- Email: [email protected]
Modulation of a viscerosomatic reflex by electrical and chemical stimulation of hypothalamic structures in the rat
400
Brain Re.search. 500 (I t~)'i 4()()--4t~ Elscvie~
BRES 23778
Modulation of a viscerosomat|c r lex by electrical and chemical stimulation of hypothatamic structures in the rat B.M. Lumb and E Cervero Department of Physiology, University of Bristol Medical School, Bristol (U. K. )
(Accepted 18 July 1989) Key words: Viscerosomatic reflex; Anterior hypothalamus; Preoptic area
Ventromedial forebrain structures were stimulated electrically with short (10-ms) trains of pulses to test for effects on a viscerosomatic reflex. Stimulation at many hypothalamic sites led to an attenuation or even a complete inhibition of reflex activity. The most sensitive sites, however (i.e. those requiring currents of 50 pA or less to inhibit the reflex), were located in the anterior hypothalamus/preoptic area (AH/POA) and rostrally in the diagonal band of Broca (DBB). At certain sites the effects of electrical stimulation were compared with those of microinjection of an excitatory amino acid (DL-homocysteic acid) which is known to excite neuronal cell bodies and not axons. The results of this part of the study indicated that activation of cell bodies located in the ventromedial AH/POA (from the level of the optic chiasma caudally to the level of DBB rostrally) mediate, at least in part, the inhibitory effects on visceral afferent processing. These data are discussed in relation to a possible role of AH/POA in the spinal processing of nociceptive information of visceral origin. The spinal transmission of visceral sensory information can be modulated by electrical stimulation in the rostral ventromedial medulla t'4'15. Similar inhibitory influences on the spinal transmission of information arising from somatic structures are well documented (see refs. 2, 16 for recent reviews). It is generally believed that this inhibitory modulation participates in the antinociceptive effects of brainstem stimulation observed in behavioural experiments and which extends to pain of visceral origin 6. In the present experiments stimulation at more rostral sites, in particular the anterior hypothalamus/ preoptic area ( A H / P O A ) of the ventromedial forebrain, has been tested for effects on reflex activity evoked by stimulation of visceral afferent fibres. Several lines of evidence have implicated the A H / P O A in antinociceptive mechanisms. For instance, it represents an area sensitive to microinjections of morphine in the production of analgesia 12 and conversely, electrolytic lesions placed in this region have been reported to attenuate the analgesic actions of systemic morphine in rats 13. In awake
animals electrical stimulation in A H / P O A has been shown to attenuate behaviourat responses to noxious somatic stimuli n and the available evidence suggests that these antinociceptive effects are mediated, at least in part, by activation of descending pathways which modulate nociceptive transmission in the dorsal horn of the spinal cord 3 and in the trigeminal nucleus 1°. The aims of the present experiments were two-fold; first, to investigate the influences of descending pathways from hypothalamic structures on reflex responses to visceral afferent stimulation and secondly, to locate the cell bodies of neurones contributing to this descending system. The effects of visceral afferent stimulation were assessed by monitoring reflex activity in the first or second lumbar nerves (L 1 or Lz) evoked by electrical stimulation of visceral afferent fibres in the splanchnic nerve (SPLN). This viscerosomatic reflex, described in the cat by D o w n m a n in 19555, can only be evoked by stimulation of SPLN at intensities which recruit C- and/or A6-fibres and is thought to be largely mediated by propriospinal mechanisms. To
Correspondence: B.M. Lumb, Department of Physiology, University of Bristol Medical School, University Walk, Bristol BS8 1TD, U.K.
