Reflex changes in thermogenesis in the interscapular brown adipose tissue in response to thermal stimulation of the skin via sympathetic efferent nerves in anesthetized rats

Reflex responses of both temperature in the interscapular brown adipose tissue (IBAT) and activity of the sympathetic efferent nerves innervating the IBAT to cutaneous thermal stimulation were examined in anesthetized rats. Thermal stimulation of the ear at 4 degrees C for 20 s produced a transient decrease in the temperature of the IBAT during this period, followed by a prolonged increase (lasting 7 min). This response was completely abolished in animals whose IBAT sympathetic nerves were bilaterally sectioned. Direct recording of IBAT sympathetic efferent activity revealed that thermal stimulation of the ear at 4 degrees C for 20 s increased activity for 80 s after the onset of stimulation. The threshold temperature of the cold stimulation for eliciting increased nerve activity was around 22 degrees C. However, warm stimulation up to 49 degrees C had no significant effect on activity. In addition to stimulation of the ear, cold stimulation of the face and neck at 4 degrees C also increased nerve activity, while warm stimulation at 49 degrees C of any skin area had no influence. The present results demonstrate that excitation of cutaneous cold receptors, but not warm or heat receptors, reflexly increases thermogenesis in the IBAT via excitation of sympathetic nerve activity, and that cold receptor afferents to cranial and cervical segments effectively increase nerve activity in the IBAT.     

A neurophysiological evidence of capsaicin-sensitive nerve components innervating interscapular brown adipose tissue

Neurophysiological basis for the heterogeneity of the nerve components in the brown adipose tissue (BAT) was examined in this experiment. Efferent nerve signals were recorded from the central cut end of the small nerve filament dissected from the nerve fibers innervating the interscapular BAT (IBAT). By focusing on qualitative aspects of observed compound action potentials (spikes), we found two distinctive types of spikes exhibited by the intercostal nerves innervating IBAT. The spikes mainly appeared upon sympathetic stimulations (cold stimulation and glucose administration) were characterized by low amplitude with relatively short duration (small spike) and their sensitivity to the ganglion blocker, hexamethonium (C6). On the other hand, the spikes seen throughout the experiments were characterized by high amplitude with long duration (large spike) and their insensitivity to C6. Since BAT is activated by cold and feeding via sympathetic nervous system, the small spikes seemed to be exhibited by sympathetic fibers. On the other hand, appearance of the large C6-insensitive spikes was strongly attenuated in capsaicin-desensitized rats. Even though the functional link between IBAT and C6 insensitive fibers remains unanswered, our results suggest that IBAT is under control of various nerve types including capsaicin-sensitive fibers in addition to the control of sympathetic nervous system.     

Effect of galanin and enterostatin on sympathetic nerve activity to interscapular brown adipose tissue

The effects of galanin and enterostatin on sympathetic activity have been examined in rats using electrophysiological techniques. Galanin, in doses of 25–300 pmol, and enterostatin, in doses of 0.5–10 nmol, were injected into the third ventricle of anesthetized male Sprague-Dawley rats in 1-μl volumes. Galanin produced a dose-dependent suppression (ranging between 20 and 80%) of sympathetic firing rate of nerves innervating interscapular brown adipose tissue. In rats fed a chow diet, injection of enterostatin produced only a transient 10% rise in firing rate which returned to baseline within 10–15 min. In contrast, animals fed a high-fat diet showed a dose-dependent increase in firing rate lasting for 60 min. The results of this experiment are consistent with the hypothesis that food intake and sympathetic nervous system activity have a reciprocal relationship. The implications of this relationship are discussed.     

Apelin-13 microinjection into the paraventricular nucleus increased sympathetic nerve activity innervating brown adipose tissue in rats

The aim of present study is to clarify the role of apelin in regulating energy homeostasis in brown adipose tissue (BAT). We examined the central effects of apelin-13 on the brain c-fos like immunoreactivity (c-FLI), BAT temperature and the activity of the sympathetic nerve activity innervating BAT in rats. In the hypothalamus, central infusion into the third cerebral ventricle (i3vt) of apelin-13 caused induction of c-FLI in the paraventricular nucleus (PVN) compared with the controls (PBS-treated) group. In addition, microinjection of apelin-13 into the PVN produced significant increases in BAT temperature. Furthermore, microinjection of apelin-13 treatment increased BAT sympathetic nerve activity compared with controls. We conclude that apelin-13 microinjection into PVN increases sympathetic nerve activity innervating BAT.     

