Inhibitory Effects Evoked From the Anterior Hypothalamus Are Selective for the Nociceptive Responses of Dorsal Horn Neurons With High- and Low-Threshold Inputs

B. J. Workman and B. M. Lumb

Department of Physiology, School of Medical Sciences, Bristol BS8 1TD, United Kingdom

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Workman, B. J. and B. M. Lumb. Inhibitory effects evoked from the anterior hypothalamus are selective for the nociceptive responses of dorsal horn neurons with high- and low-threshold inputs. J. Neurophysiol. 77: 2831-2835, 1997. The aim of the present study was to examine the selectivity of descending control of nociceptive information in the spinal dorsal horn following neuronal activation at "pressor" sites in the anterior hypothalamus. Extracellular single-unit activity was recorded from 11 dorsal horn neurons in the lower lumbar spinal cord of anesthetized rats. Neurons selected for investigation were those that responded to noxious (pinch and radiant heat >46°C) and nonnoxious (prod, stroke, and/or brush) stimulation within their cutaneous receptive fields on the ipsilateral hind paw. These are referred to as Class 2 neurons. Micropipettes were inserted stereotaxically into the anterior hypothalamus at sites where injection of the excitatory amino acidL-homocysteic acid (L-HCA) evoked increases in arterial blood pressure. The effects of microinjection of L-HCA at "pressor" sites in the anterior hypothalamus were then tested on the responses of Class 2 neurons to noxious and nonnoxious stimulation of their excitatory receptive fields. The high-threshold (pinch and/or radiant heat) responses of 7/7 Class 2 neurons tested were inhibited by an average of 66.3 ± 8.8% (mean ± SE) by neuronal activation at hypothalamic pressor sites. The low-threshold (prod) responses of 10/10 Class 2 neurons tested were not inhibited by neuronal activation at hypothalamic pressor sites; in 6 of these cells the response to low-intensity stimulation was increased by between 4 and 20%. Control injections of the inhibitory amino acid gamma -aminobutyric acid (GABA) at the same hypothalamic pressor sites had no significant effects on arterial blood pressure or neuronal activity. With regard to sensory processing in the spinal cord, these data suggest that descending inhibitory control that originates from neurons in pressor regions of the anterior hypothalamus is highly selective for nociceptive inputs to Class 2 neurons.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Clinical observations in addition to anatomic, electrophysiological, pharmacological, and behavioral studies suggest that systems controlling the perception of pain are closely coupled to systems modulating cardiovascular function (for reviews see Randich and Gebhart 1992; Randich and Maixner 1984). Initially, attention focused on the periaqueductal gray (PAG) region of the midbrain as the site of integration of autonomic and antinociceptive functions (see Duggan and Morton 1983; Lovick 1985). However, recent studies indicate that the descending control of nociceptive processing that can be evoked from sites in the anterior hypothalamus is also associated with specific patterns of autonomic changes (Lumb and Lovick 1993).

An emerging view is that descending control of nociceptive transmission evoked from areas of the PAG that also support sympathoexcitation and cardiovascular defense responses may be selective for nociceptive responses of spinal dorsal horn neurons (Duggan and Morton 1983). In contrast, adjacent areas of the PAG that support sympathoinhibition may be the source of a less selective descending control (see Lovick 1993). Interestingly, the integrated response that can be evoked from some anterior hypothalamic sites includes pressor responses and other autonomic changes characteristic of a defense response (Lumb and Lovick 1993).

The aim of the present study was to determine whether or not the reported association between selective inhibition of nociceptive reponses of spinal dorsal horn neurons and sympathoexcitation extends to descending control of spinal processing evoked from anterior hypothalamic pressor sites. A preliminary account of this work has been published in abstract form (Workman and Lumb 1994).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Anesthesia was induced in six adult male rats (290-330 g) with halothane (3% in 100% O2) and then maintained with Saffan (alphaxalone-alphadolone; 9-12 mg·kg-1·h-1 iv). The animals were paralyzed with pancuronium bromide (0.3-0.45 mg/kg as required) and ventilated. Arterial blood pressure, end-tidal CO2, and rectal temperature were monitored and maintained within physiological limits. A laminectomy exposed the lower lumbar spinal cord, and the animals were mounted in a rigid frame with the head positioned in a stereotaxic instrument. Extracellular recordings of single-unit activity were made in the dorsal horn using glass micropipettes filled with 4 M NaCl. Single units were characterized according to Menetrey et al. (1977) by their responses to natural stimulation with hand-held instruments (nonnoxious brushing, prodding or stroking, and noxious pinching); Class 1 cells responded to nonnoxious stimulation alone, Class 2 neurons responded to noxious and nonnoxious stimuli, and Class 3 neurons to noxious stimulation alone. To study any selectivity in descending influences from the hypothalamus, this study has focused on effects on Class 2 neurons.

