Physiological Studies of Spinohypothalamic Tract Neurons in the Lumbar Enlargement of Monkeys

X. Zhang, H. N. Wenk, A. P. Gokin, C. N. Honda, and G. J. Giesler, Jr.

Department of Neuroscience and Graduate Program in Neuroscience, University of Minnesota, Minneapolis, Minnesota 55455


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Zhang, X., H. N. Wenk, A. P. Gokin, C. N. Honda, and G. J. Giesler Jr.. Physiological Studies of Spinohypothalamic Tract Neurons in the Lumbar Enlargement of Monkeys. J. Neurophysiol. 82: 1054-1058, 1999. Recent anatomic results indicate that a large direct projection from the spinal cord to the hypothalamus exists in monkeys. The aim of this study was to determine whether the existence of this projection could be confirmed unambiguously using electrophysiological methods and, if so, to determine the response characteristics of primate spinohypothalamic tract (SHT) neurons. Fifteen neurons in the lumbar enlargement of macaque monkeys were antidromically activated using low-amplitude current pulses in the contralateral hypothalamus. The points at which antidromic activation thresholds were lowest were found in the supraoptic decussation (n = 13) or in the medial hypothalamus (n = 2). Recording points were located in the superficial dorsal horn (n = 1), deep dorsal horn (n = 10), and intermediate zone (n = 4). Each of the 12 examined neurons had cutaneous receptive fields on the ipsilateral hindlimb. All neurons responded exclusively or preferentially to noxious stimuli, suggesting that the transmission of nociceptive information is an important role of primate SHT axons. Twelve SHT neurons were also antidromically activated from the thalamus. In all cases, the antidromic latency from the thalamus was shorter than that from the hypothalamus, suggesting that the axons pass through the thalamus then enter the hypothalamus. These results confirm the existence of a SHT in primates and suggest that this projection may contribute to the production of autonomic, neuroendocrine, and emotional responses to noxious stimuli in primates, possibly including humans.


    INTRODUCTION
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INTRODUCTION
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The hypothalamus is involved in the production of autonomic, neuroendocrine, and motivational/emotional responses to somatosensory and visceral stimuli (reviewed in Giesler et al. 1994). Several lines of evidence indicate that the spinohypothalamic tract (SHT) carries somatosensory and visceral information directly from the spinal cord to the hypothalamus in rats (Burstein et al. 1990; Dado et al. 1994a,b; Katter et al. 1996; Malick and Burstein 1998; Zhang et al. 1995). Anatomic studies indicate that an SHT exists in primates and that its organization is similar to that in rats (Chang and Ruch 1949; Morin et al. 1951; Newman et al. 1996). Recently, we found that injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the hypothalamus of monkeys labeled large numbers of neurons throughout the length of the spinal cord (Zhang et al. 1997). These findings suggest that nociceptive information may reach the hypothalamus of primates in part through a direct projection from the spinal cord. In this study, we have 1) attempted to confirm unequivocally the existence of this projection in primates and 2) tested the idea that primate SHT neurons are capable of conveying nociceptive information.


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INTRODUCTION
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All procedures followed the guidelines of the International Association for the Study Pain and were approved by the institutional animal care and use committee.

