of the response to SPLNstimulation were completely abolished. At an intensity of 20,sA there is someevidence of the early response but still no late activity. With a conditioning stimulusintensity of 10 ,sA the early component is almost back to control levels althoughthere is still a considerable reduction of the late response whereas at 5 gA the lateresponse is comparable to the control situation.

B C

A Skin ASPLN

A CC

FX~1~j~ A Skin ASPLN

°C *AHA 10 uA NAHA 10 uA

50 ns 50 ms

A Skin A SPLNIMAHA 50uA I AHA 50 uA

Fig. 4. Viscero-somatic neurone in the thoracic spinal cord whose responses to stimulationof somatic and visceral afferents were inhibited by conditioning stimulation in AH-POA.This is the same cell as Fig. 1. A, transverse section 1-2 mm rostral to bregma illustratingthe location of the conditioning stimulation site in the anterior hypothalamic area (AHA).Key to abbreviations: CC, corpus callosum; FX, fornix; OC, optic chiasma; SC,suprachiasmatic nucleus; SO, supraoptic nucleus. B and C, superimposed oscilloscopetraces illustrating control responses of the cell to electrical stimulation of its somaticreceptive field and to stimulation of the SPLN (upper traces in B and C respectively), andthe inhibitory effects of conditioning stimulation (10 ms trains at 250 Hz) at intensities of10 and 50 ,uA (middle and lower traces respectively). Each trace represents the responseto five consecutive stimulus presentations. A time of application of the test stimuli, Utiming of the conditioning stimuli.

A second feature of the effects of AH-POA stimulation (also illustrated in Fig. 5)is that at any given- conditioning stimulus intensity the inhibitory effects on theneurone 's responses to both the somatic and visceral test stimuli are of similarmagnitude and show similar thresholds. For the entire population of viscero-somaticneurones tested (n = 12), the percentage reduction of responses to SPLN stimulationranged from 45-100% (mean 76-6 %). This is similar to the observed reductions of

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434 B. M. LUMB

24-92% (mean 71'6 %) of the responses to electrical stimulation of the somaticreceptive fields of the same cells.The effective inhibitory sites on the AH-POA were very localized. This is

illustrated in Fig. 6 which shows the responses of a cell to simulation of somatic andvisceral afferents and their reductions following conditioning stimulation of four

A B

100 5HAL rn 100 5uAD

375 OAiA] 75bA]75 - 10,upAi '75 -10,uiAE2 iEIC 20uyA] 20 pAnCL 50 W . 50

an 50 pA 50 pA]25 50 25

100]lo

01 r 00 50 100 150 200 ms 0 50 100 150 200 msAHAA A Skin AHA A A Skin

Fig. 5. Viscero-somatic neurone in the thoracic spinal cord whose responses to somatic andvisceral afferent stimulation were inhibited by conditioning stimulation in AH-POA. Thisis the same cell as Figs 1 and 4. This figure illustrates that the degree of inhibition isrelated to the intensity of the conditioning stimulus and that responses to somatic andvisceral afferent stimulation are influenced to a similar degree. A and B, dot-rasterdisplays illustrating the response of the neurone to electrical stimulation within itssomatic receptive field (A) and to stimulation of the SPLN (B). These plots show theinhibitory effects of conditioning stimulation in the anterior hypothalamic area (AHA) atdifferent stimulus intensities. The arrows mark the time of application of the test andconditioning stimuli. Open boxes indicate the sweeps where the test stimuli wereconditioned with a train of stimuli in AHA and the adjacent numbers indicate theconditioning stimulus intensity.

different sites (each 0-5 mm apart) and at the same stimulus intensity. A furtherfeature illustrated in Fig. 6 is that the most sensitive sites, i.e. those resulting in thegreatest reduction in the responses to test stimuli, were the same for both somaticand visceral evoked activities.

Excitatory effects from the ventromedial forebrainOnly one viscero-somatic neurone was found in the present study showing an

excitatory response to ventromedial forebrain stimulation (Fig. 7). The exceptionalresult was obtained, however, at a relatively high current intensity (200 gA) from arostral electrode placement in the diagonal band of Broca. It is worth noting,however, that 25% of somatic neurones tested (n = 16) were driven by electricalstimulation at ventromedial forebrain sites including some within the AH-POA. Inthe one viscero-somatic cell (and in all somatic neurones) the initial excitatoryresponse was followed by a decrease in on-going activity (see Fig. 7) and reducedresponsiveness to test stimuli.


