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Cortical spreading depression reduces paraventricular activation induced by hippocampal neostigmine injection
Brain Research 824 Ž1999. 119–124
Research report
Cortical spreading depression reduces paraventricular activation induced by hippocampal neostigmine injection M. Monda ) , A. Viggiano, A. Sullo, V. De Luca Dipartimento di Fisiologia Umana e Funzioni Biologiche Integrate ‘Filippo Bottazzi’, Seconda UniÕersita` di Napoli, Via Costantinopoli 16, 80138 Napoli, Italy Accepted 9 February 1999
Abstract The firing rate of the neurons of the hypothalamic paraventricular nucleus, the temperatures of the interscapular brown adipose tissue and of the colon ŽT IBAT and Tc . were monitored in 24 urethane-anesthetized male Sprague–Dawley rats divided into four groups. These variables were measured before and after hippocampal injection of neostigmine Ž5 = 10y7 mol. in the 1st and 2nd groups or of saline in the 3rd and 4th groups. The hippocampal injection was preceded by cortical spreading depression in the 1st and 3rd groups, while the cortical depression was not induced in the 2nd and 4th groups. The results show an increase of firing rate, T IBAT and Tc after neostigmine injection in the rats without cortical depression. Cortical spreading depression significantly reduces these enhancements. These findings demonstrate that: Ž1. the paraventricular nucleus plays a significant role in the hyperthermia induced by neostigmine injection into the hippocampus; and Ž2. the cerebral cortex is involved in the control of the paraventricular activity. q 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Cortical spreading depression; Hippocampus; Hyperthermia; Paraventricular nucleus; Unit activity; Rat
1. Introduction The paraventricular nucleus of the hypothalamus ŽPVN. serves as the crossroads of integrative physiology. This discrete hypothalamic area receives neural, humoral, and endocrine input concerning the state of the cardiovascular, endocrine, and immune systems, as well as fluid, electrolyte and energy balances w7,11,29x. Integration of afferent inputs results in efferent neural or hormonal regulation of specific organ systems w2x. The PVN, like the anterior w10,27x and posterior w17x hypothalamus, plays a role in the control of body temperature. The variety of physiological variables influenced by the PVN indicates that this cell population is functionally complex. Several studies have shown that neurons of the PVN project to a number of areas causally involved in the regulation of autonomic functions w30x, and stimulation of the PVN activates neuronal outflow to a number of brain stem nuclei w14,23x and peripheral organs w32x. In rodents, a major tissue for heat production related to non-shivering thermogenesis is inter-
) Corresponding [email protected]
author.
Fax:
q 39-81-566-5820;
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scapular brown adipose tissue ŽIBAT. w9x. IBAT is known to be under the peripheral neurogenic control of the sympathetic nervous system w13x. Stimulation of the PVN by local glutamate microinjections results in an increase in IBAT temperature in urethane-anaesthetized rats w1x, associated with increased electrical activity in the sympathetic fibers innervating this tissue w32x. In contrast, the firing rate of nerve filaments terminating in IBAT was reduced after acute lesions of the rat PVN, indicative of reduced PVN-mediated sympathetic activity w24x. 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 cold-sensitive units in the anterior hypothalamic area w12x. We have demonstrated the involvement of the neocortex in the control of heat production. In fact, functional decortication induced by cortical spreading depression ŽCSD. reduces thermogenesis in overfed rats w6x and blocks the hyperthermic reaction induced by an intracerebroventricular injection of prostaglandin E 1 w19x. Excitatory neurotransmitters increase in the frontal cortex during prostaglandin E 1 hyperthermia w21x. Stimulation of the
0006-8993r99r$ - see front matter q 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 2 2 7 - 5
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M. Monda et al.r Brain Research 824 (1999) 119–124
frontal cortex raises IBAT temperature w5x, and this increase is blocked by a lesion of the septal nuclei w16x. This last point suggests that the limbic pathway is involved in the thermogenesis. As a component of the limbic system, the hippocampus has been linked to the mechanisms of emotion w3x, and hippocampal cholinergic neurons have been reported to be stimulated by some forms of stress w8x. The hippocampus also affects autonomic system activity. Uemura et al. w31x have shown an involvement of the hippocampus in central nervous system-mediated glucoregulation. We have recently demonstrated that neostigmine injection into the hippocampus activates thermogenesis by an increase in sympathetic activity and T4-to-T3 conversion w18x. The aim of this experiment was to evaluate: Ž1. the effects of hippocampal injection of neostigmine on the firing rate of the paraventricular neurons, and Ž2. the role of the neocortex in these phenomena.