Electronically controlled stimuli were delivered for 10-s periods every minute using an electromagnetic prodder for nonnoxious stimulation and a pneumatic pincher and/or a thermostatically controlled radiant heat device for noxious stimulation. Pinch stimuli ranged from 3 to 6 N, heat stimuli between 46 and 50°C, and prod stimuli were delivered at frequencies between 10 and 30 Hz. Parameters of peripheral stimuli were adjusted so that, in any experimental "run," the responses evoked by the different forms of stimulation were of similar magnitudes. The aim of this approach was to reduce the possibility that the efficacy of any descending influences was determined by the degree of depolarization of the spinal cord neuron.

Triple-barreled glass micropipettes were inserted stereotaxically (Paxinos and Watson 1986) into the anterior hypothalamus ipsilateral to the spinal cord recording sites. The pipettes were suitable for making electrolytic lesions (through a barrel containing Woods metal and indium) and pressure injection of L-HCA (pH 7.4, 0.2 M, 50-100 nl) to selectively activate neuronal cell bodies. To control for any effects of L-HCA being due to depolorizing block, and a cessation of activity at the site of injection, a third barrel contained the inhibitory amino acid gamma -aminobutyric acid (GABA; 0.2 M) that could be pressure injected at the same site to hyperpolarize neurons. It should be borne in mind, however, that, in addition to any block of activity in descending pathways, this treatment may disinhibit neurons that give rise to descending pathways.

Experimental protocol

The stimulating electrode was introduced into the ventromedial forebrain and advanced in 200-µm steps until ejection of L-HCA evoked a pressor response. The electrode was then left in position so that the effects of L-HCA could be tested on responses of single dorsal horn neurons. In each experimental run the dorsal horn neuron was activated by one of the forms of controlled peripheral stimulation for 10 s every minute. After two or three responses to a given stimulus to establish the baseline response of the cell, L-HCA or GABA was injected at the pressor site in the anterior hypothalamus 5-10 s before the onset of the next period of peripheral stimulation. Several cycles of peripheral stimulation were then repeated to establish recovery of the response from any descending influences evoked from the hypothalamus. The effects of chemical stimulation in the hypothalamus were then repeated on responses to different forms of peripheral stimulation depending on thecell type.

Histological methods

Recording sites were marked by iontophoretic deposition of Pontamine Sky Blue dye (PSB) in the last track of the experiment. The centers of the sites of chemical stimulation in the hypothalamus were marked by electrolytic lesions or by pressure ejection ofPSB. The stimulating and recording sites were later recovered in50-µm transverse sections of the brain and spinal cord stained with Neutral Red.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Extracellular single-unit activity was recorded from 14 neurons in the dorsal horn of the lower lumbar spinal cord; 11 Class 2, two Class 1, and one Class 3. Only the data from the Class 2 cells are reported here.

Descending influences from the anterior hypothalamus on the activities of class 2 neurons

Eleven Class 2 neurons were tested with stimulation at pressor sites in the hypothalamus. In six of the Class 2 neurons recorded from, a complete investigation was made of any descending influences on low- (prod) and high- (pinch and/or radiant heat) threshold responses. Incomplete data were obtained from a further five cells; in one cell, effects on high-threshold responses alone were tested, and, in four cells, only low-threshold events were fully investigated. Regardless of the combination of tests that were carried out, when the data obtained from Class 2 neurons were pooled, the following picture emerged.

In all instances, the high-threshold responses of these neurons were depressed following neuronal activation at sites in the anterior hypothalamus. The magnitude of the inhibitions ranged from 23 to 92% (66.3 ± 8.8%; mean ± SE), and they persisted for up to 250 s. Statistical analysis using a paired nonparametric test (Wilcoxon signed-rank test) revealed the inhibitory effects to be very significant (P = 0.0078). The available data suggest that effects on radiant heat were more potent (81.7 ± 8.4%; n = 3) than those on noxious pinch (57.2 ± 11.7%; n = 7).