0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
401 test any influences of hypothalamic structures on viscerosomatic reflex activity, the test stimulus to SPLN was conditioned with electrical stimulation at a variety of hypothalamic sites. To determine whether any hypothalamic influences were due to the activation of neuronal cell bodies in this region, the effects of electrical stimulation at certain sites were then compared with those of injection of an excitatory amino acid (DL-homocysteic acid; D L H ) which is known to act only on the cell bodies or dendritic processes of central neurones and not on axons of passage ~4. Experiments were carried out in 16 male rats anaesthetised with a-chloralose (100 mg/kg i.v.), paralysed with pancuronium bromide and artificially ventilated. Arterial blood pressure, end-tidal CO2 and rectal temperature were monitored and maintained within physiological limits in all animals. The SPLN was exposed unilaterally at its bifurcation into the greater and lesser divisions, prepared with bipolar stimulating electrodes and cut peripherally. The ipsilateral L~ and L 2 spinal nerves were dissected free from supporting tissues as they emerged from the paravertebral muscles. In each experiment one of these nerves, generally L 1, was prepared with bipolar recording electrodes and cut peripherally. A pool was made with skin flaps and the nerves immersed in warm paraffin oil. The filtered and amplified signal from the recording electrode was displayed on an oscilloscope and averaged responses to SPLN stimulation were computed on-line. The animals were then positioned in a stereotaxic instrument and a small area of the contralateral frontal bone was removed so that stimulating electrodes could be introduced into hypothalamic structures. Stimulating electrodes were 3-barrelled glass micropipettes (overall tip diameter 20-30 ~m) suitable for electrical stimulation and pressure injection of D L H (McAllen and Woolard, personal communication). The electrode barrel used for electrical stimulation was filled with a mixture of Woods metal and indium. A second barrel contained D L H solution (pH 7.4, 0.2 M) saturated with Pontamine sky blue (PSB). This barrel was attached to a pressure injection pump which allowed small amounts of the D L H solution to be ejected with 0.5-s pulses at pressures between 5 and 30 p.s.i. The third barrel
was also attached to the pressure injection pump and contained solutions for control injections of either NaC1 (pH 7.4, 0.2 M) or the inhibitory amino acid x-aminobutyric acid ( G A B A ; pH 7.4, 0.2 M). An inhibitory amino acid was used as a control in some experiments because microinjections of D L H have been reported to produce a depolarising block of neuronal activity at the centre of injections ~ and a cessation of activity might contribute to any effects observed on viscerosomatic reflex activity. The stimulating electrode was mounted in a micromanipulator and introduced into the brain between 0.5 and 2.0 mm lateral to the midline. An area of the ventromedial forebrain (0.8-3.0 mm rostral to bregma) was then mapped using short (10-ms) trains of electrical stimuli (0.2-ms pulses at 300 Hz) for influences on viscerosomatic reflex activity. From an initial position within a few mm of the ventral surface of the brain, the electrode was advanced ventrally and sites were tested every 500 1~m to determine the threshold current intensity necessary to attenuate reflex activity. At the end of each track the electrode was returned to the site from which the lowest threshold had been obtained and, if the threshold was unchanged, the site was then tested for the effects of D L H microinjection on reflex activity. In an attempt to localise the sites sensitive to D L H , other injections were made at sites which had required higher intensities of electrical stimulation to influence the viscerosomatic reflex. At the end of each experiment, the brain was removed and fixed in formal saline. The locations and an indication of the extent of D L H injections were then determined from the spread of PSB as seen in serial 60-ym transverse sections. Sites of electrical stimulation and D L H injections, together with their effects on viscerosomatic reflex activity, are summarised in the transverse sections of Fig. 1. For clarity, sites of electrical stimulation are depicted on the right of each of the sections and locations of D L H injections on the left, although in practice both forms of stimulation were made contralateral to the spinal nerve from which recordings were made. In the case of electrical stimulation, each of the filled circles represents a site that was tested and the size of each symbol gives an indication of the current intensity necessary to depress reflex activity. The most sensitive sites, that is those requiring the
402 least current (50 HA or less), were situated ventrally and medially in the A H / P O A and extended rostrally to the diagonal band of Broca (DBB). Stimulation at sites more dorsal or more lateral to this, and stimulation of more caudal hypothalamic structures, generally required higher current intensities to produce similar depressions of the reflex. From the symbols on the left side of each of the sections in Fig. 1, it can be seen that effective DLH injection sites largely correspond with those sites that were most sensitive when tested with electrical stimulation, that is, they were situated ventrally within the A H / P O A and more rostrally in DBB. Fig. 2 gives examples from one experiment of the effects of D L H and G A B A on viscerosomatic reflex activity following their microinjection at two discrete hypothalamic sites. From the lower traces it can be
seen that injection of D L H at a site in the ventromedial anterior hypothalamus completely abolished viscerosomatic reflex activity whereas, after recovery of the reflex, injection of G A B A at the same site had no effect. A subsequent injection of D L H at this site (bottom trace) produced similar inhibitory effects on the reflex which suggests that the inefficacy of G A B A was not due to previous tissue damage in the region of the microinjections. Sites sensitive to D L H were generally situated ventrally in this region and this is illustrated in the upper traces of Fig. 2 where, in the same electrode track, a more dorsal injection of D L H had little or no influence on reflex activity. It is worth noting that, at many of the sites tested, microinjections of D L H and G A B A evoked changes (either increases or decreases) in arterial blood pressure. However, the direction and amplitude of
/
iL., [ 'i I! ".: m'I~;
/ s~ / ~c\
1.2
0
. I ';:V~'. ,~";. ~Ii
~
'"
: I • I
• < , ..