Hepatic portal injection of glucose elevates efferent sympathetic discharges of interscapular brown adipose tissue

Efferent sympathetic discharges of interscapular brown adipose tissue were recorded after three different concentrations of glucose (138, 277, and 416 mM) and 154 mM NaCl were injected into the portal vein or into the right jugular vein. When injected into the portal vein there was a significant increase in the discharge in response to both concentrations of glucose (277 and 416 mM), whereas only 416 mM glucose solution could cause an increase in the discharge when injected into the right jugular vein. There was no appreciable change in the discharge following the NaCl injections into the portal and jugular veins, and the portal glucose responses in the discharge were abolished by transection of the hepatic branch of the vagus nerve. Since stimulated sympathetic activity has been shown to elevate thermogenesis of the adipose tissue, these findings suggest that vagal glucose signals derived from the portal vein may be involved in the regulation of heat production of this tissue.     

Effects of cholecystokinin on sympathetic activity to interscapular brown adipose tissue.

The effects of injecting cholecystokinin (CCK) into the third ventricle or into selected hypothalamic sites on electrical firing rate of sympathetic nerves to interscapular brown fat (IBAT) has been investigated in anesthetized rats. The hypothesis for these experiments was that there was a reciprocal relationship between sympathetic activity and food intake. Since CCK reduces food intake we predicted that CCK would stimulate sympathetic activity to IBAT. Following the injection of CCK into the third ventricle there was an increase in firing rate of sympathetic nerves to IBAT. When the peptide was injected into either the ventromedial hypothalamic nucleus (VMH) or lateral hypothalamic area (LHA), there was likewise an increase in sympathetic firing rate. The injection of CCK into the paraventricular nucleus produced a small decrease in sympathetic firing rate. In contrast, no effect was seen following injection of CCK into the preoptic area or dorsomedial hypothalamic nucleus. Thus, the VMH or LHA appear to be the principal hypothalamic areas mediating the stimulation of sympathetic activity to IBAT which is observed following the third ventricular injection of CCK. These studies support the hypothesis of a reciprocal relationship between the effects of CCK on the thermogenic component of the sympathetic nervous system and food intake and identify the VMH and LHA as the primary sites for this effect.     

Studies on the sympathetic efferent nerves of brown adipose tissue of lean and obese Zucker rats

Previous studies have suggested that the sympathetic tone to brown adipose tissue (BAT) is reduced in the genetically obese (fa/fa) rat. The following experiments were designed to examine with electrophysiological techniques the activity of the sympathetic nerve innervating the interscapular BAT. The spontaneous activity of the efferent nerves was reduced in the obese (fa/fa) rat compared with the lean control. The activity of the nerve showed a linear relationship with changes in core temperature in both genotypes. Electrical stimulation of the ventromedial hypothalamus resulted in similar heat increments in BAT temperature for lean and obese, but this was associated with a smaller increase in nerve firing in the obese rat. Intracerebroventricular administration of glucose enhanced the nerve activity, whereas 2-deoxy-D-glucose reduced the nerve activity in both lean and obese rats. These data suggest that the sympathetic tone is suppressed in the genetically obese rat, but the response to temperature and central glucose metabolism is intact.     

Interaction of intracerebroventricular insulin and glucose in the regulation of the activity of sympathetic efferent nerves to brown adipose tissue in lean and obese Zucker rats

The effects of injection of insulin and glucose into the third cerebral ventricle on the firing rate of the sympathetic efferent nerves to interscapular brown adipose tissue was investigated in anaesthetized lean and obese Zucker rats. Injection of insulin resulted in a dose-dependent (70–480 pmol) inhibition of nerve firing rate, whereas in combination with glucose (140 pmol of insulin and 139 nmol of glucose), insulin strongly potentiated the increase in firing rate seen with glucose alone. Although basal levels of nerve firing rates were lower in the obese rat, responses to insulin, glucose, and insulin plus glucose were qualitatively similar to those seen in the lean rat. These results are consistent with the hypothesis that insulin acts in the central nervous system as a physiological signal in the control of thermogenesis after feeding, and that this effector system is intact in the obese rat.     

Inhibition of renal sympathetic nerve activity by electrical stimulation of the hypothalamic paraventricular nucleus in anesthetized rats

In an attempt to obtain direct evidence for involvement of sympathetic outflow in paraventricular nucleus (PVN)-induced depressor response, effects of electrical stimulation of the PVN on renal sympathetic nerve activity were examined in urethane-chloralose anesthetized rats. Low intensity stimulation of the PVN inhibited renal sympathetic nerve activity, whereas the high intensity stimulation evoked an excitation followed by long-lasting inhibition in renal sympathetic nerve activity. Latencies of the inhibitory and the excitatory responses were about 200 ms and 50-100 ms, respectively. The result suggests that a decrease in blood pressure induced by low intensity stimulation of the PVN is due to inhibiting the sympathetic outflow.     

