- Email: [email protected]
Procaine injection into the paraventricular nucleus reduces sympathetic and thermogenic activation induced by frontal cortex stimulation in the rat
Brain Research Bulletin, Vol. 47, No. 6, pp. 657– 662, 1998 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/99/$–see front matter
PII S0361-9230(98)00138-5
Procaine injection into the paraventricular nucleus reduces sympathetic and thermogenic activation induced by frontal cortex stimulation in the rat M. Monda,* A. Sullo, V. De Luca and A. Viggiano Department of Human Physiology and Integrated Biological Functions, “Filippo Bottazzi,” Second University of Naples, Naples, Italy [Received 23 June 1998; Accepted 21 August 1998] ABSTRACT: These experiments were designed to test the effect of procaine injection into the paraventricular nucleus on the sympathetic and thermogenic changes induced by frontal cortex stimulation. Oxygen consumption, firing rate of the sympathetic nerves to interscapular brown adipose tissue (IBAT), along with IBAT and colonic temperatures (TIBAT and TC) were monitored in fasted male Sprague-Dawley rats before and 25 min after an electrical stimulation of the frontal cortex. The same variables were monitored in rats with administration of procaine into the paraventricular nucleus. The results show that cortical stimulation raises oxygen consumption, sympathetic neuron firing rates, TIBAT, and TC. This increase is reduced by procaine injection. These findings suggest that the paraventricular nucleus plays a key role in the sympathetic and thermogenic changes induced by cortical stimulation. © 1999 Elsevier Science Inc.
[1], associated with increased electrical activity in the sympathetic fibers innervating this tissue [31]. In contrast, the firing rate of nerve filaments terminating at IBAT was reduced after acute lesions of the rat PVN, indicative of reduced PVN-mediated sympathetic activity [27]. These studies indicate a possible role of the PVN in the neural control of thermogenesis. Other cerebral areas are involved in the control of body temperature, including the cerebral cortex. Electrical stimulation of the frontal cortex activates warm-sensitive units and suppresses coldsensitive units in the anterior hypothalamic area [13]. Excitatory neurotransmitters increase in the frontal cortex during prostaglandin E1-induced hyperthermia [24]. The aim of the present experiment was: (a) to evaluate the effect of procaine injection into the paraventricular nucleus on the sympathetic and thermogenic changes induced by the cortical stimulation and (b) to test the hypothesis that the PVN is involved in the neural pathway connecting the frontal cortex to the sympathetic nerves of IBAT.
KEY WORDS: Body temperature, Cerebral cortex, Paraventricular nucleus, Sympathetic nervous system.
MATERIALS AND METHODS
INTRODUCTION Animals
The hypothalamus is a key structure in thermoregulation. The anterior hypothalamus regulates responses related to body temperature changes [10,28], probably by controlling the posterior hypothalamus, which, in turn, modulates heat production related to shivering and nonshivering thermogenesis [22]. Also the hypothalamic paraventricular nucleus (PVN) plays a role in the control of body temperature. The variety of physiological variables influenced by the PVN has revealed this cell population as functionally complex. Several studies have shown that neurones of the PVN project to a number of areas causally involved in the regulation of autonomic functions [30], and stimulation of the PVN activates neuronal outflow to a number of brain stem nuclei [18,26] and peripheral organs [31]. In rodents, a major tissue for heat production related to nonshivering thermogenesis is interscapular brown adipose tissue (IBAT) [9]. IBAT is known to be under the peripheral neurogenic control of the sympathetic nervous system [16]. Stimulation of the PVN by local glutamate microinjections results in an increase in IBAT temperature of urethane-anesthetized rats
We used male inbred Sprague-Dawley rats (n 5 24), 3 months old and weighing 280 –320 g. These were housed in pairs at controlled temperature (22° 6 1°C) and humidity (70%) with a 12 h light:12 h dark cycle from 0700 to 1900 h. The experiments conformed to the European Convention for the Protection of Vertebrate Animals Used for Experimental and Scientific Purpose (Council of Europe No. 123, Strasbourg 1985). Apparatus The firing rate of nerves to IBAT was recorded by a pair of silver wire electrodes. The electrical pulses were amplified by a condenser-coupled amplifier and were filtered by band-pass filters (NeuroLog System, Digitimer, UK). The raw pulses were displayed on a oscilloscope (Tektronix, USA) and sent to a window discriminator. Square waves from the discriminator were sent to an analog-digital converter (DAS system, Keithley, USA) and stored
* Address for correspondence: Dr. Marcellino Monda, Dipartimento di Fisiologia Umana, Via Costantinopoli 16, 80138 Napoli, Italy, Fax 139-815665820.
657
658
MONDA ET AL.
FIG. 1. Means 6 standard error of changes in sympathetic firing rate of stimulated or sham-stimulated rats with injection of saline or procaine. Cerebral injection at time 0. Stimulation of cerebral cortex was made 2 min after cerebral injection.
on a computer (Personal Computer AT, IBM, USA) every 5 s. Furthermore, a rate meter with a reset time of 5 s was used to observe the time course of the nerve activity recorded by pen recorder (Dynograph, Beckman, USA). Because signal-to-noise ratio depended on the number of nerve filaments and the condition of contact between nerve and electrodes, the basal burst rates were different for each rat. The threshold level of the event detector was fixed during the experiment at 50% of the peaks of the largest pulses and above background noise. Thermocouples (Ellab) were used to monitor colonic and IBAT temperatures (Tc and TIBAT) and the values were stored on a chart recorder. Resting oxygen (O2) consumption was determined with an indirect calorimeter. The closed circuit apparatus was an adaptation of Benedict and MacLeod’s calorimeter. Air was continuously circulated through a drying column (CaSO4 Drierite), a respiratory chamber 2.5 l and CO2 trap (soda lime), by a peristaltic pump at a rate of 2 l z min21. A 1-l cylindrical metal bell, fitted in a concentric cylinder filled with water forming an air-tight seal, served as the O2 reservoir. The O2 percentage was 21% in the respiratory chamber and in the reservoir. The 5-ml graduated cylinder was connected to the respiratory chamber. Respiratory chamber temperature was maintained constant at 29°C by circulating water, and was monitored by an internal thermometer. The volume of O2 consumed by each animal was corrected to standard temperature and pressure and was expressed as ml z min-1 z kg b. wt.2.75 [14]. Drug Procaine hydrochloride was purchased from Sigma Chemical Co. (St. Louis, MO, USA).