~
O ~,', .. ~
1.0
:
I/
~
s0~A
• so-,~a .>
zoop~
...<
[] DLH -re 08
Fig. 1. Outline representations of transverse sections through the ventromedial forebrain to illustrate sites of electrical stimulation (symbols on the right of each section) and locations of microinjections of DLH (symbols on the left of each section) which were tested for their effects on viscerosomatic reflex acitvity. Electrical conditioning stimulus intensities necessary to attenuate reflex activity are indicated by the size of solid circles (see key). Sites sensitive to DLH are shown as solid symbols and sites where DLH had no influence on reflex activity as hatched symbols. A, arcuate nucleus; AC, anterior commissure; FX, fornix; MS, medial septal nucleus; OC, optic chiasma; OT, optic tract; PVH, parav'entricular hypothalamic nucleus; SC, suprachiasmatic nucleus; SO, supraoptic nucleus. Numbers indicate the distance in mm rostral to bregma.
403
.~
~,,~,,~,~.,.~
J~7
L l
.., .......
....
4
.........................
..... ~"'\,,i...... - . . . . . . . . . . . . . . . . . . . . . . . . . . .
SPLN control
SPLN +DLH SPLN control
%W,'A~'" .............................
SPLN control
z. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SPLN+DLH
5 ..................
SPLN + DLH
1"2 ~.. . . . . . . . . . . . . . .
~JlOO}JV SPLN
10ms
Fig. 2. Example from one experiment to illustrate the effects of DLH and GABA microinjected at two discrete hypothalamic sites on reflex activity recorded from a spinal nerve (Lt) in response to stimulation of visceral afferent fibres in the SPLN. Transverse section on the left shows the locations of microinjections (solid circles). Abbreviations: see Fig. 1. Each of the traces on the right represent averaged activity recorded from the L~ spinal nerve in response to l0 single shock stimuli of the SPLN in either control situations or immediately following injections of GABA and/or DLH. Arrow marks the timing of the stimulus artifacts. Numbers indicate the sequence the recordings were made in.
these changes were not consisent with those of the changes in viscerosomatic reflexes. Furthermore, changes in arterial blood pressure were not observed following the short trains of electrical conditioning stimuli necessary to depress reflex activity. These experiments have demonstrated that reflex activity evoked by activity in visceral afferent fibres can be attenuated by electrical stimulation at a number of sites in the ventromediai forebrain. The sites that required the least current to produce these effects were located in the ventromedial A H / P O A and DBB. These low currents suggest that the cell bodies of origin of a descending modulatory system are located in this region and this is supported by the observation that similar inhibitory effects were produced by local microinjection of an excitatory amino acid into the A H / P O A . Other authors 3"m have reported inhibitory influences from the A H / P O A on the spinal and trigeminal transmission of nociceptive information of somatic origin. The present study has extended these observations by demonstrating that the inhibitory influences extend to the processing of visceral information. Furthermore, this study is the first to
provide evidence that neurones whose cell bodies are located in the A H / P O A participate in these effects. The present study cannot, however, offer precise information about the site of action of the descending modulation. First, although this viscerosomatic reflex is thought to be mediated largely by propriospinal mechanisms 5, the inhibitory effects observed might operate, at least in part, at supraspinal levels. Secondly, the inhibitory effects of hypothalamic stimulation might reflect modulation of thoracic motor output rather than inhibition of transmission in the dorsal horn. For instance, excitatory and inhibitory effects of A H / P O A stimulation on thoracic preganglionic sympathetic outflow have been described previously 7"9. In on-going experiments these questions are being addressed by determining the effects of hypothalamic stimulation on the responses of individual neurones in the thoracic dorsal horn to stimulation of visceral and somatic afferent fibres. The reflex recorded in the present study is thought to be the electrophysiological correlate of the sustained contractions of abdominal muscles produced