2. Materials and methods 2.1. Animals We used male inbred Sprague–Dawley rats Ž N s 24., 3 months old and weighing 270–300 g. These were housed in pairs at controlled temperature Ž22 " 18C. and humidity Ž70%. with a 12:12 h light–dark cycle from 0700 to 1900 h. The experiments conformed with the European Convention for the Protection of Vertebrate Animals used for Experimental and Scientific Purpose ŽCouncil of Europe No. 123, Strasbourg 1985..
2.2. Apparatus Extracellular single unit activities were recorded using a tungsten electrode ŽClark Electromedical Instruments. with tip diameter of 10 mm and impedance of 12 M V. The tip of the microelectrode reached the PVN stereotaxically at the following coordinates: 0.6 mm anterior to the bregma, 0.4 mm lateral to the midline and 7.7–7.9 mm from the surface w22x. The electrical pulses were amplified by a condenser-coupled amplifier and were filtered by band-path filters ŽNeuroLog System, Digitimer.. The raw pulses were displayed on an oscilloscope ŽTektronix. and sent to a window discriminator. Square waves from the discriminator were sent to an analog–digital converter ŽDAS system, Keithley. and stored on a computer ŽPersonal Computer AT, IBM. every 5 s. Furthermore, a rate meter with a reset time of 5 s was used to observe the time course of the unit activity recorded by a pen recorder ŽVitatron.. The threshold level of the event detector was fixed during the experiment at 3–4 times above background noise. Electrocorticographic activity and slow potential changes were recorded with two wick calomel cell electrodes applied on the parietal cortex. The reference electrode was placed on the nasal bone. The three electrodes were connected to a polygraph ŽDynograph, Beckman.. Thermocouples ŽEllab. were used to monitor colonic and IBAT temperatures and the values were stored on a chart recorder. The cannula for drug injections was 0.4 mm longer than the guide cannula. 2.3. Drug Neostigmine was purchased from Sigma ŽSt. Louis, MO. and dissolved in a pyrogen-free saline solution Ž5 = 10y7 mol of neostigmine in 1 ml of saline..
Fig. 1. Means" S.E. of changes in the firing rate of the neurons of the PVN. The neostigmine or saline was injected at time 0. The KCl or NaCl was applied on the cortex at time y5.
M. Monda et al.r Brain Research 824 (1999) 119–124 Table 1 Absolute values"S.E. of firing rate Žspikesr5 s. at time of neostigmine injection Group
Firing rate
1st
2nd
3rd
4th
21.43"3.78
21.76"5.15
22.25"6.11
19.42"4.23
121
4th group were used as controls: 2.0 M NaCl solution was applied on the cerebral cortex and saline was injected into the hippocampus. The baseline values of Tc from all animals used were maintained constant by a heating pad. The electrical energy supplied to the pad was not altered during the experimental period. In the other words, a servo system for controlling the animal’s temperature was not used.
2.4. Surgery
2.6. Histology
All animals were anesthetized with intraperitoneal pentobarbital sodium Ž50 mgrkg b.wt., and a 20-gauge stainless guide cannula was positioned stereotaxically above the dorsal hippocampus at the following coordinates: 1.5 mm lateral to the midline, 2.0 mm posterior to the bregma, 2.2 mm from the surface w22x. The guide cannula was secured to the skull by screws and dental cement. A stylet was inserted into the guide tube and removed only during drug administration. In addition, two symmetrical craniotomies Ž5 mm in diameter. were performed in the frontal bone. The centers of the trephine openings were located at 3 mm anterior to the bregma and at 3 mm lateral to midline. Two polyethylene wells Žwith internal diameter of 1.5 mm and volume of 30 ml. were placed upon the frontal cortex in the animals. These wells were filled with saline solution and capped until the experiment began. Rats were given 7–10 days to recover from surgery as judged by recovery of preoperative body weight.