Monkeys (Macaca fascicularis or mulatta) were anaesthetized initially with ketamine (100 mg/kg im), followed by alpha -chloralose (60 mg/kg iv) and maintained with a continuous infusion of nembutal (2-4 mg · kg-1 · h-1 iv). Animals were placed in a stereotaxic frame, paralyzed, and artificially ventilated. Body temperature, end-tidal CO2, and blood pressure were monitored and kept within physiological limits. Pneumothoraces were performed to improve mechanical stability. Laminectomies and large craniotomies were made. Multiunit recordings of somatosensory responses in the thalamus were made to locate the ventral posterior lateral nucleus (VPL). The initial placement of the stimulating electrode (stainless steel) in the hypothalamus was made using the location of VPL as a reference point. The search stimulus consisted of cathodal pulses (200 µs, 750 µA) delivered within the contralateral hypothalamus. Single-unit recordings were made in the lumbosacral spinal cord using stainless steel microelectrodes (5-10 MOmega ). After isolating an antidromically activated action potential (criteria in Fig. 1A), the stimulating electrode was moved systematically through the hypothalamus until a point was located at which the antidromic threshold was <= 30 µA (Fig. 1A) (Dado et al. 1994a). We determined whether each of the examined SHT neurons could also be antidromically activated from the contralateral thalamus using search pulses of 500-750 µA. If a neuron was activated, the thalamic stimulating electrode was moved until a low-threshold point (<= 30 µA) was located. Collision of action potentials evoked from the electrodes in the hypothalamus and thalamus was demonstrated (Dado et al. 1994a) to ensure that the same neuron was activated from both locations. Cutaneous receptive fields and response characteristics of recorded cells were determined using innocuous and noxious mechanical stimuli (Dado et al. 1994b). At the end of each experiment, electrolytic lesions were made at each low-threshold stimulation point and at the recording site. Monkeys were perfused with 0.9% saline followed by 10% Formalin containing 1% potassium ferrocyanide (Prussian blue reaction). The areas of the brain and spinal cord containing lesions were removed, cut transversely, and stained with neutral red. The locations of lesions were reconstructed with a microscope equipped with a camera lucida drawing attachment.



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Fig. 1. Antidromic activation of a primate spinohypothalamic tract (SHT) neuron. A: electrode penetrations were made throughout the contralateral hypothalamus. Antidromic thresholds are indicated with the low-threshold point in supraoptic decussation (SoD) circled. The response had a stable latency (a1, overlapping traces), followed high-frequency pulses (a2), and collided with orthodromic spikes (a3). This SHT neuron was also antidromically activated from the contralateral thalamus (latency, 4.0 ms; location not illustrated). Spikes elicited from the hypothalamus and thalamus collided (a4 and a5). B: For 2 SHT cells, multiple stimulation tracks were made in the ipsilateral hypothalamus to determine whether the examined SHT axons continued into the ipsilateral hypothalamus. This figure illustrates the one case in which a low-threshold point was found in ipsilateral SoD. Note the longer latency (b1) and collision (b2 and b3), indicating that the examined axon ascended contralaterally, then entered the ipsilateral hypothalamus. Solid arrows indicate stimulation times. Open arrows indicate the times at which antidromic spikes would have occurred. C: recording site. CF, column fornix; F, fornix; GP, globus pallidus; IC, internal capsule; LH, lateral hypothalamus; PvN, paraventricular nucleus; OT, optic tract; RT, reticular thalamic nucleus; 3V, third ventricle.


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INTRODUCTION
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Fifteen SHT neurons were antidromically activated using current pulses <= 30 µA delivered in the contralateral hypothalamus. Two examples of such neurons are illustrated in Figs. 1 and 2. In Fig. 1, an SHT neuron in the lateral reticulated area of L4 (Fig. 1C) was antidromically activated from the supraoptic decussation (SoD) of the contralateral hypothalamus (Fig. 1A) at a latency of 4.2 ms (Fig. 1, a1-a3). The axon was also activated in the contralateral thalamus at a latency of 4.0 ms. Spikes elicited from the hypothalamus and thalamus collided within a critical period (Fig. 1, a4 and a5), indicating that action potentials from both locations traveled in the same axon. This axon was also activated from the SoD of the hypothalamus ipsilateral to the recording site at a latency of 5.0 ms (Fig. 1B), and spikes elicited from this location also collided with spikes elicited in the contralateral thalamus (Fig. 1, b2 and b3). The antidromic latencies at these three points indicate that the SHT axon ascended through the contralateral thalamus to the hypothalamus and crossed the midline from the contralateral to the ipsilateral hypothalamus, as frequently occurs in rats (Dado et al. 1994a). Figure 2 illustrates an SHT neuron in the marginal zone that was activated antidromically from a low-threshold point in the contralateral medial hypothalamus (Fig. 2, A and C). This axon was also activated using small-amplitude current pulses in contralateral VPL at a shorter latency (Fig. 2B). Action potentials elicited from these two points collided (Fig. 2, b2 and b3). This cell had a receptive field on the ipsilateral hind limb (Fig. 2D), and it responded preferentially to noxious stimuli (wide dynamic range, WDR, Fig. 2E).