The effects of anterior hypothalamic stimulation were tested on the low-threshold responses of 10 Class 2 neurons. This includes six of the cells that were also tested for any effects on high-threshold responses. In four cells there was no apparent effect, the magnitude of the responses remaining within 1% of baseline values. In the remaining six cells, responses to peripheral prod stimuli were enhanced by between 4 and 20% (7.6 ± 2.6%). Statistical analysis ofthe effects of hypothalamic stimulation on the prod-evoked responses of all 10 neurons tested revealed the mean facilitatory effect to be 4.6 ± 1.9%, which was significantly different from control responses (P = 0.0156, Wilcoxon signed-rank test). An example of the effects of anterior hypothalamic stimulation on the responses of a Class 2 neuron to low-intensity (prod) and high-intensity (pinch and radiant heat) stimuli is illustrated in Fig. 1. Figure 1 also includes the arterial blood pressure record that was taken over the same time course as the ratemeter record in Fig. 1A and shows the pressor responses to the two microinjections of L-HCA.


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FIG. 1. Ratemeter records (binwidth 1 s) illustrating the effects of neuronal activation in the anterior hypothalamus on the responses of a single Class 2 neuron to pinch (A), radiant heat (B), and prod (C) stimuli. Bars indicate the periods of peripheral stimulation. Arrows indicate the timing of microinjections of L-homocysteic acid (L-HCA) and the inhibitory amino acid gamma -aminobutyric acid (GABA). The transverse section through the forebrain indicates the site of microinjections in the anterior hypothalamus. The transverse section of the L6 segment of the spinal cord illustrates the location of the recording site in lamina V. The arterial blood pressure trace is taken over the same time course as the ratemeter record in A and illustrates the pressor responses to microinjection of L-HCA and the failure of a similar injection of GABA to affect arterial pressure. A1.0, 1.0 mm rostral to bregma; AHA, anterior hypothalamic area; OC, optic chiasma; SO, supraoptic nucleus.

Facilitatory effects of anterior hypothalamic stimulation on responses to low-intensity prod stimuli are shown in Fig. 1C. Figure 1 also illustrates the failure of GABA to influence arterial blood pressure or neuronal activity when injected at the same site. This was a consistent finding at all sites where GABA was tested (n = 5).

Locations of neurons recorded in the dorsal horn and of hypothalamic stimulation sites

The laminar distributions of all neurons tested with hypothalamic stimulation are shown in Fig. 2A. All cells were recorded in the deep dorsal horn (laminae IV, V, and VI). It should be noted that, in all cases where the low-threshold responses of Class 1 or 2 neurons were not inhibited, these data are only included if stimulation at the same site in the same animal was shown to inhibit the nociceptive responses of Class 3 and/or Class 2 neurons.


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FIG. 2. Representative transverse sections through the L6 segment of the spinal cord (A) and the ventral forebrain (B) to illustrate the locations of recording sites in the dorsal horn and of stimulation sites in the anterior hypothalamus, respectively. A: locations of all cells tested with hypothalamic stimulation. Filled symbols indicate that responses of neurons were inhibited by hypothalamic stimulation and open symbols that responses of neurons were not inhibited by hypothalamic stimulation. Class 2 neurons are represented by circles where the left hemisphere represents the reponse to low-intensity stimulation and the right that to high-intensity stimulation. Incomplete circles represent Class 2 neurons where the effects of hypothalamic stimulation were tested on responses to one form of stimulation alone. B: locations of sites in the anterior hypothalamus where microinjections of L-HCA evoked pressor responses and which were tested for their effects on dorsal horn neurons. Numbers indicate the distance in mm rostral to bregma. OC, optic chiasma; PVN, paraventricular nucleus; SC, suprachiasmatic nucleus; SO, supraoptic nucleus; VMH, ventromedial hypothalamus.