404 by noxious stimulation of abdominal viscera. Modulation of such a reflex supports the proposal that
of n e u r o n e s influenced by hypothalamic stimulation in terms of their peripheral inputs and ascending
descending inhibitory influences from A H / P O A are concerned with mechanisms of antinociception as
projections,
suggested previously~'m. Recordings in the dorsal horn of the thoracic spinal cord will help clarify this issue since it will be possible to determine the classes 1 Ammons, W.S., Blair, R.W. and Foreman, R.D., Raphe magnus inhibition of primate T~-T2 spinothalamic cells with cardiopulmonary visceral input, Pain. 20 (1984) 247-260. 2 Besson, J.M. and Chaouch, A., Peripheral and central mechanisms of nociception, Physiol. Rev., 67 (1987) 67-187. 3 Carstens, E., Mackinnon, J.D. and Guinan, M.J., Inhibition of spinal dorsal horn neuronal responses to noxious skin heating by medial preoptic and septal stimulation in the cat, J. Neurophysiol., 48 (1982) 981-991. 4 Chapman, C.D., Ammons, W.S. and Foreman, R.D., Raphe magnus inhibition of feline T~-T4 spinoreticular tract cell responses to visceral and somatic inputs, J. Neurophysiol., 53 (1985) 773-785. 5 Downman, C.B.B., Skeletal muscle reflexes of splanchnic and intercostal nerve origin in acute spinal and decerebrate cats, J. Neurophysiol., 18 (1955) 217-235. 6 Giesler, G.J. and Liebeskind, J.C., Inhibition of visceral pain by electrical stimulation in the periaqueductal grey matter, Pain, 2 (1976) 43-48. 7 Jansson, G., Lisander, B. and Martinson, J., Hypothalamic control of adrenergic outflow to the stomach in the cat, Acta Physiol. Scand., 75 (1%9) 176-186. 8 Lipski, J., Bellingham, M.C., West, M.J. and Pilowsky, P., Limitations of the technique of pressure microinjection of excitatory amino acids for evoking responses from localized regions of the CNS, J. Neurosci. Methods, 26 (1988) 169-179.
This work was supported by a grant from the M R C (U.K.). 9 Lisander, B. and Delbro, D., Hypothalamic stimulation counteracts sympathetically mediated gastrointestinal inhibition in chloralose-anaesthetised cats, J. Auton. Nerv. Syst., 20 (1987) 147-153. 10 Mokha, S.S., Goldsmith, G.E., Hellon, R.F. and Puri, R.. Hypothalamic control of nocireceptive and other neurones in the marginal layer of the dorsal horn of the medulla (trigeminal nucleus caudalis) in the rat, Exp. Brain Res., 65 (1987) 427-436. 11 Oleson, T.D., Kirkpatrick, D.B. and Goodman, S.J., Elevation of pain threshold to tooth shock by brain stimulation in primates, Brain Res., 194 (1980) 79-95. 12 Pert, A. and Yaksh, T., Sites of morphine-induced analgesia in the primate brain: relation to pain pathways, Brain Res., 80 (1974) 135-140. 13 Pottoff, P., Valentino, D. and Lal, H., Attenuation of morphine analgesia by lesions of the preoptic forebrain region in the rat, Life Sci., 24 (1979) 421-424. 14 Stone, T.W., Microiontophoresis and pressure injection, IBRO Handbook Series: Methods in the Neurosciences, Vol. 8, 1985, p. 214. 15 Tattersall, J.E.H., Cervero, E and Lumb, B.M., Viscerosomatic neurones in the lower thoracic spinal cord of the cat: excitations and inhibitions evoked by splanchnic nerve volleys and by stimulation of brainstem nuclei, J. Neurophysiol., 56 (1986) 1411-1423. 16 Willis, W.D., Control of nociceptive transmission in the spinal cord. In D. Ottoson (Ed.), Progress in Sensory Physiology, Vol. 3, Springer, New York.