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Fig. 2. Primate SHT neuron with an axon that projected to contralateral hypothalamus and thalamus. A: low-threshold points in contralateral hypothalamus (a1-a3) and thalamus (b1, note shorter latency). Action potentials elicited from these 2 points collided (b2 and b3). C: recording point in superficial dorsal horn. D: receptive field. E: graded responses to innocuous and noxious mechanical stimuli. Br, brush; Cm, central medial nucleus; LG, lateral geniculate nucleus; LP, lateral posterior thalamic nucleus; MD, medial dorsal thalamic nucleus; Pi, pinch; Pr, pressure; Sq, Squeeze; VPL, ventral posterior lateral thalamic nucleus; VPM, ventral posterior medial thalamic nucleus; ZI, zona incerta.

The points at which antidromic activation thresholds were lowest for the examined neurons (Fig. 3, A, b1, and b2) were located in the SoD (n = 13) and medial hypothalamus (n = 2), areas in which many SHT axons were seen in anterograde tracing experiments in monkeys (Chang and Ruch 1949; Morin et al. 1951; Newman et al. 1996). Recording points (Fig. 3, C and D) were located in the marginal zone (n = 1), deep dorsal horn (n = 10), and lateral intermediate zone (n = 4), areas in which the majority of labeled SHT neurons were found in our retrograde tracing experiments (Zhang et al. 1997). The mean antidromic conduction velocity was 45 m/s (range, 17-75 m/s), indicating that information is carried rapidly in SHT axons.



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Fig. 3. A: photomicrograph of a low-threshold stimulation point in hypothalamus. B: low-threshold stimulation points in hypothalamus and thalamus. C: photomicrograph of a recording site in lumbar enlargement. D: recording sites in lumbar enlargement. E: receptive fields. F: histograms of graded responses of 2 additional SHT neurons to innocuous and noxious mechanical stimuli. The receptive fields for these 2 SHT neurons are indicated in E. Both of these neurons were recorded in the deep dorsal horn. The stars in B3 indicate the 3 low-threshold points where 1 SHT neuron was activated within the thalamus. Triangles in B3 indicate the 2 points at which another SHT neuron was activated. Aq, aqueduct; MN, mammillary nucleus; Put, putamen; SN substantia nigra; VA, ventral anterior thalamic nucleus; III, oculomotor nucleus.

Spike amplitudes were sufficient to determine cutaneous receptive fields (RF) for 12 of the 15 antidromically identified SHT neurons. Each RF was restricted to the ipsilateral hind limb (Fig. 3E). The smallest RF covered one toe, and the largest covered the entire hind limb. Each of the tested neurons was nociceptive (Figs. 2E and 3F); nine were WDR neurons and three responded specifically to noxious stimuli (high-threshold neurons, HT). Responses of two additional SHT neurons to cutaneous mechanical stimuli are illustrated in Fig. 3F. One was classified WDR (Fig. 3F1), the other HT (Fig. 3F2).