FIG. 2. Actual sympathetic firing rate changes in a stimulated rat receiving saline (panel A) or procaine (panel B) and in a sham-stimulated rat receiving saline (panel C) or procaine (panel D). Cerebral injection at time 0. Stimulation of cerebral cortex was made 2 min after cerebral injection.
Surgery All animals were anesthetized with intraperitoneal (i.p.) pentobarbital (50 mg z kg b. wt.21) and two 20-gauge stainless guide cannulae were positioned stereotaxically [25] 0.1 mm above the PVN at the following coordinates: 0.5 mm lateral to midline, 0.6 mm anterior to the bregma, 6.8 mm from the surface. Furthermore, a twisted stainless steel wire (125 mm in diameter), coated with Teflon and soldered to a tow-pin connector, was inserted stereotaxically at following coordinates: 3.7 mm lateral to midline, 4.5 mm anterior to the bregma, 3.5 mm from the surface. Guide
PVN AND CORTICAL STIMULATION
659
FIG. 3. Means 6 standard error. of changes in interscapular brown adipose tissue (IBAT) temperature of stimulated or sham-stimulated rats with injection of saline or procaine. Cerebral injection at time 0. Stimulation of cerebral cortex was made 2 min after cerebral injection.
FIG. 4. Means 6 standard error of changes in colonic temperature of stimulated or sham-stimulated rats with injection of saline or procaine. Cerebral injection at time 0. Stimulation of cerebral cortex was made 2 min after cerebral injection.
cannulae and a stimulating electrode were secured to the skull by screws and dental cement. Stylets were inserted into the guide tubes and removed only during drug administration. Rats were given 7–10 days to recover from surgery, as judged by recovery of preoperative body weight.
animals (second group), but saline was substituted for procaine. The same procedure used with the first group was carried out with six other animals (third group), but cortical stimulation was not performed. Saline was injected into the PVN in six rats (fourth group) without cortical stimulation and the same variables were monitored. The baseline values of TC of all animals used were maintained constant by a heating pad. The electrical energy supplied to pad was not altered during the experimental period.
Procedure After recovery, six animals (first group) were anesthetized with urethane (1.2 g z kg b. wt.21) and mounted in a stereotaxic instrument (Stoelting, USA). The level of anesthesia was kept constant as evaluated by skeletal muscle relaxation, eye and palpebral responses to stimuli [29]. Nerve activity was recorded by small nerve bundles dissected from the nerve branches innervating the right side of IBAT. Nerve filaments were isolated from the central cut end of these nerve bundles under a dissecting microscope to record the efferent activity with a pair of silver wire electrodes. The nerve filaments were covered with a mixture of Vaseline and liquid petroleum at 37°C to avoid dehydration. The firing rate was recorded over 25 min (baseline period) before and 25 min after injection of drug. Procaine hydrochloride (0.5 ml, 20% wt. vol.21) were injected bilaterally in the PVN, over a 40-s period. Two minutes after procaine injection, sine wave stimuli were delivered by an autotransformer, and the current was monitored with an oscilloscope indicating the voltage drop across a known resistor placed in series with the animal. Parameters of stimulation were: sine wave current (50 Hz, 150 mA for 15 s). Furthermore, TC, TIBAT, and O2 consumption were monitored at the same time as the nerve activity record. TC was measured by inserting the thermocouple into the colon at 7 cm from the anus, while TIBAT was monitored by inserting the thermocouple in the left side of IBAT. The same variables were recorded in six other
HISTOLOGY At the end of the experiment, the location of the cannulae was identified. A stain (Bromophenol Blue) was injected in the PVN at the same volume (0.5 ml) used for drug administration. The rats were then injected with an overdose of pentobarbital (200 mg z kg b. wt.21). They were perfused with 0.9% NaCl followed by phosphate-buffered formalin containing 5% (wt. vol.21) potassium forrocyanide to reveal the site of electrical stimulation. The brain was removed and stored in formalin solution. After a few days, 50 mm coronal sections of the fixed brain were cut and stained with cresyl violet. Statistical Analysis The values are presented as means 6 standard error (SE). Statistical analysis was performed using analysis of variance (ANOVA). Multiple comparisons were performed by NewmanKeuls’ post hoc test. RESULTS Figure 1 shows the percentage changes in firing rate of nerves to IBAT. The cortical stimulation increased the firing rate, which
660
MONDA ET AL.
FIG. 5. Means 6 standard error of changes in oxygen consumption of stimulated or sham-stimulated rats with injection of saline or procaine. Cerebral injection at time 0. Stimulation of cerebral cortex was made 2 min after cerebral injection.