Brain Re.search. 500 (I t~)'i 4()()--4t~ Elscvie~
BRES 23778
Modulation of a viscerosomat|c r lex by electrical and chemical stimulation of hypothatamic structures in the rat B.M. Lumb and E Cervero Department of Physiology, University of Bristol Medical School, Bristol (U. K. )
(Accepted 18 July 1989) Key words: Viscerosomatic reflex; Anterior hypothalamus; Preoptic area
Ventromedial forebrain structures were stimulated electrically with short (10-ms) trains of pulses to test for effects on a viscerosomatic reflex. Stimulation at many hypothalamic sites led to an attenuation or even a complete inhibition of reflex activity. The most sensitive sites, however (i.e. those requiring currents of 50 pA or less to inhibit the reflex), were located in the anterior hypothalamus/preoptic area (AH/POA) and rostrally in the diagonal band of Broca (DBB). At certain sites the effects of electrical stimulation were compared with those of microinjection of an excitatory amino acid (DL-homocysteic acid) which is known to excite neuronal cell bodies and not axons. The results of this part of the study indicated that activation of cell bodies located in the ventromedial AH/POA (from the level of the optic chiasma caudally to the level of DBB rostrally) mediate, at least in part, the inhibitory effects on visceral afferent processing. These data are discussed in relation to a possible role of AH/POA in the spinal processing of nociceptive information of visceral origin. The spinal transmission of visceral sensory information can be modulated by electrical stimulation in the rostral ventromedial medulla t'4'15. Similar inhibitory influences on the spinal transmission of information arising from somatic structures are well documented (see refs. 2, 16 for recent reviews). It is generally believed that this inhibitory modulation participates in the antinociceptive effects of brainstem stimulation observed in behavioural experiments and which extends to pain of visceral origin 6. In the present experiments stimulation at more rostral sites, in particular the anterior hypothalamus/ preoptic area ( A H / P O A ) of the ventromedial forebrain, has been tested for effects on reflex activity evoked by stimulation of visceral afferent fibres. Several lines of evidence have implicated the A H / P O A in antinociceptive mechanisms. For instance, it represents an area sensitive to microinjections of morphine in the production of analgesia 12 and conversely, electrolytic lesions placed in this region have been reported to attenuate the analgesic actions of systemic morphine in rats 13. In awake
animals electrical stimulation in A H / P O A has been shown to attenuate behaviourat responses to noxious somatic stimuli n and the available evidence suggests that these antinociceptive effects are mediated, at least in part, by activation of descending pathways which modulate nociceptive transmission in the dorsal horn of the spinal cord 3 and in the trigeminal nucleus 1°. The aims of the present experiments were two-fold; first, to investigate the influences of descending pathways from hypothalamic structures on reflex responses to visceral afferent stimulation and secondly, to locate the cell bodies of neurones contributing to this descending system. The effects of visceral afferent stimulation were assessed by monitoring reflex activity in the first or second lumbar nerves (L 1 or Lz) evoked by electrical stimulation of visceral afferent fibres in the splanchnic nerve (SPLN). This viscerosomatic reflex, described in the cat by D o w n m a n in 19555, can only be evoked by stimulation of SPLN at intensities which recruit C- and/or A6-fibres and is thought to be largely mediated by propriospinal mechanisms. To
Correspondence: B.M. Lumb, Department of Physiology, University of Bristol Medical School, University Walk, Bristol BS8 1TD, U.K.
