Identity of muscle efferents activated by skin cooling
It is clear that the cold-activated efferent activity recorded in these experiments was of fusimotor fibres. This is supported by arguments against it being caused by any other efferent fibre type, in that it was not accompanied by evidence of α-motoneuron activity (movement, or electromyographic activity in innervated muscles), and it was unaffected by ganglion-blocking doses of hexamethonium. The positive argument for fusimotor fibre activation is that there was an accompanying sensitization of muscle spindle afferent activity. Although changes in muscle temperature secondary to skin cooling (via blood) could theoretically have influenced spindle afferent discharge, the disappearance of the sensitization once the efferent nerve supply was cut shows that any such direct effect was negligible.
The absence of EMG changes during cooling suggests that β-motoneurons, if present (Andrew et al. 1978), were not activated. The patterns of spindle afferent sensitization seen during cooling (steady increases in tonic firing with little or no adaptation to a maintained stretch, and de novo tonic activation) are characteristics of spindle sensitization by static but not dynamic fusimotor neurons (Andrew et al. 1978). Our protocol was not set up to detect dynamic fusimotor fibre actions, nor did we isolate single units and measure conduction velocities. We therefore conclude that while static fusimotor fibres were activated by skin cooling, we cannot say whether or not dynamic fusimotor fibres were activated as well.
Not all muscle afferent discharge was sensitized by skin cooling. There are at least three reasons why this may have happened in those cases: first, the fusimotor supply to the spindle under study may not have been activated, or activated sufficiently, by cooling; second, the process of dissecting afferent filaments could have damaged the efferent supply to that spindle; and third, the tonic afferent activity could have been caused by tendon organ rather than spindle discharge. We therefore cannot be sure about the proportion of fusimotor fibres activated by skin cooling; but the sensitization of afferents by cooling was common enough to be found in every experimen.
In order to study the central control of fusimotor fibres, previous studies needed to use preparations in which fusimotor neurons were spontaneously active. This was achieved by using lightly anaesthetized or unanaesthetized preparations (von Euler & Söderberg, 1957; Schäfer & Schäfer, 1973). Under these conditions, fusimotor discharge is influenced strongly by local spinal reflexes (Hunt & Paintal, 1958; Alnaes et al. 1965) and by postural factors (Burton & Bronk, 1937). A change in fusimotor discharge may be detected by its effect on the muscle spindle afferent response to a standard stretch (von Euler & Söderberg, 1957; Sato, 1984). On this basis, von Euler & Söderberg (1957) stated that there was strong, tonic discharge from muscle spindles in the ‘lightly anaesthetised animal slightly below thermal balance’, but this was diminished by warming the preoptic/hypothalamic region with a thermode; Sato (1984) later confirmed this principle by showing increased muscle spindle responses when the preoptic region was cooled. The influence of cutaneous thermoreceptors was not investigated.
The present study, by contrast, used rats that were anaesthetized sufficiently deeply to prevent spontaneous fusimotor activity. We were then able to make the novel finding that fusimotor activity is generated as a reflex response to skin cooling, without any direct contribution from falls in core temperature. An inhibitory effect of a warm core temperature was confirmed, but only by its ability to block or attenuate responses to skin cooling.
von Euler & Söderberg (1957) considered that the fusimotor discharge was directly linked to cortical arousal, because they found parallel changes in fusimotor activity and EEG state, in that moderate hypothalamic warming caused EEG synchronization and decreased fusimotor activity, while strong heating desynchronized the EEG and increased fusimotor activity. Although our experiments confirmed that fusimotor discharge and EEG desynchronization commonly occurred together, our evidence does not support a direct, causal relationship. First, their timing did not accurately coincide, in that fusimotor activation during cooling episodes generally led EEG desynchronization, and finished earlier. Second, we were able to dissociate the two responses by using scrotal warming to desynchronize the EEG without activating fusimotor discharge. We were also able to block the fusimotor response to cooling, without blocking the EEG response, by inhibiting raphé neurons. This last point indicates that the brainstem pathways for the two effects are separate, and that the abolition of the fusimotor response by raphé glycine injections was not caused by interruption of an ascending ‘arousal’ pathway. We therefore conclude that, while skin cooling drives both responses, fusimotor activation is not a simple consequence of cortical arousal. Rather, fusimotor activation appears to be an independent reflex response to skin cooling. The inhibitory effect of core temperature on the fusimotor response to skin cooling further supports the concept that it is a thermoregulatory reflex.