peaked at 5 min in the rats with saline injection. This rise was reduced in the animals with procaine administration. Neither saline nor procaine injection produced any modification in the rats without cortical stimulation. ANOVA showed significant effects for cortical stimulation [F(1,20) 5 22.324, p , 0.01], for drug [F(1,20) 5 11.864, p , 0.01], for time [F(5,100) 9.648, p , 0.01], for interaction cortical stimulation 3 drug [F(1,20) 5 7.703, p , 0.05], cortical stimulation 3 time [F(5,100) 5 8.853, p , 0.01], and drug 3 time [F(5,100) 5 2.660, p , 0.05]. The post hoc test showed that saline 1 stimulation group was different from other groups by 5–20 min. Differences were demonstrated between procaine 1 stimulation group and other groups at 5 min. Examples of changes in actual firing rate of the nerves to IBAT are shown in Fig. 2. Figure 3 illustrates TIBAT changes. Cortical stimulation caused
a rise that peaked at 5 min in the rats injected with saline. This increase was reduced in the animals injected with procaine. In the sham-stimulated rats, neither saline nor procaine caused any modification. ANOVA showed significant effects for cortical stimulation [F(1,20) 5 11.439, p , 0.01], for time [F(5,100) 5 4.224, p , 0.01], for interaction cortical stimulation 3 drug [F(1,20) 5 5.683, p , 0.05], and cortical stimulation 3 time [F(5,100) 5 2.726, p , 0.05]. The post hoc test showed that saline 1 stimulation group was different from other groups by 5–25 min. Differences were demonstrated between procaine 1 stimulation group and other groups at 5 min. Figure 4 illustrates TC changes. Cortical stimulation induced a rise with a peak at 10 min in the rats injected with saline. Procaine reduced the increase due to cortical stimulation. No changes resulted in the sham-stimulated animals with saline or procaine administration. ANOVA showed significant effects for cortical stimulation [F(1,20) 5 9.198, p , 0.01], for time [F(5,100) 5 4.206, p , 0.01], for interaction cortical stimulation 3 time [F(5,100) 5 3.206, p , 0.01]. The post hoc test showed that saline 1 stimulation group was different from other groups between 5 and 15 min. Differences were demonstrated between procaine 1 stimulation group and other groups at 10 min. Figure 5 shows changes in O2 consumption. Cortical stimulation induced an increase with a peak at 5 min in the animal injected with saline. Procaine reduced this rise in the stimulated animals. Neither saline nor procaine administration caused any change in the rats without cortical stimulation. ANOVA showed significant effects for cortical stimulation [F(1,20) 5 35.046, p , 0.01], for drug [F(1,20) 5 21.111, p , 0.01], for time [F(5,100) 5 21.620, p , 0.01], for interaction cortical stimulation 3 drug [F(1,20) 5 24.483, p , 0.01], cortical stimulation 3 time [F(5,100) 5 19.784, p , 0.01], drug 3 time [F(5,100) 5 10.420, p , 0.01], and cortical stimulation 3 drug 3 time [F(5,100) 5 10.519, p , 0.01]. The post hoc test showed that saline 1 stimulation group was different from other groups between 5 and 25 min. Differences were demonstrated between procaine 1 stimulation group and other groups at 5 min. There were no differences in four baseline variables of all groups, as reported in the Table 1. DISCUSSION The changes in physiological functions after procaine injection into the medial hypothalamus have also been studied by other authors. They showed that procaine modifies feeding behavior and the sympathetic activity [2,7]. The present findings are the first to indicate that procaine injection in the PVN reduces heat production due to the stimulation of the prefrontal cortex. This suggests that the PVN is an important station in the
TABLE 1 ABSOLUTE VALUES 6 STANDARD ERROR OF FIRING RATE, INTERSCAPULAR BROWN ADIPOSE TISSUE (TIBAT) AND COLONIC TEMPERATURES (TC), AND OXYGEN (O2) CONSUMPTION AT THE TIME OF INJECTION INTO THE PARAVENTRICULAR NUCLEUS
Stimulation 1 saline Stimulation 1 procaine Sham stimulation 1 saline Sham stimulation 1 procaine
Firing Rate (Spikes 5 s21)
TIBAT(°C)
TC (°C)
O2 Consumption (ml z min21 z kg b.wt.20.75)
37.25 6 5.71 39.28 6 5.12 40.62 6 5.59 38.91 6 6.58
37.29 6 0.08 37.35 6 0.11 37.41 6 0.13 37.34 6 0.07
36.79 6 0.09 36.85 6 0.10 36.9 6 10.12 36.76 6 0.09
10.98 6 1.66 11.21 6 1.99 11.65 6 1.72 11.39 6 1.54
PVN AND CORTICAL STIMULATION neural network connecting the prefrontal cortex to structures driving the responses related to nonshivering thermogenesis. Anesthesia with urethane maintains the rat’s ability to develop fever [19], and removes the possible interference due to motor activity. With the evidence from this experiment, we cannot draw any conclusion about heat loss, but we can say that procaine injection in the PVN influences the thermogenic events evoked by the cortical stimulation. These findings show that procaine in the PVN reduces the expected rise in “core” body temperature through reduced neuronal activation of BAT as a means of reducing O2 consumption. Depression of the PVN acts on the thermogenic response by reducing the activity of the sympathetic nervous system. This effect on the sympathetic activity has also been shown with other experimental models. Lesions of the PVN cause a marked decrease in heat production related to postprandial thermogenesis, and this phenomenon is mediated by the adrenergic system. Indeed, PVN lesion blocks sympathetic activation induced by food intake in rat fed with “cafeteria” diet [4]. In the present experiment, procaine does not modify body temperature and metabolic rate under basal conditions, the effect occurs only after the cortical stimulation. This suggests a phasic influence of the PVN in rising thermogenesis due to the cortical stimulation, and seems to exclude a tonic activity on thermogenic mechanisms. The increase in O2 consumption confirms that the cerebral cortex is involved in the control of metabolic rate, in accord with other evidence. Indeed, cortical spreading depression reduces the metabolic rate induced by prostaglandin E1, and this effect depends on the pyrogen dose [15]. Furthermore, bilateral lesions of hypothalamic nuclei cause a marked increase in metabolic rate with hyperthermia for many hours [11], and this phenomenon is mediated by the adrenergic system [3]. It has been shown that, like b-blockers, cortical spreading depression impairs the O2 consumption gain following electrolytic damage of the lateral hypothalamus in rats [6]. The present experiment emphasizes that the cerebral cortex needs the PVN to influence metabolic rate. Other experiments demonstrated that a septal lesion blocks O2 consumption gain due to cortical stimulation [5,21], indicating that septal nuclei take part in the pathway controlling metabolic rate. The present experiment suggests a need to include the PVN in this pathway. The present data indicate that the PVN controls the discharge of sympathetic nerves to IBAT. IBAT is the organ responsible for evoking 25– 65% of the total increase in metabolic heat production during various experimental manipulations in rodents [12,23]. IBAT is supplied by a mixed nerve, which provides five separate branches to the individual lobes [8]. IBAT activity is controlled by the sympathetic nervous system [17], and factors that influence thermogenesis appear to act centrally to modify the sympathetic outflow to IBAT [20]. The significant role of IBAT in the thermogenic changes due to the cortical stimulation is confirmed by these findings. In conclusion, we can affirm that the PVN plays a key role in the sympathetic and thermogenic changes induced by stimulation of the prefrontal cortex, although further investigations are needed to evaluate the role of other cerebral areas involved in these phenomena. ACKNOWLEDGEMENT
This study has been supported by grants from the Italian National Research Council and Regione Campania.