Microneurography in anesthetized rats for the measurement of sympathetic nerve activity in the sciatic nerve

Microneurography is widely used for the measurement of human peripheral sympathetic nerve activity (SNA) in conscious subjects by virtue of its low invasive nature, but has rarely been employed in animal experiments. Because the low invasive nature sometimes is very useful even in animal experiments, we tested its feasibility for the measurement of SNA in the sciatic nerve of the anesthetized rat, aiming in particular to establish a methodology for measurement. A tungsten microelectrode was inserted into the nerve exposed at the thigh level to detect the "spontaneous, intermittent burst" signal that is one of the main characteristics of compound SNA. Such signals were found in more than 70% of experiments after surgical operators became accustomed to the method. Whenever such signals were detected, electrical stimulation of the sympathetic chain resulted in induced action potentials detected with the microelectrode after a reasonable conduction period. The spikes were successfully reduced after administration of hexamethonium bromide, a sympathetic ganglion blocker. On the other hand, induced spikes were never observed when we failed to find the "spontaneous, intermittent burst" signal. The results demonstrate the feasibility of microneurography in evaluating SNA in anesthetized small animals, and that the "spontaneous, intermittent burst" signal may be sufficient to identify SNA.     

Metabolic and morphological alterations of brown adipose tissue after sympathetic denervation in rats

Effects of sympathetic denervation on biochemical and morphological changes of brown adipose tissue (BAT) were studied in rats after severing 5 intercostal nerve bundles that enter the unilateral interscapular BAT, the contralateral BAT being used as the control. Four weeks after denervation, there was no appreciable change in BAT weight or its DNA and protein contents, whereas the triglyceride content was increased significantly. However, the rate of fatty acid synthesis was decreased to about half the rate in controls. The denervated BAT was much paler than controls and contained lots of adipocytes filled with large lipid droplets, some of which were unilocular. The results are discussed with reference to changes in BAT seen after bilateral lesions of the ventromedial hypothalamus.     

Long-range correlation of renal sympathetic nerve activity in both conscious and anesthetized rats

In this study we employed both detrended fluctuation analysis (DFA) and multiscale entropy (MSE) measurements to compare the long-range temporal correlation (LRTC) of multifibre renal sympathetic nerve activity (RSNA) between conscious and anesthetized Wistar rats. It was found that both methods showed the obvious LRTC properties in conscious state. Moreover, the scaling exponent of the RSNA in conscious rats was significantly higher than that in anesthetized rats. The results of MSE analysis showed that the entropy values, derived from the conscious group, increased on small time scales and then stabilized to a relatively constant value whereas the entropy measure, derived from anesthetized animals, almost monotonically decreased. This suggests that the fractal properties of underlying dynamics of the system have been reduced by anesthesia. The results demonstrate that apparently random fluctuations in multifibre RSNA are dictated by a complex deterministic process that imparts "long-term" memory to the dynamic system. However, this memory is significantly weakened by anesthesia.     

Effect of chemoreceptor stimulation on the periodicity of renal sympathetic nerve activity in anesthetized cats.

The effect of chemoreceptor stimulation, with asphyxia (1 min), hypoxia (2 min) or hypercapnia (2 min), on the periodicity of synchronized renal sympathetic nerve activity (RNA) was examined in anesthetized cats before and after peripheral chemoreceptor and baroreceptor denervation. RNA was filtered between 50-3000 Hz, rectified and integrated. Time intervals, less than 500 ms, between synchronized interburst intervals were measured and used to produce periodicity histograms. Under control normoxia two major periodicities were evident, a Tc rhythm between 6 and 17 c/s comprising 34 +/- 5% (+/- SE) of measured intervals and a Tb rhythm between 2 and 6 c/s with a 66% probability. The mean periods of Tc and Tb were 110 +/- 6 ms and 299 +/- 7 ms respectively. The periodicity distribution and mean Tc and Tb rhythms for RNA discharge under various chemoreceptor stimulations were not significantly changed despite significant increases in arterial blood pressure in all cases. The amplitude and overall number of synchronized RNA peaks were however increased with chemoreceptor stimulation. When asphyxia was applied under a constant arterial pressure the periodicity of synchronized RNA still was not significantly altered. Baroreceptor and peripheral chemoreceptor denervation led to an increase in the probability of the Tc mode and reduction in the Tb mode, once again the application of chemoreceptor stimulation did not significantly alter the frequency distribution of synchronized RNA. The results indicate that chemoreceptor stimulation does not affect the 10 c/s fundamental rhythm and the stability of gate operators altering Tc/Tb proportions, although it can alter the number of active fibres and interacts with the baroreflexes to maintain RNA at elevated blood pressures. The results support our model that the Tc mode reflects a fundamental periodicity of central origin and the Tb mode a periodicity of cardiac related RNA, which is produced by the opening and closing of gate operators to the fundamental rhythm.     