Local cooling of the paw pad has been used in lightly anaesthetized cats to activate fusimotor fibres supplying muscles in the same limb (Lupandin, 1983; Lupandin & Kuz'mina, 1985). These effects are also prominent in spinal animals (Sato & Hasegawa, 1977; Sato, 1981, 1983), and may thus best be interpreted as segmental or local spinal reflexes. Cooling such a small body region is unlikely to provide much drive for thermoregulation. Under conditions where spinal reflexes provide the dominant drive, one may expect reflex responses to differ between the supplies to flexor and extensor muscles. Indeed, Lupandin reported such differences on cooling the paw pad (Lupandin, 1983; Lupandin & Kuz'mina, 1985). In line with that interpretation, we found, by contrast, that trunk skin cooling activated the fusimotor fibres to both extensor (the gastrocnemius) and flexor muscle (peroneal nerve. Tanaka M, Owens NC, Nagashima K, Kanosue K, McAllen RM, unpublished observations), suggesting that this was a generalized thermoregulatory response affecting at least the limbs, and perhaps the whole body.
The medullary raphé and fusimotor activity
The link between the medullary raphé and fusimotor activity was first addressed by Sato et al. (1990), who recorded from the ventral roots of anaesthetized rats whose preoptic regions had been destroyed by electrolytic lesions. In response to brief, tetanic electrical stimulation of the nucleus raphé magnus, these workers found that some fusimotor neurons were facilitated but most were inhibited, often for several minutes (Sato et al. 1990). Only the minority of facilitatory responses, however, could have perhaps involved the raphé–fusimotor pathway implicated in the present study.
It is believed that neurons in the ventral medullary raphé, level with the caudal part of the facial nucleus, are an essential synaptic relay mediating cold defence responses such as cutaneous vasoconstriction and non-shivering thermogenesis by interscapular brown adipose tissue (BAT; Blessing et al. 1999; Morrison, 1999; Morrison et al. 1999; Rathner & McAllen, 1999; Blessing & Nalivaiko, 2000; Tanaka et al. 2002), and it has been demonstrated that inhibition of neurons in the medullary raphé can block the sympathetic cutaneous vasoconstrictor (tail sympathetic nerve) responses to skin and core cooling (Ootsuka et al. 2004). Our results indicate that the fusimotor neuron activation by trunk skin cooling is also prevented when raphé neurons are inhibited. One hypothesis, then, might be that the same raphé neurons drive three responses to cold: cutaneous vasoconstriction, thermogenesis by BAT and fusimotor activation. We consider that this is unlikely, because the firing properties of fusimotor fibres (this study) differ significantly from those of cutaneous vasoconstrictor fibres in the rat's tail (Owens et al. 2002). Most strikingly, cutaneous vasocontrictor fibres are strongly activated by falls in core temperature as well as (less strongly) by falls in skin temperature (Owens et al. 2002); by contrast, under matching experimental conditions, fusimotor neurons did not respond at all to falls in core temperature. Interestingly, non-shivering thermogenesis, as measured by oxygen consumption in anaesthetized rats, is also activated by skin cooling rather than core cooling (Osaka, 2004), although a minor component of activity attributable to core cooling may also be observed in recordings from the nerve to interscapular BAT (Tanaka & McAllen, 2005). The different activation patterns of cutaneous vasoconstrictor, BAT and fusimotor fibres strongly suggest that the medullary raphé neurons controlling these cold-defence responses are separate.
The results of this study provide evidence for a thermoregulatory reflex which activates fusimotor fibres in response to skin cooling. This response is not secondary to cortical ‘arousal’ and, like several other cold-defence responses, it depends upon neurons in the medullary raphé.
We speculate that this reflex acts to increase the gain of the stretch reflex during cold exposure. Before shivering occurs, this increased gain would cause an increase in muscle tone, by enhancing tonic activity in (for example) postural muscles (Burton & Bronk, 1937; von Euler, 1961; Meigal et al. 2003). It would also help sustain or amplify shivering, the magnitude of which depends heavily upon the stretch reflex (Perkins, 1945; Lippold et al. 1959).