At the end of the experiment, a 2 mA cathodal current was passed through the recording electrode for 40 s to produce an electrolytical lesion for identifying the locus of registration. A stain Žbromophenol blue. was injected in the hippocampus at the same volume used for drug administration to identify the location of the cannula. The rats
2.5. Procedure After recovery, 6 animals Ž1st group. were anesthetized with urethane Ž1.2 grkg b.w. i.p.. and mounted in a stereotaxic instrument ŽStoelting.. The level of anesthesia was kept constant as evaluated by skeletal muscle relaxation, eye and palpebral responses to stimuli w28x. The content of wells was removed and the parietal cortex was previously exposed before the experiment. 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 estimated region. Firing rate, TIBAT and Tc were recorded for 30 min before and for 40 min after injection of neostigmine Ž1 ml of 0.5 M solution. into the hippocampus. An application of KCl Ž10 ml of 2.0 M solution. upon the frontal cerebral cortex Žwhich causes several waves of CSD. w4x preceded the neostigmine injection by 5 min. In addition, electrocorticographic activity and slow potential changes of the parietal cortex were recorded at the same time. The same variables were recorded in another 6 rats Ž2nd group., but the KCl solution applied to the cortex was substituted with 2.0 M NaCl solution Žwhich has no effect on cortical function. w4x. The same procedure used with the 1st group was carried out with another 6 animals Ž3rd group. except than saline was injected into the hippocampus. The rats of the
Fig. 2. Firing rate changes in a non-decorticated Žpanel A. or decorticated Žpanel B. rat receiving neostigmine and in a non-decorticated Žpanel C. or decorticated Žpanel D. rat receiving saline. The arrow indicates the hippocampal injection of neostigmine or saline.
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Fig. 3. Means " S.E. of changes in interscapular brown adipose tissue temperature. The neostigmine or saline was injected at time 0. The KCl or NaCl was applied on the cortex at time y5.
were then injected with an overdose of pentobarbital Ž150 mgrkg b.wt. and were perfused with 0.9% NaCl followed by 10% Žvolrvol. formalin solution. 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 neutral red.
3. Results Fig. 1 shows the percentage changes in firing rate of the neurons of the PVN. Neostigmine injection caused a rise
that peaks at 10 min in the rats. This increase was reduced by CSD. In the control rats, the NaCl or KCl applied upon the cortex did not cause any modification. Three-way ANOVA Ždrug = CSD = days, 2 = 2 = 9. with repeated measures on the last factor showed significant main effects of neostigmine w F Ž1,20. s 115.305, p - 0.01x, of CSD w F Ž1,20. s 15.256, p - 0.01x, of days w F Ž8,160. s 60.961, p - 0.01x, as well as significant first order interactions, neostigmine = CSD w F Ž1,20. s 15.185, p - 0.01x, neostigmine= days w F Ž8,160. s 59.821, p - 0.01x, CSD = time w F Ž8,160. s 6.586, p - 0.01x, and second order interaction Ždrug = CSD = days. w F Ž8,160. s 6.303,
Fig. 4. Means" S.E. of changes in colonic temperature. The neostigmine or saline was injected at time 0. The KCl or NaCl was applied on the cortex at time y5.
M. Monda et al.r Brain Research 824 (1999) 119–124
p - 0.01x. Newman–Keuls post hoc test showed that the neostigmine group without CSD and the neostigmineq CSD were different from other groups at 10 and 15 min. Differences were demonstrated between the neostigmineq CSD group and other groups at 10 and 15 min. There were no differences in the baseline absolute values of all groups, as reported in Table 1. Examples of the changes in firing rate are shown in Fig. 2. Fig. 3 illustrates the T IBAT changes. Neostigmine injection caused a rise that peaked at 20 min in the rats without CSD. This increase was reduced by CSD. In the control rats, the NaCl or KCl applied upon the cortex did not cause any modification. Three-way ANOVA Ždrug = CSD = days, 2 = 2 = 9. with repeated measures on the last factor showed significant main effects of neostigmine w F Ž1,20. s 16.543, p - 0.01x, of CSD w F Ž1,20. s 5.533, p - 0.01x, and of days w F Ž8,160. s 4.778, p - 0.01x, and as well as significant first order interactions, neostigmine = CSD w F Ž1,20. s 4.654, p - 0.01x, and neostigmine= days w F Ž8,160. s 4.476, p - 0.01x. The second order interaction was not significant. Newman–Keuls post hoc test showed that the neostigmine group without CSD was different from other groups at 10 to 40 min. Differences were demonstrated between the neostigmineq CSD group and other groups at 15 and 20 min. Fig. 4 illustrates the Tc changes. Neostigmine injection caused a rise that peaks at 20 min. This increase was reduced by CSD. In the control rats, the NaCl or KCl applied upon the cortex did not cause any modification. Three-way ANOVA Ždrug = CSD = days, 2 = 2 = 9. with repeated measures on the last factor showed significant main effects of neostigmine w F Ž1,20. s 20.697, p - 0.01x, of CSD w F Ž1,20. s 5.983, p - 0.01x, of days w F Ž8,160. s 5.864, p - 0.01x, as well as significant first order interactions, neostigmine= CSD w F Ž1,20. s 5.368, p - 0.01x, and neostigmine= days w F Ž8,160. s 4.846, p - 0.01x. The second order interaction was not significant. Newman– Keuls post hoc test showed that the neostigmine group without CSD was different from other groups at 15 to 40 min. Differences were demonstrated between the neostigmine q CSD group and other groups at 15 and 20 min.