661 REFERENCES 1. Amir, S. Stimulation of the paraventricular nucleus with glutamate activates interscapular brown adipose tissue thermogenesis in rats. Brain Res. 508:152–155; 1990. 2. Berthous, M., Jeanrenaud, B. Changes of insulinemia, glycemia and feeding behavior induced by VMH-procainization in the rat. Brain Res. 174:184 –187; 1979. 3. Corbett, S. W.; Kaufman, L. M.; Keesey, E. Effects of b-adrenergic blockade on lateral hypothalamic lesion-induced thermogenesis. Am. J. Physiol. 245:E535–E541; 1983. 4. De Luca, B.; Monda, M.; Amaro, S.; Pellicano, M. P.; Cioffi, L. A Lack of diet-induced thermogenesis following lesions of paraventricular nucleus in rats. Physiol. Behav. 4:685– 691; 1989. 5. De Luca, B.; Monda, M.; Amaro, S.; Pellicano, M. P.; Cioffi, L. A. Thermogenic changes following frontal neocortex stimulation. Brain Res. Bull. 22:1003–1007; 1989. 6. De Luca, B.; Monda, M.; Pellicano, M. P.; Zenga, A. Cortical control of thermogenesis induced by lateral hypothalamic lesion and overeating. Am. J. Physiol. 253:R626 –R633; 1987. 7. Epstein, A. N. Reciprocal changes in feeding behavior produced by intrahypothalamic chemical injection. Am. J. Physiol., 199:969 –974; 1960. 8. Foster, D. O.; Depocas, F.; Zaror-Behrens, G. Unilaterality of the sympathetic innervation of each pad of rat interscapular brown adipose tissue. Can. J. Physiol. Pharmacol. 60:107–113; 1982. 9. Fyda, D. F.; Cooper, K. E.; Veale, W. L. Contribution of brown adipose tissue to central PGE1-evoked hyperthermia in rats. Am. J. Physiol. 260:R59 –R66; 1991. 10. Gollman, H. M.; Rudy, T. A. Comparative pyrogenic potency of endogenous prostanoids and of prostanoid-mimetics injected into the anterior/preoptic region of the cat. Brain Res. 449: 281293; 1988. 11. Grijalva, C.V. Aphagia, gastric pathology, hyperthermia and sensomotor dysfunction following lateral hypothalamic lesions: Effects of insulin pretreatment. Physiol. Behav. 25:931–937; 1980. 12. Himms-Hagen, J. Non-shivering thermogenesis. Brain. Res. Bull. 12: 151–160; 1984. 13. Hori, T.; Shibata, M.; Kyohara, T.; Nakashima, T. Prefrontal cortical influences on behavioural thermoregulation and thermosensitive neurons. J. Therm. Biol. 9:27–31; 1984. 14. Kleiber, M. The fire of life. An introduction to animal energetics. Huntington: Kreiger Publishing Company; 1975. 15. Komaromi, I.; Malkinson, T. J.; Veale, W. L.; Rosenbaum, G.; Cooper, K. E.; Pittman, Q. J. Effect of potassium-induced cortical spreading depression on prostaglandin-induced fever in conscious and urethane-anesthetized rats. Can. J. Physiol. Pharmacol. 72:716 –721, 1994. 16. Landsberg, L.; Saville, M. E.; Young, J. B. Sympathoadrenal system and regulation of thermogenesis. Am. J. Physiol. 247:E181–E189; 1984. 17. Landsberg, L.; Young, J. B. Autonomic regulation of thermogenesis. In: Girardier, L.; Stock, M. J. eds. Mammalian thermogenesis. London: Chapman and Hall; 1983:99 –140. 18. Lawrence, D.; Pittman. Q. J. Interaction between descending paraventricular neurons and vagal motor neurons. Brain Res. 332:158 –160; 1985. 19. Malkinson, T. J.; Cooper, K. E.; Veale, W. L. Physiological changes during thermoregulation and fever in urethan-anaesthetized rats, Am. J. Physiol. 255:R73–R81; 1988. 20. Monda, M.; Amaro, S.; De Luca, B. Non-shivering thermogenesis during prostaglandin E1 fever in rats: role of cerebral cortex. Brain. Res. 651:148 –154; 1994. 21. Monda, M.; Amaro, S.; De Luca, B. Septal lesions block sympathetic activation following frontal cortex stimulation in the rat. Acta Physiol. Scand. 155:275–282; 1995. 22. Monda, M.; Amaro, S.; Sullo, A.; De Luca, B. Posterior hypothalamic activity and cortical control during the PGE1-hyperthermia. Neuroreport 6: 135–139; 1994. 23. Monda, M.; Sullo, A.; De Luca, E.; Pellicano, M. P. Lysine acetylsalycilate modifies aphagia and thermogenic changes induced by lateral hypothalamic lesion. Am. J. Physiol. 271:R1638 –R1642; 1996. 24. Monda, M.; Viggiano, A.; Sullo, A.; De Luca, V. Aspartic and glu-
662 tamic acids increase in the frontal cortex during pge1 hyperthermia. Neuroscience 83:1239 –1243; 1998. 25. Pellegrino, L. J.; Pellegrino, A. S.; Cushman, A. J. A stereotaxic atlas of the rat brain. New York: Plenum Press; 1979. 26. Pittman, Q. J.; Franklin, L. G. Vasopressin antagonist in nucleus tractus solitarius/vagal area reduces pressor and tachycardia responses to paraventricular nucleus stimulation in rats. Neurosci. Lett. 56:155– 160; 1985. 27. Sakaguchi, T.; Bray, G.A.; Eddlestone, G. Sympathetic activity following paraventricular or ventromedial hypothalamic lesions in rats. Brain Res. Bull. 20:461– 465; 1988.
MONDA ET AL. 28. Simpson, C. W.; Ruwe, W. D.; Myers, R. D. Prostaglandins and hypothalamic neurotransmitter receptors involved in hyperthermia: a critical evaluation. Neurosci. Biobehav. Rev.18:1–20; 1994. 29. Soma, L. R. Textbook of veterinary surgery, depth of general anesthesia. Baltimore, MD: Williams and Wilkins; 1971:178 –187. 30. Swanson, L. W.; Sawchenko, P. E. Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms. Neuroendocrinology 31:410 – 417; 1980. 31. Yoshimatsu, H.; Egawa, M.; Bray, G.A. Sympathetic nerve activity after discrete hypothalamic injections of L-glutamate. Brain Res. 601: 121–128; 1993.