A B

E 12.0 12.0 E1.1r 9.0 9.0

6.0 C 6.0

n3.0 3.30

DELr0.04-- 0.0U/) 0 30 6Omrs 0 30 6mrnsA A

Skin SPLN

D Control ~~~~~~~~~~Control E

Control Control

8 6 4 2 00 2 4 6Spikes per response OC Spikes per response

Fig. 6. Viscero-somatic neurone recorded in lamina V of the thoracic spinal cord showingdifferent degrees of inhibition following conditioning stimulation at four separate sites(0 5 mm apart) in a single vertical track through the preoptic area. A and B, peristimulustime histograms (10 sweeps) illustrating the control responses of the cell to electricalstimulation of its somatic receptive field (A) and to stimulation of the SPLN (B). Arrowsmark the time of application of the stimuli. C, transverse section 1-8 mm rostral tobregma (the relevant area is shown enlarged below) illustrating the locations ofconditioning stimulation sites (@). Key to abbreviations: AC, anterior commissure; MS,medial septal nucleus; OC, optic chiasma; PO, preoptic area. D and E, histogramsshowing the mean number of spikes in response to skin (D) and SPLN (E) stimulation incontrol tests and following conditioning stimulation (10 ms trains at 250 Hz and 100 ,sA)at each of four preoptic sites. Each of the histogram bars represent the mean value afterten trials.

Cells unaffected by ventromedial forebrain stimulationSeven cells were unaffected by stimulation in the ventromedial forebrain, often at

sites which produced inhibition in other cells during the same experiment. Further-more, there were no apparent differences in the input properties and laminarlocations of cells with and without descending inhibitory influences from theAH-POA. These observations suggest that the descending influences describedabove may not be widespread and that the activity of any one spinal neurone isinfluenced by neurones in a relatively restricted region of the hypothalamus.

Location of electrical stimulation sitesFigure 8A illustrates the locations of electrical stimulation sites and gives an

indication of the threshold intensities of conditioning stimulation necessary todepress responses to somatic and visceral stimulation. Inhibitory effects wereobserved following stimulation over a relatively large rostrocaudal extent of theregion under study. The most sensitive sites, however, i.e. those requiring currentsof 50 ,tA or less to depress neuronal responses, were located ventrally in theAH-POA. Stimulation at more rostral and more caudal sites generally requiredslightly higher currents to produce similar effects.

435

B. M. LUMB

D 10.0 a

EA 7.5

50Aim

10. - b

u)

.< 2.5Touch/pinch '

00.0 - 1, 10 51 100 150 200 250 ms

SPLN

100-7

EIV I ~~~~~7.5

C,)

DBB X) 2-5

0.00 s50 100 150 200 250 ms

DBB

Fig. 7. Viscero-somatic neurone in the thoracic spinal cord with an excitatory drive fromthe ventromedial forebrain. A, location of the recording site of the neurone in lamina V.B. this cell responded to noxious (pinch) and innocuous (touch/brush) stimulation of itssomatic receptive field (shaded area). C, transverse section 2-4 mm rostral to bregmaillustrating the location of the conditioning stimulation site. Key to abbreviations: AC,anterior commissure; CC, corpus callosum; DBB, diagonal band of Broca; MS, medialseptal nucleus; POA, preoptic area. D, peristimulus time histograms, each taken over tensweeps, illustrating the responses of the cell to (i) electrical stimulation of its somaticreceptive field (Da), (ii) electrical stimulation of the SPLN nerve (Db) and (iii) stimulationwithin DBB with a single 0 5 ms shock at 200 ,uA (Dc).