0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
401 test any influences of hypothalamic structures on viscerosomatic reflex activity, the test stimulus to SPLN was conditioned with electrical stimulation at a variety of hypothalamic sites. To determine whether any hypothalamic influences were due to the activation of neuronal cell bodies in this region, the effects of electrical stimulation at certain sites were then compared with those of injection of an excitatory amino acid (DL-homocysteic acid; D L H ) which is known to act only on the cell bodies or dendritic processes of central neurones and not on axons of passage ~4. Experiments were carried out in 16 male rats anaesthetised with a-chloralose (100 mg/kg i.v.), paralysed with pancuronium bromide and artificially ventilated. Arterial blood pressure, end-tidal CO2 and rectal temperature were monitored and maintained within physiological limits in all animals. The SPLN was exposed unilaterally at its bifurcation into the greater and lesser divisions, prepared with bipolar stimulating electrodes and cut peripherally. The ipsilateral L~ and L 2 spinal nerves were dissected free from supporting tissues as they emerged from the paravertebral muscles. In each experiment one of these nerves, generally L 1, was prepared with bipolar recording electrodes and cut peripherally. A pool was made with skin flaps and the nerves immersed in warm paraffin oil. The filtered and amplified signal from the recording electrode was displayed on an oscilloscope and averaged responses to SPLN stimulation were computed on-line. The animals were then positioned in a stereotaxic instrument and a small area of the contralateral frontal bone was removed so that stimulating electrodes could be introduced into hypothalamic structures. Stimulating electrodes were 3-barrelled glass micropipettes (overall tip diameter 20-30 ~m) suitable for electrical stimulation and pressure injection of D L H (McAllen and Woolard, personal communication). The electrode barrel used for electrical stimulation was filled with a mixture of Woods metal and indium. A second barrel contained D L H solution (pH 7.4, 0.2 M) saturated with Pontamine sky blue (PSB). This barrel was attached to a pressure injection pump which allowed small amounts of the D L H solution to be ejected with 0.5-s pulses at pressures between 5 and 30 p.s.i. The third barrel
was also attached to the pressure injection pump and contained solutions for control injections of either NaC1 (pH 7.4, 0.2 M) or the inhibitory amino acid x-aminobutyric acid ( G A B A ; pH 7.4, 0.2 M). An inhibitory amino acid was used as a control in some experiments because microinjections of D L H have been reported to produce a depolarising block of neuronal activity at the centre of injections ~ and a cessation of activity might contribute to any effects observed on viscerosomatic reflex activity. The stimulating electrode was mounted in a micromanipulator and introduced into the brain between 0.5 and 2.0 mm lateral to the midline. An area of the ventromedial forebrain (0.8-3.0 mm rostral to bregma) was then mapped using short (10-ms) trains of electrical stimuli (0.2-ms pulses at 300 Hz) for influences on viscerosomatic reflex activity. From an initial position within a few mm of the ventral surface of the brain, the electrode was advanced ventrally and sites were tested every 500 1~m to determine the threshold current intensity necessary to attenuate reflex activity. At the end of each track the electrode was returned to the site from which the lowest threshold had been obtained and, if the threshold was unchanged, the site was then tested for the effects of D L H microinjection on reflex activity. In an attempt to localise the sites sensitive to D L H , other injections were made at sites which had required higher intensities of electrical stimulation to influence the viscerosomatic reflex. At the end of each experiment, the brain was removed and fixed in formal saline. The locations and an indication of the extent of D L H injections were then determined from the spread of PSB as seen in serial 60-ym transverse sections. Sites of electrical stimulation and D L H injections, together with their effects on viscerosomatic reflex activity, are summarised in the transverse sections of Fig. 1. For clarity, sites of electrical stimulation are depicted on the right of each of the sections and locations of D L H injections on the left, although in practice both forms of stimulation were made contralateral to the spinal nerve from which recordings were made. In the case of electrical stimulation, each of the filled circles represents a site that was tested and the size of each symbol gives an indication of the current intensity necessary to depress reflex activity. The most sensitive sites, that is those requiring the
402 least current (50 HA or less), were situated ventrally and medially in the A H / P O A and extended rostrally to the diagonal band of Broca (DBB). Stimulation at sites more dorsal or more lateral to this, and stimulation of more caudal hypothalamic structures, generally required higher current intensities to produce similar depressions of the reflex. From the symbols on the left side of each of the sections in Fig. 1, it can be seen that effective DLH injection sites largely correspond with those sites that were most sensitive when tested with electrical stimulation, that is, they were situated ventrally within the A H / P O A and more rostrally in DBB. Fig. 2 gives examples from one experiment of the effects of D L H and G A B A on viscerosomatic reflex activity following their microinjection at two discrete hypothalamic sites. From the lower traces it can be
seen that injection of D L H at a site in the ventromedial anterior hypothalamus completely abolished viscerosomatic reflex activity whereas, after recovery of the reflex, injection of G A B A at the same site had no effect. A subsequent injection of D L H at this site (bottom trace) produced similar inhibitory effects on the reflex which suggests that the inefficacy of G A B A was not due to previous tissue damage in the region of the microinjections. Sites sensitive to D L H were generally situated ventrally in this region and this is illustrated in the upper traces of Fig. 2 where, in the same electrode track, a more dorsal injection of D L H had little or no influence on reflex activity. It is worth noting that, at many of the sites tested, microinjections of D L H and G A B A evoked changes (either increases or decreases) in arterial blood pressure. However, the direction and amplitude of
/
iL., [ 'i I! ".: m'I~;
/ s~ / ~c\
1.2
0
. I ';:V~'. ,~";. ~Ii
~
'"
: I • I
• < , ..