PII S0361-9230(98)00138-5
Procaine injection into the paraventricular nucleus reduces sympathetic and thermogenic activation induced by frontal cortex stimulation in the rat M. Monda,* A. Sullo, V. De Luca and A. Viggiano Department of Human Physiology and Integrated Biological Functions, “Filippo Bottazzi,” Second University of Naples, Naples, Italy [Received 23 June 1998; Accepted 21 August 1998] ABSTRACT: These experiments were designed to test the effect of procaine injection into the paraventricular nucleus on the sympathetic and thermogenic changes induced by frontal cortex stimulation. Oxygen consumption, firing rate of the sympathetic nerves to interscapular brown adipose tissue (IBAT), along with IBAT and colonic temperatures (TIBAT and TC) were monitored in fasted male Sprague-Dawley rats before and 25 min after an electrical stimulation of the frontal cortex. The same variables were monitored in rats with administration of procaine into the paraventricular nucleus. The results show that cortical stimulation raises oxygen consumption, sympathetic neuron firing rates, TIBAT, and TC. This increase is reduced by procaine injection. These findings suggest that the paraventricular nucleus plays a key role in the sympathetic and thermogenic changes induced by cortical stimulation. © 1999 Elsevier Science Inc.
[1], associated with increased electrical activity in the sympathetic fibers innervating this tissue [31]. In contrast, the firing rate of nerve filaments terminating at IBAT was reduced after acute lesions of the rat PVN, indicative of reduced PVN-mediated sympathetic activity [27]. These studies indicate a possible role of the PVN in the neural control of thermogenesis. Other cerebral areas are involved in the control of body temperature, including the cerebral cortex. Electrical stimulation of the frontal cortex activates warm-sensitive units and suppresses coldsensitive units in the anterior hypothalamic area [13]. Excitatory neurotransmitters increase in the frontal cortex during prostaglandin E1-induced hyperthermia [24]. The aim of the present experiment was: (a) to evaluate the effect of procaine injection into the paraventricular nucleus on the sympathetic and thermogenic changes induced by the cortical stimulation and (b) to test the hypothesis that the PVN is involved in the neural pathway connecting the frontal cortex to the sympathetic nerves of IBAT.
KEY WORDS: Body temperature, Cerebral cortex, Paraventricular nucleus, Sympathetic nervous system.
MATERIALS AND METHODS
INTRODUCTION Animals
The hypothalamus is a key structure in thermoregulation. The anterior hypothalamus regulates responses related to body temperature changes [10,28], probably by controlling the posterior hypothalamus, which, in turn, modulates heat production related to shivering and nonshivering thermogenesis [22]. Also the hypothalamic paraventricular nucleus (PVN) plays a role in the control of body temperature. The variety of physiological variables influenced by the PVN has revealed this cell population as functionally complex. Several studies have shown that neurones of the PVN project to a number of areas causally involved in the regulation of autonomic functions [30], and stimulation of the PVN activates neuronal outflow to a number of brain stem nuclei [18,26] and peripheral organs [31]. In rodents, a major tissue for heat production related to nonshivering thermogenesis is interscapular brown adipose tissue (IBAT) [9]. IBAT is known to be under the peripheral neurogenic control of the sympathetic nervous system [16]. Stimulation of the PVN by local glutamate microinjections results in an increase in IBAT temperature of urethane-anesthetized rats
We used male inbred Sprague-Dawley rats (n 5 24), 3 months old and weighing 280 –320 g. These were housed in pairs at controlled temperature (22° 6 1°C) and humidity (70%) with a 12 h light:12 h dark cycle from 0700 to 1900 h. The experiments conformed to the European Convention for the Protection of Vertebrate Animals Used for Experimental and Scientific Purpose (Council of Europe No. 123, Strasbourg 1985). Apparatus The firing rate of nerves to IBAT was recorded by a pair of silver wire electrodes. The electrical pulses were amplified by a condenser-coupled amplifier and were filtered by band-pass filters (NeuroLog System, Digitimer, UK). The raw pulses were displayed on a oscilloscope (Tektronix, USA) and sent to a window discriminator. Square waves from the discriminator were sent to an analog-digital converter (DAS system, Keithley, USA) and stored
* Address for correspondence: Dr. Marcellino Monda, Dipartimento di Fisiologia Umana, Via Costantinopoli 16, 80138 Napoli, Italy, Fax 139-815665820.
657
658
MONDA ET AL.
FIG. 1. Means 6 standard error of changes in sympathetic firing rate of stimulated or sham-stimulated rats with injection of saline or procaine. Cerebral injection at time 0. Stimulation of cerebral cortex was made 2 min after cerebral injection.
on a computer (Personal Computer AT, IBM, USA) every 5 s. Furthermore, a rate meter with a reset time of 5 s was used to observe the time course of the nerve activity recorded by pen recorder (Dynograph, Beckman, USA). Because signal-to-noise ratio depended on the number of nerve filaments and the condition of contact between nerve and electrodes, the basal burst rates were different for each rat. The threshold level of the event detector was fixed during the experiment at 50% of the peaks of the largest pulses and above background noise. Thermocouples (Ellab) were used to monitor colonic and IBAT temperatures (Tc and TIBAT) and the values were stored on a chart recorder. Resting oxygen (O2) consumption was determined with an indirect calorimeter. The closed circuit apparatus was an adaptation of Benedict and MacLeod’s calorimeter. Air was continuously circulated through a drying column (CaSO4 Drierite), a respiratory chamber 2.5 l and CO2 trap (soda lime), by a peristaltic pump at a rate of 2 l z min21. A 1-l cylindrical metal bell, fitted in a concentric cylinder filled with water forming an air-tight seal, served as the O2 reservoir. The O2 percentage was 21% in the respiratory chamber and in the reservoir. The 5-ml graduated cylinder was connected to the respiratory chamber. Respiratory chamber temperature was maintained constant at 29°C by circulating water, and was monitored by an internal thermometer. The volume of O2 consumed by each animal was corrected to standard temperature and pressure and was expressed as ml z min-1 z kg b. wt.2.75 [14]. Drug Procaine hydrochloride was purchased from Sigma Chemical Co. (St. Louis, MO, USA).