Twelve of the 15 tested SHT neurons were also antidromically activated from the thalamus using 500- to 750-µA pulses. In each case, antidromic activity elicited from the thalamus and hypothalamus collided, and the antidromic latency from the thalamus was less than that from the hypothalamus. In seven cases, the stimulating electrode was moved dorsal-ventrally and medial-laterally within the thalamus until the threshold for antidromic activation was <= 30 µA (Figs. 2B and 3B3). Four SHT neurons were antidromically activated from such low-threshold points in VPL and one from the white matter dorsomedial to the lateral geniculate nucleus (Fig. 3B3). In the other two cases, SHT neurons were antidromically activated at multiple low-threshold points (each at a different antidromic latency, Fig. 3B3).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The 15 examined SHT neurons were antidromically activated by stimulus pulses <= 30 µA delivered within the hypothalamus. These small-amplitude pulses have an effective spread of <= 400 µm (Dado et al. 1994a; Ranck 1975). Each of the low-threshold points was located in the ventral half of the hypothalamus, at a considerable distance from areas such as the thalamus, zona incerta, or midbrain that are known to receive a direct input from spinal cord neurons (Apkarian and Hodge 1989; Cliffer et al. 1991). Indeed, with the exception of one low-threshold point that was ~4 mm from the thalamus, low-threshold points in the hypothalamus were at least 6 mm from these areas. Therefore the axons of the examined neurons were activated within the hypothalamus and not as a result of current that spread into the zona incerta, thalamus, or midbrain. Thus the present findings strongly confirm the existence of SHT in primates.

All tested SHT neurons responded preferentially or exclusively to noxious mechanical stimulation, indicating that conveying nociceptive information is an important function of primate SHT neurons. The physiological characteristics of SHT neurons, including conduction velocities, sizes of cutaneous receptive fields, and responses to mechanical stimuli, were similar to those of spinothalamic tract (STT) neurons in monkeys (Willis 1985; Willis et al. 1974).

In this study, 12 of the 15 examined SHT neurons could also be antidromically activated from thalamus. In each case, the antidromic latency from the thalamus was less than that from the hypothalamus, demonstrating that the current had not spread from the electrode in the thalamus to the axon in the hypothalamus. These findings suggest that a significant number of SHT axons either pass through the thalamus before reaching the hypothalamus or give rise to collateral branches that enter thalamus. In four cases, SHT neurons were activated using <= 30 µA from points within VPL. VPL is generally thought to play an important role in localization of somatosensory stimuli (Willis 1985). The present results suggest that, through collateral projections to VPL, SHT axons may also contribute to the localization of noxious stimuli.

In many previous studies, either antidromic activation or retrograde labeling from injections of tracer into thalamus was used to identify "STT" cells. The present findings suggest that some of these neurons may have had axons that continued rostrally and medially into the hypothalamus.

The present findings demonstrate that primate SHT neurons are nociceptive. Previous anterograde tracing experiments in rats and monkeys (Cliffer et al. 1991; Newman et al. 1996) indicate that SHT axons terminate densely and widely throughout many regions of the hypothalamus. Our preliminary retrograde tracing experiments also indicate that large numbers of SHT neurons located throughout the length of the spinal cord of monkeys project directly to the hypothalamus (Zhang et al. 1997). The previous and present results suggest that the SHT is an important source of sensory information to neurons in the hypothalamus that underlie the production or modification of neuroendocrine, autonomic, and affective responses to painful stimuli in primates, including humans.


    ACKNOWLEDGMENTS

We thank H. Truong for valuable technical assistance, and we are grateful to Dr. Martin Wessendorf for critically reading an early version of this manuscript.

This work was supported by National Institutes of Health Grants NS-25932 to G. J. Giesler, DA-09641 to C. N. Honda, and by NIH Training Grant DE-07288 to H. N. Wenk.

Present address of A. P. Gokin: Dept. of Anesthesiology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115.


    FOOTNOTES

Address for reprint requests: G. J. Giesler, Dept. of Cell Biology and Neuroanatomy, 4-102 Owre Hall, University of Minnesota, Minneapolis, MN 55455.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 September 1998; accepted in final form 1 April 1999.


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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society