~
O ~,', .. ~
1.0
:
I/
~
s0~A
• so-,~a .>
zoop~
...<
[] DLH -re 08
Fig. 1. Outline representations of transverse sections through the ventromedial forebrain to illustrate sites of electrical stimulation (symbols on the right of each section) and locations of microinjections of DLH (symbols on the left of each section) which were tested for their effects on viscerosomatic reflex acitvity. Electrical conditioning stimulus intensities necessary to attenuate reflex activity are indicated by the size of solid circles (see key). Sites sensitive to DLH are shown as solid symbols and sites where DLH had no influence on reflex activity as hatched symbols. A, arcuate nucleus; AC, anterior commissure; FX, fornix; MS, medial septal nucleus; OC, optic chiasma; OT, optic tract; PVH, parav'entricular hypothalamic nucleus; SC, suprachiasmatic nucleus; SO, supraoptic nucleus. Numbers indicate the distance in mm rostral to bregma.
403
.~
~,,~,,~,~.,.~
J~7
L l
.., .......
....
4
.........................
..... ~"'\,,i...... - . . . . . . . . . . . . . . . . . . . . . . . . . . .
SPLN control
SPLN +DLH SPLN control
%W,'A~'" .............................
SPLN control
z. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SPLN+DLH
5 ..................
SPLN + DLH
1"2 ~.. . . . . . . . . . . . . . .
~JlOO}JV SPLN
10ms
Fig. 2. Example from one experiment to illustrate the effects of DLH and GABA microinjected at two discrete hypothalamic sites on reflex activity recorded from a spinal nerve (Lt) in response to stimulation of visceral afferent fibres in the SPLN. Transverse section on the left shows the locations of microinjections (solid circles). Abbreviations: see Fig. 1. Each of the traces on the right represent averaged activity recorded from the L~ spinal nerve in response to l0 single shock stimuli of the SPLN in either control situations or immediately following injections of GABA and/or DLH. Arrow marks the timing of the stimulus artifacts. Numbers indicate the sequence the recordings were made in.
these changes were not consisent with those of the changes in viscerosomatic reflexes. Furthermore, changes in arterial blood pressure were not observed following the short trains of electrical conditioning stimuli necessary to depress reflex activity. These experiments have demonstrated that reflex activity evoked by activity in visceral afferent fibres can be attenuated by electrical stimulation at a number of sites in the ventromediai forebrain. The sites that required the least current to produce these effects were located in the ventromedial A H / P O A and DBB. These low currents suggest that the cell bodies of origin of a descending modulatory system are located in this region and this is supported by the observation that similar inhibitory effects were produced by local microinjection of an excitatory amino acid into the A H / P O A . Other authors 3"m have reported inhibitory influences from the A H / P O A on the spinal and trigeminal transmission of nociceptive information of somatic origin. The present study has extended these observations by demonstrating that the inhibitory influences extend to the processing of visceral information. Furthermore, this study is the first to
provide evidence that neurones whose cell bodies are located in the A H / P O A participate in these effects. The present study cannot, however, offer precise information about the site of action of the descending modulation. First, although this viscerosomatic reflex is thought to be mediated largely by propriospinal mechanisms 5, the inhibitory effects observed might operate, at least in part, at supraspinal levels. Secondly, the inhibitory effects of hypothalamic stimulation might reflect modulation of thoracic motor output rather than inhibition of transmission in the dorsal horn. For instance, excitatory and inhibitory effects of A H / P O A stimulation on thoracic preganglionic sympathetic outflow have been described previously 7"9. In on-going experiments these questions are being addressed by determining the effects of hypothalamic stimulation on the responses of individual neurones in the thoracic dorsal horn to stimulation of visceral and somatic afferent fibres. The reflex recorded in the present study is thought to be the electrophysiological correlate of the sustained contractions of abdominal muscles produced