123
we showed that the PVN is involved in the sympathetic activation induced by a cortical stimulation. Indeed, procaine injection into the PVN reduces the sympathetic and thermogenic changes due to stimulation of frontal cortex w20x. The present experiment is the first to show that a cholinergic hippocampal stimulation induces an increase in the firing rate of the paraventricular neurons. The modification of the firing rate precedes temperature changes. This suggests the hippocampus may control body temperature through the PVN. Furthermore, these findings indicate that the cerebral cortex is involved in these complex phenomena. In fact, CSD reduces the increase in the paraventricular activity, suggesting a cortical control on the hippocampus andror PVN. CSD does not modify the firing rate of the PVN in the control rats, and this confirms previous data showing a phasic influence of the cerebral cortex on subcortical structures w6,15x. In other words, CSD modifies the thermogenic response only during the hippocampal stimulation, suggesting that the cerebral cortex does not influence body temperature under basal conditions. Shibata et al. w25x indicate that in urethane anesthetized rats firing rate of warm-sensitive and cold sensitive neurons in preoptic arearanterior hypothalamus is decreased and increased, respectively, during invasion of a CSD wave into frontal cortex. Furthermore, CSD activates in non-anesthetized rats behavioral thermoregulation and metabolic heat production, increasing colonic temperature w26x. The difference of our results can be due to procedural factors ŽPVN vs. preoptic arearanterior hypothalamus, several vs. single CSD waves, etc... In conclusion, the neurons of the PVN are stimulated by the hippocampal injection of neostigmine and functional decortication induced by CSD reduces this stimulation.
Acknowledgements The Italian National Research Council.
References 4. Discussion The findings of the present experiment emphasize: Ž1. the role of the PVN in the thermogenic activation induced by hippocampal cholinergic stimulation; and Ž2. the effects of CSD on the neuronal activity of the PVN. Uemura et al. w31x have shown that a neostigmine injection in the hippocampus produces hyperglycemia by epinephrine secretion. This demonstrates that the hippocampus is involved in the control of metabolic variables. We demonstrated that hippocampal cholinergic stimulation by neostigmine injection increases the firing rate of sympathetic nerves to IBAT and the temperature of this tissue w18x. Furthermore,
w1x S. Amir, Stimulation of the paraventricular nucleus with glutamate activates interscapular brown adipose tissue thermogenesis in rats, Brain Res. 508 Ž1990. 152–155. w2x C. Boudaba, L.A. Schrader, J.G. Tasker, Physiological evidence for local excitatory synaptic circuits in the rat hypothalamus, J. Neurophysiol. 77 Ž1977. 3396–3400. w3x C.M. Brooks, The autonomic nervous system, molder and integrator of function. Review of a concept, Braz. J. Med. Biol. Res. 14 Ž1981. 151–160. w4x J. Bures, O. Buresova, Inducing cortical spreading depression, in: R.D. Myers ŽEd.., Method in Psychology, Academic Press, New York, 1972, pp. 319–343. w5x B. De Luca, M. Monda, S. Amaro, M.P. Pellicano, L.A. Cioffi, Thermogenic changes following frontal neocortex stimulation, Brain Res. Bull. 22 Ž1989. 1003–1007.