436


A

0 <50 yA* 50-l100 A* 100-200 MA* No effect

B

_ DLH +ve

M DLH -ve

1 2 3 mm

Fig. 8. Locations of stimulation sites in the ventromedial forebrain. Locations are plottedon a representative sagittal section taken 10 mm lateral to the mid-line. A shows thelocations of electrical stimulation sites, and the size of the symbols gives an indicationof the threshold stimulus intensities necessary to depress responses of thoracic dorsalhorn neurones to somatic and visceral test stimuli (see key in figure). B shows thelocations of microinjections of DLH solution. *, sites where injections of DLH resultedin a depression of dorsal horn neuronal responses to somatic and visceral test stimuli; 6,sites where DLH had no detectable influence on the activities of dorsal horn cells. Key toabbreviations: AC, anterior commissure; AHA, anterior hypothalamic area; FX, fornix;OT, optic track; POA, preoptic area.

Effects of chemical stimulation of ventromedial forebrain sitesAt certain ventromedial forebrain sites the effects of electrical conditioning

stimulation on the responses of viscero-somatic neurones were compared with thoseof local microinjection of DLH. Electrical stimulation at all sites tested (n = 7) ledto an inhibition of on-going and evoked neuronal activity. At two sites, both locatedin the ventral part of the POA (see Fig. 8B), microinjection of DLH resulted in acomplete abolition of responses to test stimuli and a cessation of any on-going

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438 B. M. LUMB

activity. Microinjection at the remaining five sites (see Fig. 8B) had no detectableinfluences on dorsal horn activity.An example of the inhibitory influences on a thoracic viscero-somatic neurone

following electrical and chemical stimulation at a single preoptic site is given inFig. 9. The responses of this cell to stimulation of somatic and visceral afferents weredepressed by conditioning stimulation with current intensities as low as 5 j#A. Aftera short delay, microinjection of DLH at the same site abolished the response of thecell to SPLN stimulation. This inhibition persisted for about 3 min and resulted inan almost complete cessation of on-going activity with the same time course.

A B C CC

XIII '(\Vll~~~~oVMS

Touch/pinch

K-P0A500Mm oc

D

LSkinC SPLN 175 j°

Fi.9. Vseosmtcnurn ntetaic pnlcr hs epne osomai n

E125U) c ~~~~~~~~~100

visceral 75imuliweredpesdyeetcladhmcl stDLH6 35,

(5 ~~~ ~ ~ ~ ~ ~~~20 3020 15 10 521 0 20 40 6080 120 160 200

Conditioning stimulus intensity (aA) A 100 140 180SPLN

Time (ins)Fig. 9. Viscero-somatic neurone in the thoracic spinal cord whose responses to somatic andvisceral stimuli were depressed by electrical and chemical (DLH) stimulation at a site inthe preoptic area. A, location of the recording site of the neurone in lamina V. B, this cellresponded to noxious (pinch) and innocuous (touch/brush) stimulation of its somaticreceptive field (shaded area). C, transverse section 2-0 mm rostral to bregma showing thelocation of the electrical and chemical stimulation sites. Key to abbreviations: see Fig. 6.DA histogram illustrating the inhibitory effects of electrical stimulation (10 ms trains at250 Hz at the current intensities indicated) of the POA on the responses of the cell tosomatic and visceral afferent stimulation. E, dot-raster display of the response of the cellto supramaximal stimulation of the SPLN to illustrate the inhibitory effect of DLHmicroinjection in POA. Arrows mark the time of the application of the stimulus to theSPLN and the microinjection of DLH.

DISCUSSION

The results presented in this paper include the first description of the inputproperties of viscero-somatic neurones in the lower thoracic spinal cord of the rat.Viscero-somatic neurones have also been characterized in terms of their laminarlocations within the spinal gray matter. The results demonstrate that the responses


of these cells to stimulation of visceral and somatic afferent fibres can be powerfullyinhibited by activation of descending pathways that originate in the AH-POA of theventromedial forebrain.

Samples of neuronesThe general properties of thoracic viscero-somatic neurones reported here do not

differ substantially from those recorded in previous studies in other species or atother levels of the spinal cord (see Cervero & Tattersall, 1986; Foreman, Blair &Ammons, 1986; Ness & Gebhart (1990) for recent reviews). For instance, viscero-somatic neurones comprise a large proportion of all the neurones recorded in thedorsal horn (> 50 %). The somatic receptive fields of viscero-somatic neurones weregenerally larger than those of somatic neurones and in the vast majority of viscero-somatic neurones their somatic drives included a noxious input (either exclusively orin addition to low-threshold inputs).With regard to their laminar locations, viscero-somatic neurones were recorded in

the superficial dorsal horn and in laminae IV and V. No viscero-somatic neurones wererecorded in inner lamina II or in lamina III. This is again in agreement with previousreports in the cat and monkey (see above) and is consistent with anatomicaldescriptions of the spinal distribution of SPLN afferents to the lower thoracic cordin the rat (Neuhuber, Sandoz & Fryscak, 1986).No attempt was made in the present study to identify viscero-somatic neurones

with respect to their efferent projections. As such, when interpreting the data itshould be borne in mind that they may not represent a homogeneous population.