FIG. 2. Actual sympathetic firing rate changes in a stimulated rat receiving saline (panel A) or procaine (panel B) and in a sham-stimulated rat receiving saline (panel C) or procaine (panel D). Cerebral injection at time 0. Stimulation of cerebral cortex was made 2 min after cerebral injection.
Surgery All animals were anesthetized with intraperitoneal (i.p.) pentobarbital (50 mg z kg b. wt.21) and two 20-gauge stainless guide cannulae were positioned stereotaxically [25] 0.1 mm above the PVN at the following coordinates: 0.5 mm lateral to midline, 0.6 mm anterior to the bregma, 6.8 mm from the surface. Furthermore, a twisted stainless steel wire (125 mm in diameter), coated with Teflon and soldered to a tow-pin connector, was inserted stereotaxically at following coordinates: 3.7 mm lateral to midline, 4.5 mm anterior to the bregma, 3.5 mm from the surface. Guide
PVN AND CORTICAL STIMULATION
659
FIG. 3. Means 6 standard error. of changes in interscapular brown adipose tissue (IBAT) temperature of stimulated or sham-stimulated rats with injection of saline or procaine. Cerebral injection at time 0. Stimulation of cerebral cortex was made 2 min after cerebral injection.
FIG. 4. Means 6 standard error of changes in colonic temperature of stimulated or sham-stimulated rats with injection of saline or procaine. Cerebral injection at time 0. Stimulation of cerebral cortex was made 2 min after cerebral injection.
cannulae and a stimulating electrode were secured to the skull by screws and dental cement. Stylets were inserted into the guide tubes and removed only during drug administration. Rats were given 7–10 days to recover from surgery, as judged by recovery of preoperative body weight.
animals (second group), but saline was substituted for procaine. The same procedure used with the first group was carried out with six other animals (third group), but cortical stimulation was not performed. Saline was injected into the PVN in six rats (fourth group) without cortical stimulation and the same variables were monitored. The baseline values of TC of all animals used were maintained constant by a heating pad. The electrical energy supplied to pad was not altered during the experimental period.
Procedure After recovery, six animals (first group) were anesthetized with urethane (1.2 g z kg b. wt.21) and mounted in a stereotaxic instrument (Stoelting, USA). The level of anesthesia was kept constant as evaluated by skeletal muscle relaxation, eye and palpebral responses to stimuli [29]. Nerve activity was recorded by small nerve bundles dissected from the nerve branches innervating the right side of IBAT. Nerve filaments were isolated from the central cut end of these nerve bundles under a dissecting microscope to record the efferent activity with a pair of silver wire electrodes. The nerve filaments were covered with a mixture of Vaseline and liquid petroleum at 37°C to avoid dehydration. The firing rate was recorded over 25 min (baseline period) before and 25 min after injection of drug. Procaine hydrochloride (0.5 ml, 20% wt. vol.21) were injected bilaterally in the PVN, over a 40-s period. Two minutes after procaine injection, sine wave stimuli were delivered by an autotransformer, and the current was monitored with an oscilloscope indicating the voltage drop across a known resistor placed in series with the animal. Parameters of stimulation were: sine wave current (50 Hz, 150 mA for 15 s). Furthermore, TC, TIBAT, and O2 consumption were monitored at the same time as the nerve activity record. TC was measured by inserting the thermocouple into the colon at 7 cm from the anus, while TIBAT was monitored by inserting the thermocouple in the left side of IBAT. The same variables were recorded in six other
HISTOLOGY At the end of the experiment, the location of the cannulae was identified. A stain (Bromophenol Blue) was injected in the PVN at the same volume (0.5 ml) used for drug administration. The rats were then injected with an overdose of pentobarbital (200 mg z kg b. wt.21). They were perfused with 0.9% NaCl followed by phosphate-buffered formalin containing 5% (wt. vol.21) potassium forrocyanide to reveal the site of electrical stimulation. The brain was removed and stored in formalin solution. After a few days, 50 mm coronal sections of the fixed brain were cut and stained with cresyl violet. Statistical Analysis The values are presented as means 6 standard error (SE). Statistical analysis was performed using analysis of variance (ANOVA). Multiple comparisons were performed by NewmanKeuls’ post hoc test. RESULTS Figure 1 shows the percentage changes in firing rate of nerves to IBAT. The cortical stimulation increased the firing rate, which
660
MONDA ET AL.
FIG. 5. Means 6 standard error of changes in oxygen consumption of stimulated or sham-stimulated rats with injection of saline or procaine. Cerebral injection at time 0. Stimulation of cerebral cortex was made 2 min after cerebral injection.