The locations of stimulation sites in the anterior hypothalamus are shown in Fig. 2B. These sites are located close to the ventral surface of the brain at the level of the optic chiasma. In addition to influences on the evoked resposes in the spinal dorsal horn, activation of neurons at these sites also evoked pressor responses (e.g., Fig. 1). Increases in mean arterial blood pressure evoked from these sites ranged from 12 to 32.5 mmHg (17.08 ± 3.2 mmHg), and the duration of responses ranged from 80 to 250 s (135 ± 2.3 s).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The results presented in this paper demonstrate that activation of neuronal perikarya, at pressor sites in the anterior hypothalamus exerts a selective inhibitory effect on nociceptive responses of Class 2 dorsal horn neurons. In contrast, responses to low-intensity stimulation (prod) were left intact or even enhanced. The failure of GABA to influence arterial blood pressure or neuronal activity suggests that the effects of L-HCA were not due to a cessation of activity at the site of injection and argue against GABA disinhibiting a source of descending control. It is possible that changes in arterial blood pressure themselves may affect the properties of peripheral receptors and hence the responses of the dorsal horn neurons recorded from. This possibility was not tested directly; however, in other studies (Hudson and Lumb 1995) we have observed inhibition of nociceptive spinal reflexes evoked by pinch stimulation regardless of the direction of blood pressure changes evoked by chemical stimulation in the anterior hypothalamus. Also, the evoked increases in arterial pressure and any inhibition of neuronal activity did not always occur over the same time course. Precise measurement of the time course of effects on neuronal activity was limited by the intermittent nature of the peripheral stimulus, but in two cases it was possible to say with certainty that effects on arterial blood pressure outlasted any effects on neuronal activity, and in a further two cases the opposite was observed.

Descending inhibitory control from the anterior hypothalamus

Several studies have examined descending influences from the anterior hypothalamus on the processing of nociceptive information in the spinal cord (Carstens et al. 1982; Lumb 1990; Lumb and Cervero 1989) and trigeminal system (Mokha et al. 1987). With the exception of previous investigations from this laboratory, these studies used electrical stimulation at forebrain sites to activate descending pathways, which activates all neuronal elements at the site of stimulation and, as a consequence, makes the results difficult to interpret.

The studies of Carstens et al. (1982) and Mokha et al. (1987) suggested that the anterior hypothalamus may exert selective effects on nociceptive compared with nonnoxious inputs to the dorsal horn. However, in neither of these studies were the effects of hypothalamic stimulation on the low- and high-threshold inputs to individual Class 2 neurons tested systematically nor were correlations made with any autonomic changes. Both of these issues were addressed in the current investigation. In the current study, only sites that supported pressor responses were tested for their effects on the activities of dorsal horn neurons. As such, our observations are consistent with reports (Duggan and Morton 1983; see Lovick 1993) that in the midbrain, descending inhibitory control that can be evoked from pressor sites is associated with a selective inhibition of dorsal horn neurons, whereas, in contrast, stimulation at depressor sites evokes a nonselective inhibition of dorsal horn activity (however see, Waters and Lumb 1997).

Descending facilitation from the anterior hypothalamus

Descending excitation of dorsal horn neurons from supraspinal sites has been reported previously (Cervero and Lumb 1988; Dubuisson and Wall 1980; Light et al. 1986; McMahon and Wall 1988). However, to our knowledge this is the first report of facilitation following chemical stimulation at supraspinal sites and the first of a selective facilitatory effect on the low-threshold responses of Class 2 neurons.

Functional significance

A role for other hypothalamic regions in the integration of affective defensive behavior after noxious stimulation has been suggested previously (e.g., Bester et al. 1995). The hypothalamic sites selected for investigation in the present study were those from which pressor responses could be evoked, and it has been shown previously (Lumb and Lovick 1993) that other cardiovascular adjustments associated with defense behavior are evoked from anterior hypothalamic pressor sites. Interestingly, recent behavioral studies in rats suggest that states of high arousal, often associated with a decreased perception of pain, can be produced by neuronal activation in the anterior hypothalamus (Adams et al. 1993; Roeling et al. 1993). This raises the possibility that the highly selective descending control system described here may operate as part of an integrated response of an animal to stressful or life-threatening stimuli. From a survival perspective this system would allow an animal to respond in an appropriate manner to a life-threatening situation without the distraction caused or reflexes evoked by noxious stimulation. Survival would be further protected because responses to other, nonnoxious, cues would be left intact or even enhanced.

    ACKNOWLEDGEMENTS

  The authors are grateful to S. W. Lishman for expert technical help.

  The work was supported by the Wellcome Trust.

    FOOTNOTES

  Address for reprint requests: B. M. Lumb, Dept. of Physiology, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.

  Received 18 November 1996; accepted in final form 5 February 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society