Effects of electrical stimulation in AH-POAElectrical stimulation at many sites in and adjacent to AH-POA led to changes in

thoracic dorsal horn neuronal activity. In the majority of viscero-somatic neurones(14/22) conditioning stimulation (consisting of short trains of pulses at currentintensities of 200 ,uA or less) produced a marked reduction in neuronal activity.

These inhibitory effects were not selective for any particular peripheral input. Inany one cell, responses to somatic and visceral test stimuli were depressed to a similardegree and indeed, where present, any on-going activity was also attenuated orabolished. Furthermore, stimulation in AH-POA reduced both the early (presumedA fibre) and late (presumed C fibre) evoked activity of viscero-somatic neurones. Notonly were all forms of activity influenced in a similar manner but the thresholdconditioning intensities necessary to inhibit responses to somatic and visceral teststimuli were generally the same and, where tested, the most sensitive sites inAH-POA (that is those with the lowest thresholds) were the same for both forms ofperipheral input. Taken together, these observations suggest that the inhibitoryeffects observed are mediated ultimately by a common postsynaptic mechanism.The inhibitory effects of AH-POA stimulation observed in the present study are

similar to those described by other authors on the somatic inputs of dorsal neuronesat other spinal levels in the cat (Carstens et al. 1982) and in the marginal zone of thetrigeminal nucleus caudalis in the rat (Mokha et al. 1987). The present study hasextended these investigations by demonstrating that this descending modulationextends to the processing of visceral afferent information in the thoracic cord.

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Effects of chemical stimulation in AH-POANine sites in AH-POA from which inhibition of dorsal horn neurones could be

evoked by electrical stimulation were tested for the effects of microinjections of theexcitatory amino acid DLH. At two sites this treatment attenuated or abolisheddorsal horn neuronal responses to somatic and visceral test stimuli and depressed anyon-going activity over the same time course. This result suggests that the inhibitoryeffects on dorsal horn neurones are mediated, at least in part, by descendingpathways with cell bodies located in this region of the ventromedial forebrain.

A

Effects ondorsal hornneurones

B

Effects on aviscero-somaticreflex

_ DLH +ve

m DLH -ve

I I I1 2 3 mm

Fig. 10. Comparison of the locations of ventromedial forebrain sites sensitive tomicroinjections ofDLH when tested for (A) their effects on thoracic dorsal horn neuronesand (B) their effects on a viscero-somatic reflex (see Lumb & Cervero, 1989). Locations areplotted on a representative sagittal section taken 1 0 mm lateral to the mid-line. Numbersindicate the distance (mm) rostral to bregma. *, sites where injections of DLH resultedin a depression of neuronal or reflex activity; @ sites where DLH had no effect. Key toabbreviations: see Fig. 8.

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These data are largely consistent with the findings of a previous study (Lumb &Cervero, 1989) which demonstrated that activation of cell bodies in the AH-POAproduced an inhibition of a nociceptive viscero-somatic reflex. An importantdifference between these two studies is that the area of the ventromedial forebrainsensitive to microinjection of DLH was more extensive in the reflex study whencompared with the present results (see Fig. 10).There are several possible explanations for this apparent discrepancy. First, with

regard to the reflex, the inhibitory effects observed might operate at multiple sitesin the reflex pathway. For instance, although the present study suggests that at leastpart of the inhibition operates on the transmission of visceral afferent information inthe thoracic dorsal horn it does not exclude the possibility that modulation ofthoracic motor output contributes further to the observed effects on reflex activity.Indeed, both excitatory and inhibitory effects of AH-POA stimulation on thoracicpreganglionic sympathetic outflow have been described previously (Jansson, Lisan-der & Martinson, 1969; Lisander & Delbro, 1987). If this is the case, effects onafferent and efferent transmission could well be mediated by different populations ofneurones with a wider distribution in the ventromedial forebrain.A second consideration is that although the viscero-somatic reflex recorded in the

first study is thought to be mediated largely by propriospinal mechanisms theinhibitory effects observed might operate, at least in part, at supraspinal levels. Forexample, inhibitory effects of AH-POA stimulation on nociceptor-evoked activityin the medullary reticular formation has been reported previously (Lumb &Wolstencroft, 1983). Again, effects on spinal and supraspinal transmission could bemediated by different populations of ventromedial forebrain neurones.