Thermogenesis of interscapular brown adipose tissue selectively influences pontine and preoptic-hypothalamic temperatures during sleep in the rat

The thermal influence of dorsal cervical and interscapular brown adipose tissue (ISBAT) on pontine and preoptic-hypothalamic temperatures was studied in unrestrained adult male rats across the non-rapid eye movement sleep (NREM) and rapid eye movement sleep (REM) sleep states. The animals had chronically implanted electrodes and thermistors for electroencephalographic and temperature recordings. After acclimation to either low (4 degrees C) or neutral (30 degrees C) ambient temperatures, the rats were studied before and after ISBAT excision. The warming influence of ISBAT thermogenesis activated by low ambient temperature reached the pontine and the preoptic-hypothalamic areas. ISBAT thermogenesis is necessary to maintain the homeothermy of the preoptic-hypothalamic area in NREM sleep at low ambient temperature. Such an ISBAT thermal effect may contribute to the integrated control of non-shivering and shivering thermogenesis.     

Well-maintained reflex responses of sympathetic nerve activity to stimulation of baroreceptor, chemoreceptor and cutaneous mechanoreceptors in neonatal capsaicin-treated rats

Neonatal treatment with capsaicin (CAP) reduced the content of substance P-like immunoreactivity in the area of the nucleus tractus solitarii and the dorsal horn of the spinal cord in rats. The resting values of the mean arterial blood pressure and heart rate of CAP-treated rats were not significantly different from those of control rats. Reflex responses of the renal sympathetic nerve activity to stimulation of baroreceptor, chemoreceptor and cutaneous mechanoreceptor were compared in CAP- and vehicle-treated rats. Under these conditions the reflex responses were quite well maintained in CAP-treated rats.     

Afferent activity in fine myelinated and unmyelinated nerve fibers of a cutaneous nerve upon mechanical stimulation of cutaneous receptors]

The activity in fine myelinated and nonmyelinated nerve fibres of a cat cutaneous nerve was analyzed using a collision method and method improving the signal-to-noise ratio in the antidromic action potential neurogram. On the basis of the investigation of conduction velocities, electrical excitation thresholds and responses to mechanical stimulations, the following groupsare distinguished among fine myelinated and nonmyelinated fibres: group A delta1 (30-14m/s) activated mostly under light mechanical stimulation; group A delta2 (14.4.0 m/s) more readily activated by intense mechanical stimulation; group of "mixed" fibres containing myelinated and nonmyelinated nerve fibres (4-2 m/s) activated under both kinds of mechanical stimulation; group C1 (2.0-1.0 M/S) ACTIVATED MOSTLY BY LIGHT Mechanical stimulation and group C2 (1.0-0.15 M/S) CONNECTED WITh receptors that require for excitation intense mechanical stimulation.     

Tumour necrosis factor-alpha and lymphotoxin have opposite effects on sympathetic efferent nerves to brown adipose tissue by direct action in the central nervous system

Intracerebral injection of recombinant human tumour necrosis factor-alpha (rTNF alpha) decreased, whereas lymphotoxin increased the activity of the sympathetic efferent nerves to interscapular brown adipose tissue (IBAT). The changes in firing rates were accompanied by a significant dose-dependent change in IBAT and rectal temperatures (which were decreased with rTNF alpha and increased with lymphotoxin treatment). These results indicate that rTNF alpha and lymphotoxin act directly in the CNS to modulate the sympathetic outflow to IBAT. The opposite effects of the two peptides also suggest that in the CNS, separate receptor systems exist.     

Localization of efferent sympathetic neurons and afferent neurons in dorsal root ganglia innervating the heart

The retrograde transport of horseradish peroxidase (HRP) has been used to study the localization and the number of neurons innervating the heart in the right stellate ganglion and accessory cervical ganglion, spinal cord and dorsal root ganglia of the cat. HRP was applied to the central cuts of anastomose of the stellate ganglion with the vagal nerve, of the vagosympathetic trunk caudal to anastomose and of the inferior cardiac nerve. HRP-labelled neurons were detected in the stellate ganglion in the regions which give off nerves, whereas in the accessory cervical ganglion labelled neurons were distributed throughout the whole ganglion. HRP-stained cells were found in the anastomose. In the spinal cord labelled neurons were detected in the lateral horn of T1-T5 segments. In the dorsal root ganglion the greatest number of neurons was observed in T2-T4 segments.