peaked at 5 min in the rats with saline injection. This rise was reduced in the animals with procaine administration. Neither saline nor procaine injection produced any modification in the rats without cortical stimulation. ANOVA showed significant effects for cortical stimulation [F(1,20) 5 22.324, p , 0.01], for drug [F(1,20) 5 11.864, p , 0.01], for time [F(5,100) 9.648, p , 0.01], for interaction cortical stimulation 3 drug [F(1,20) 5 7.703, p , 0.05], cortical stimulation 3 time [F(5,100) 5 8.853, p , 0.01], and drug 3 time [F(5,100) 5 2.660, p , 0.05]. The post hoc test showed that saline 1 stimulation group was different from other groups by 5–20 min. Differences were demonstrated between procaine 1 stimulation group and other groups at 5 min. Examples of changes in actual firing rate of the nerves to IBAT are shown in Fig. 2. Figure 3 illustrates TIBAT changes. Cortical stimulation caused
a rise that peaked at 5 min in the rats injected with saline. This increase was reduced in the animals injected with procaine. In the sham-stimulated rats, neither saline nor procaine caused any modification. ANOVA showed significant effects for cortical stimulation [F(1,20) 5 11.439, p , 0.01], for time [F(5,100) 5 4.224, p , 0.01], for interaction cortical stimulation 3 drug [F(1,20) 5 5.683, p , 0.05], and cortical stimulation 3 time [F(5,100) 5 2.726, p , 0.05]. The post hoc test showed that saline 1 stimulation group was different from other groups by 5–25 min. Differences were demonstrated between procaine 1 stimulation group and other groups at 5 min. Figure 4 illustrates TC changes. Cortical stimulation induced a rise with a peak at 10 min in the rats injected with saline. Procaine reduced the increase due to cortical stimulation. No changes resulted in the sham-stimulated animals with saline or procaine administration. ANOVA showed significant effects for cortical stimulation [F(1,20) 5 9.198, p , 0.01], for time [F(5,100) 5 4.206, p , 0.01], for interaction cortical stimulation 3 time [F(5,100) 5 3.206, p , 0.01]. The post hoc test showed that saline 1 stimulation group was different from other groups between 5 and 15 min. Differences were demonstrated between procaine 1 stimulation group and other groups at 10 min. Figure 5 shows changes in O2 consumption. Cortical stimulation induced an increase with a peak at 5 min in the animal injected with saline. Procaine reduced this rise in the stimulated animals. Neither saline nor procaine administration caused any change in the rats without cortical stimulation. ANOVA showed significant effects for cortical stimulation [F(1,20) 5 35.046, p , 0.01], for drug [F(1,20) 5 21.111, p , 0.01], for time [F(5,100) 5 21.620, p , 0.01], for interaction cortical stimulation 3 drug [F(1,20) 5 24.483, p , 0.01], cortical stimulation 3 time [F(5,100) 5 19.784, p , 0.01], drug 3 time [F(5,100) 5 10.420, p , 0.01], and cortical stimulation 3 drug 3 time [F(5,100) 5 10.519, p , 0.01]. The post hoc test showed that saline 1 stimulation group was different from other groups between 5 and 25 min. Differences were demonstrated between procaine 1 stimulation group and other groups at 5 min. There were no differences in four baseline variables of all groups, as reported in the Table 1. DISCUSSION The changes in physiological functions after procaine injection into the medial hypothalamus have also been studied by other authors. They showed that procaine modifies feeding behavior and the sympathetic activity [2,7]. The present findings are the first to indicate that procaine injection in the PVN reduces heat production due to the stimulation of the prefrontal cortex. This suggests that the PVN is an important station in the
TABLE 1 ABSOLUTE VALUES 6 STANDARD ERROR OF FIRING RATE, INTERSCAPULAR BROWN ADIPOSE TISSUE (TIBAT) AND COLONIC TEMPERATURES (TC), AND OXYGEN (O2) CONSUMPTION AT THE TIME OF INJECTION INTO THE PARAVENTRICULAR NUCLEUS
Stimulation 1 saline Stimulation 1 procaine Sham stimulation 1 saline Sham stimulation 1 procaine
Firing Rate (Spikes 5 s21)
TIBAT(°C)
TC (°C)
O2 Consumption (ml z min21 z kg b.wt.20.75)
37.25 6 5.71 39.28 6 5.12 40.62 6 5.59 38.91 6 6.58
37.29 6 0.08 37.35 6 0.11 37.41 6 0.13 37.34 6 0.07
36.79 6 0.09 36.85 6 0.10 36.9 6 10.12 36.76 6 0.09
10.98 6 1.66 11.21 6 1.99 11.65 6 1.72 11.39 6 1.54
PVN AND CORTICAL STIMULATION neural network connecting the prefrontal cortex to structures driving the responses related to nonshivering thermogenesis. Anesthesia with urethane maintains the rat’s ability to develop fever [19], and removes the possible interference due to motor activity. With the evidence from this experiment, we cannot draw any conclusion about heat loss, but we can say that procaine injection in the PVN influences the thermogenic events evoked by the cortical stimulation. These findings show that procaine in the PVN reduces the expected rise in “core” body temperature through reduced neuronal activation of BAT as a means of reducing O2 consumption. Depression of the PVN acts on the thermogenic response by reducing the activity of the sympathetic nervous system. This effect on the sympathetic activity has also been shown with other experimental models. Lesions of the PVN cause a marked decrease in heat production related to postprandial thermogenesis, and this phenomenon is mediated by the adrenergic system. Indeed, PVN lesion blocks sympathetic activation induced by food intake in rat fed with “cafeteria” diet [4]. In the present experiment, procaine does not modify body temperature and metabolic rate under basal conditions, the effect occurs only after the cortical stimulation. This suggests a phasic influence of the PVN in rising thermogenesis due to the cortical stimulation, and seems to exclude a tonic activity on thermogenic mechanisms. The increase in O2 consumption confirms that the cerebral cortex is involved in the control of metabolic rate, in accord with other evidence. Indeed, cortical spreading depression reduces the metabolic rate induced by prostaglandin E1, and this effect depends on the pyrogen dose [15]. Furthermore, bilateral lesions of hypothalamic nuclei cause a marked increase in metabolic rate with hyperthermia for many hours [11], and this phenomenon is mediated by the adrenergic system [3]. It has been shown that, like b-blockers, cortical spreading depression impairs the O2 consumption gain following electrolytic damage of the lateral hypothalamus in rats [6]. The present experiment emphasizes that the cerebral cortex needs the PVN to influence metabolic rate. Other experiments demonstrated that a septal lesion blocks O2 consumption gain due to cortical stimulation [5,21], indicating that septal nuclei take part in the pathway controlling metabolic rate. The present experiment suggests a need to include the PVN in this pathway. The present data indicate that the PVN controls the discharge of sympathetic nerves to IBAT. IBAT is the organ responsible for evoking 25– 65% of the total increase in metabolic heat production during various experimental manipulations in rodents [12,23]. IBAT is supplied by a mixed nerve, which provides five separate branches to the individual lobes [8]. IBAT activity is controlled by the sympathetic nervous system [17], and factors that influence thermogenesis appear to act centrally to modify the sympathetic outflow to IBAT [20]. The significant role of IBAT in the thermogenic changes due to the cortical stimulation is confirmed by these findings. In conclusion, we can affirm that the PVN plays a key role in the sympathetic and thermogenic changes induced by stimulation of the prefrontal cortex, although further investigations are needed to evaluate the role of other cerebral areas involved in these phenomena. ACKNOWLEDGEMENT
This study has been supported by grants from the Italian National Research Council and Regione Campania.