404 by noxious stimulation of abdominal viscera. Modulation of such a reflex supports the proposal that
of n e u r o n e s influenced by hypothalamic stimulation in terms of their peripheral inputs and ascending
descending inhibitory influences from A H / P O A are concerned with mechanisms of antinociception as
projections,
suggested previously~'m. Recordings in the dorsal horn of the thoracic spinal cord will help clarify this issue since it will be possible to determine the classes 1 Ammons, W.S., Blair, R.W. and Foreman, R.D., Raphe magnus inhibition of primate T~-T2 spinothalamic cells with cardiopulmonary visceral input, Pain. 20 (1984) 247-260. 2 Besson, J.M. and Chaouch, A., Peripheral and central mechanisms of nociception, Physiol. Rev., 67 (1987) 67-187. 3 Carstens, E., Mackinnon, J.D. and Guinan, M.J., Inhibition of spinal dorsal horn neuronal responses to noxious skin heating by medial preoptic and septal stimulation in the cat, J. Neurophysiol., 48 (1982) 981-991. 4 Chapman, C.D., Ammons, W.S. and Foreman, R.D., Raphe magnus inhibition of feline T~-T4 spinoreticular tract cell responses to visceral and somatic inputs, J. Neurophysiol., 53 (1985) 773-785. 5 Downman, C.B.B., Skeletal muscle reflexes of splanchnic and intercostal nerve origin in acute spinal and decerebrate cats, J. Neurophysiol., 18 (1955) 217-235. 6 Giesler, G.J. and Liebeskind, J.C., Inhibition of visceral pain by electrical stimulation in the periaqueductal grey matter, Pain, 2 (1976) 43-48. 7 Jansson, G., Lisander, B. and Martinson, J., Hypothalamic control of adrenergic outflow to the stomach in the cat, Acta Physiol. Scand., 75 (1%9) 176-186. 8 Lipski, J., Bellingham, M.C., West, M.J. and Pilowsky, P., Limitations of the technique of pressure microinjection of excitatory amino acids for evoking responses from localized regions of the CNS, J. Neurosci. Methods, 26 (1988) 169-179.
This work was supported by a grant from the M R C (U.K.). 9 Lisander, B. and Delbro, D., Hypothalamic stimulation counteracts sympathetically mediated gastrointestinal inhibition in chloralose-anaesthetised cats, J. Auton. Nerv. Syst., 20 (1987) 147-153. 10 Mokha, S.S., Goldsmith, G.E., Hellon, R.F. and Puri, R.. Hypothalamic control of nocireceptive and other neurones in the marginal layer of the dorsal horn of the medulla (trigeminal nucleus caudalis) in the rat, Exp. Brain Res., 65 (1987) 427-436. 11 Oleson, T.D., Kirkpatrick, D.B. and Goodman, S.J., Elevation of pain threshold to tooth shock by brain stimulation in primates, Brain Res., 194 (1980) 79-95. 12 Pert, A. and Yaksh, T., Sites of morphine-induced analgesia in the primate brain: relation to pain pathways, Brain Res., 80 (1974) 135-140. 13 Pottoff, P., Valentino, D. and Lal, H., Attenuation of morphine analgesia by lesions of the preoptic forebrain region in the rat, Life Sci., 24 (1979) 421-424. 14 Stone, T.W., Microiontophoresis and pressure injection, IBRO Handbook Series: Methods in the Neurosciences, Vol. 8, 1985, p. 214. 15 Tattersall, J.E.H., Cervero, E and Lumb, B.M., Viscerosomatic neurones in the lower thoracic spinal cord of the cat: excitations and inhibitions evoked by splanchnic nerve volleys and by stimulation of brainstem nuclei, J. Neurophysiol., 56 (1986) 1411-1423. 16 Willis, W.D., Control of nociceptive transmission in the spinal cord. In D. Ottoson (Ed.), Progress in Sensory Physiology, Vol. 3, Springer, New York.