Descending pathways mediating inhibitionThere is no anatomical evidence for direct projections from AH-POA to the spinal

cord, although more lateral and more caudal hypothalamic structures have beenshown to be the source of spinally projecting axons (e.g. Hosoya, 1980; Holstege,1987; Cechetto & Saper, 1988). it is likely therefore that the inhibitory effectsobserved in the present study involve indirect pathways with relays at othersupraspinal sites.Anatomical studies have identified widespread projections of this region of the

ventromedial forebrain to other supraspinal structures. Amongst these are descend-ing projections to the midbrain periaqueductal gray, the medullary reticularformation including the mid-line nucleus raphe magnus (NRM) and the parabrachialregion of the dorsolateral pons (Conrad & Pfaff, 1976; Chiba & Marata, 1985;Holstege, 1987). All these areas have been implicated in mechanisms of descendingcontrol and antinociception and as such are contenders for a relay mediating theinhibitory effects of AH-POA stimulation.The study of Mokha et al. (1987) addressed this question by testing the effects of

electrolytic lesions in the periaqueductal gray, NRM and medullary reticularformation on the inhibitory effects of preoptic stimulation on trigeminal nociceptiveneurones. The results of their study indicated that all three sites could participate inthis inhibition. A possible involvement of NRM in this relay, is strengthened by thedemonstration of excitatory inputs from the AH-POA onto antidromicallyidentified raphe-spinal neurones in the rat (Lumb & Morrison, 1986).

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Functional significanceThe results of this study have demonstrated that transmission though viscero-

somatic neurones in the thoracic spinal dorsal horn can be powerfully inhibited bydescending pathways that originate in the AH-POA of the ventromedial forebrain.

Viscero-somatic neurones in the thoracic spinal cord are presumed to mediateupper abdominal pain and/or participate in reflex responses to nociceptor stimulation(Cervero & Tattersall, 1986). If this is the case then the inhibitory effects ofAH-POAstimulation reported here may indeed have a role in the antinociceptive effects ofstimulation in this region in awake animals as suggested by other authors (Carstenset al. 1982; Mokha et al. 1987).

It has been postulated that descending control systems which modulate spinaltransmission of nociceptive information may constitute the descending limb ofsupraspinal looped pathways that are themselves triggered by nociceptive stimu-lation. It is proposed that such negative feedback loops could participate in thesignalling of pain by certain spinal cord neurones (LeBars, Dickenson & Besson,1979). A role for more rostral brain structures in similar feedback loops is providedby the recent demonstration of direct projections from the spinal cord tohypothalamic regions in the rat (Burstein, Cliffer & Giesler, 1987). Furthermore,spinal neurones with nociceptive somatic inputs have been shown to contributeaxons to this projection.

It is well documented that hypothalamic structures including the AH-POAparticipate in autonomic regulatory mechanisms including cardiovascular control(see Mancia & Zanchetti, 1981 for review). Not surprisingly therefore, microinjectionsof DLH at many of the sites tested (Lumb & Cervero, 1989; see also Gelsema, Roe& Calaresu, 1989, and the present study) lead to marked alterations in arterial bloodpressure (either increases or decreases) in addition to any influences on the spinalprocessing of visceral afferent input.

These observations, and the demonstration of direct nociceptive projections tohypothalamic structures, suggest that this region of the ventromedial forebrain mayhave a role in the integration of autonomic and sensory consequences of a nociceptivevolley arriving in the CNS.

The financial support of the MRC and the expert technical assistance of S. Allen and S. W.Lishman are gratefully acknowledged. I thank Dr F. Cervero for his advice in the preparation ofthe manuscript.

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