661 REFERENCES 1. Amir, S. Stimulation of the paraventricular nucleus with glutamate activates interscapular brown adipose tissue thermogenesis in rats. Brain Res. 508:152–155; 1990. 2. Berthous, M., Jeanrenaud, B. Changes of insulinemia, glycemia and feeding behavior induced by VMH-procainization in the rat. Brain Res. 174:184 –187; 1979. 3. Corbett, S. W.; Kaufman, L. M.; Keesey, E. Effects of b-adrenergic blockade on lateral hypothalamic lesion-induced thermogenesis. Am. J. Physiol. 245:E535–E541; 1983. 4. De Luca, B.; Monda, M.; Amaro, S.; Pellicano, M. P.; Cioffi, L. A Lack of diet-induced thermogenesis following lesions of paraventricular nucleus in rats. Physiol. Behav. 4:685– 691; 1989. 5. De Luca, B.; Monda, M.; Amaro, S.; Pellicano, M. P.; Cioffi, L. A. Thermogenic changes following frontal neocortex stimulation. Brain Res. Bull. 22:1003–1007; 1989. 6. De Luca, B.; Monda, M.; Pellicano, M. P.; Zenga, A. Cortical control of thermogenesis induced by lateral hypothalamic lesion and overeating. Am. J. Physiol. 253:R626 –R633; 1987. 7. Epstein, A. N. Reciprocal changes in feeding behavior produced by intrahypothalamic chemical injection. Am. J. Physiol., 199:969 –974; 1960. 8. Foster, D. O.; Depocas, F.; Zaror-Behrens, G. Unilaterality of the sympathetic innervation of each pad of rat interscapular brown adipose tissue. Can. J. Physiol. Pharmacol. 60:107–113; 1982. 9. Fyda, D. F.; Cooper, K. E.; Veale, W. L. Contribution of brown adipose tissue to central PGE1-evoked hyperthermia in rats. Am. J. Physiol. 260:R59 –R66; 1991. 10. Gollman, H. M.; Rudy, T. A. Comparative pyrogenic potency of endogenous prostanoids and of prostanoid-mimetics injected into the anterior/preoptic region of the cat. Brain Res. 449: 281293; 1988. 11. Grijalva, C.V. Aphagia, gastric pathology, hyperthermia and sensomotor dysfunction following lateral hypothalamic lesions: Effects of insulin pretreatment. Physiol. Behav. 25:931–937; 1980. 12. Himms-Hagen, J. Non-shivering thermogenesis. Brain. Res. Bull. 12: 151–160; 1984. 13. Hori, T.; Shibata, M.; Kyohara, T.; Nakashima, T. Prefrontal cortical influences on behavioural thermoregulation and thermosensitive neurons. J. Therm. Biol. 9:27–31; 1984. 14. Kleiber, M. The fire of life. An introduction to animal energetics. Huntington: Kreiger Publishing Company; 1975. 15. Komaromi, I.; Malkinson, T. J.; Veale, W. L.; Rosenbaum, G.; Cooper, K. E.; Pittman, Q. J. Effect of potassium-induced cortical spreading depression on prostaglandin-induced fever in conscious and urethane-anesthetized rats. Can. J. Physiol. Pharmacol. 72:716 –721, 1994. 16. Landsberg, L.; Saville, M. E.; Young, J. B. Sympathoadrenal system and regulation of thermogenesis. Am. J. Physiol. 247:E181–E189; 1984. 17. Landsberg, L.; Young, J. B. Autonomic regulation of thermogenesis. In: Girardier, L.; Stock, M. J. eds. Mammalian thermogenesis. London: Chapman and Hall; 1983:99 –140. 18. Lawrence, D.; Pittman. Q. J. Interaction between descending paraventricular neurons and vagal motor neurons. Brain Res. 332:158 –160; 1985. 19. Malkinson, T. J.; Cooper, K. E.; Veale, W. L. Physiological changes during thermoregulation and fever in urethan-anaesthetized rats, Am. J. Physiol. 255:R73–R81; 1988. 20. Monda, M.; Amaro, S.; De Luca, B. Non-shivering thermogenesis during prostaglandin E1 fever in rats: role of cerebral cortex. Brain. Res. 651:148 –154; 1994. 21. Monda, M.; Amaro, S.; De Luca, B. Septal lesions block sympathetic activation following frontal cortex stimulation in the rat. Acta Physiol. Scand. 155:275–282; 1995. 22. Monda, M.; Amaro, S.; Sullo, A.; De Luca, B. Posterior hypothalamic activity and cortical control during the PGE1-hyperthermia. Neuroreport 6: 135–139; 1994. 23. Monda, M.; Sullo, A.; De Luca, E.; Pellicano, M. P. Lysine acetylsalycilate modifies aphagia and thermogenic changes induced by lateral hypothalamic lesion. Am. J. Physiol. 271:R1638 –R1642; 1996. 24. Monda, M.; Viggiano, A.; Sullo, A.; De Luca, V. Aspartic and glu-
662 tamic acids increase in the frontal cortex during pge1 hyperthermia. Neuroscience 83:1239 –1243; 1998. 25. Pellegrino, L. J.; Pellegrino, A. S.; Cushman, A. J. A stereotaxic atlas of the rat brain. New York: Plenum Press; 1979. 26. Pittman, Q. J.; Franklin, L. G. Vasopressin antagonist in nucleus tractus solitarius/vagal area reduces pressor and tachycardia responses to paraventricular nucleus stimulation in rats. Neurosci. Lett. 56:155– 160; 1985. 27. Sakaguchi, T.; Bray, G.A.; Eddlestone, G. Sympathetic activity following paraventricular or ventromedial hypothalamic lesions in rats. Brain Res. Bull. 20:461– 465; 1988.
MONDA ET AL. 28. Simpson, C. W.; Ruwe, W. D.; Myers, R. D. Prostaglandins and hypothalamic neurotransmitter receptors involved in hyperthermia: a critical evaluation. Neurosci. Biobehav. Rev.18:1–20; 1994. 29. Soma, L. R. Textbook of veterinary surgery, depth of general anesthesia. Baltimore, MD: Williams and Wilkins; 1971:178 –187. 30. Swanson, L. W.; Sawchenko, P. E. Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms. Neuroendocrinology 31:410 – 417; 1980. 31. Yoshimatsu, H.; Egawa, M.; Bray, G.A. Sympathetic nerve activity after discrete hypothalamic injections of L-glutamate. Brain Res. 601: 121–128; 1993.