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w6x B. De Luca, M. Monda, M.P. Pellicano, A. Zenga, Cortical control of thermogenesis induced by lateral hypothalamic lesion and overeating, Am. J. Physiol. 253 Ž1987. R626–R633. w7x Y.F. Duan, R. Winters, P.M. McCabe, E.J. Green, Y. Huang, N. Schneiderman, Cardiorespiratory components of defense reaction elicited from paraventricular nucleus, Physiol. Behav. 61 Ž1997. 325–330. w8x Y. Finkelstein, B. Koffier, L.M. Pabey, G.M. Gilad, Dynamic of cholinergic synaptic mechanisms in rat hippocampus after stress, Brain Res. 343 Ž1985. 314–319. w9x D.F. Fyda, K.E. Cooper, W.L. Veale, Contribution of brown adipose tissue to central PGE 1 -evoked hyperthermia in rats, Am. J. Physiol. 260 Ž1991. R59–R66. w10x H.M. Gollman, T.A. Rudy, Comparative pyrogenic potency of endogenous prostanoids and of prostanoid-mimetics injected into the anteriorrpreoptic region of the cat, Brain Res. 449 Ž1988. 281–293. w11x J.P. Herman, W.E. Cullinan, Neurocircuitry of stress: central control of the hypothalamo-pituitary-adrenocortical axis, Trends-Neurosci. 20 Ž1997. 78–84. w12x T. Hori, M. Shibata, T. Kyohara, T. Nakashima, Prefrontal cortical influences on behavioural thermoregulation and thermosensitive neurons, J. Therm. Biol. 9 Ž1984. 27–31. w13x L. Landsberg, M.E. Saville, J.B. Young, Sympathoadrenal system and regulation of thermogenesis, Am. J. Physiol. 247 Ž1984. E181– E189. w14x D. Lawrence, Q.J. Pittman, Interaction between descending paraventricular neurons and vagal motor neurons, Brain Res. 332 Ž1985. 158–160. w15x M. Monda, S. Amaro, B. De Luca, Non-shivering thermogenesis during prostaglandin E1 fever in rats: role of cerebral cortex, Brain Res. 651 Ž1994. 148–154. w16x M. Monda, S. Amaro, B. De Luca, Septal lesions block sympathetic activation following frontal cortex stimulation in the rat, Acta Physiol. Scand. 55 Ž1995. 275–282. w17x M. Monda, S. Amaro, A. Sullo, B. De Luca, Posterior hypothalamic activity and cortical control during the PGE 1 -hyperthermia, NeuroReport 6 Ž1994. 135–139. w18x M. Monda, A. Papa, G. Brizzi, B. De Luca, Neostigmine injection in the hippocampus activates thermogenesis by an increase in sympathetic activity and T4 -to-T3 conversion, Am. J. Physiol. 270 Ž1996. R1215–1219. w19x M. Monda, Q.J. Pittman, Cortical spreading depression blocks
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w28x w29x
w30x
w31x
w32x
prostaglandin El and endotoxin fever in rats, Am. J. Physiol. 264 Ž1993. R456–R459. M. Monda, A. Sullo, V. De Luca, A. Viggiano, Procaine injection into the paraventricular nucleus reduces sympathetic and thermogenic activation induced by frontal cortex stimulation in the rat. Brain Res. Bull., 1998, accepted. M. Monda, A. Viggiano, A. Sullo, V. De Luca, Aspartic and glutamic acids increase in the frontal cortex during PGE 1 hyperthermia, Neuroscience 83 Ž1998. 1239–1243. L.J. Pellegrino, A.S. Pellegrino, A.J. Cushman ŽEds.., A Stereotaxic Atlas of the Rat Brain, Plenum, New York, 1979. Q.J. Pittman, L.G. Franklin, Vasopressin antagonist in nucleus tractus solitariusrvagal area reduces pressor and tachycardia responses to paraventricular nucleus stimulation in rats, Neurosci. Lett. 56 Ž1985. 155–160. T. Sakaguchi, G.A. Bray, G. Eddlestone, Sympathetic activity following paraventricular or ventromedial hypothalamic lesions in rats, Brain Res. Bull. 20 Ž1998. 461–465. M. Shibata, T. Hori, T. Kyohara, T. Nakashima, Activity of hypothalamic thermosensitive neurons during cortical spreading depression, Brain Res. 308 Ž1984. 255–262. M. Shibata, T. Hori, T. Nagasaka, Effects of single cortical spreading depression on metabolic heat production in the rat, Physiol. Behav. 34 Ž1985. 563–567. C.W. Simpson, W.D. Ruwe, R.D. Myers, Prostaglandins and hypothalamic neurotransmitter receptors involved in hyperthermia: a critical evaluation, Neurosci. Biobehav. Rev. 18 Ž1994. 1–20. L.R. Soma ŽEd.., Textbook of Veterinary Surgery. Williams and Wilkins, Baltimore, MD, 1971, 178–187. J.Y. Summy-Long, S. Gestl, M.L. Terrell, G. Wolz, M. Kadekaro, Osmoregulation of the magnocellular neuroendocrine system during lactation, Am. J. Physiol. 272 Ž1977. R275–288. L.W. Swanson, P.E. Sawchenko, Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms, Neuroendocrinology 31 Ž1980. 410–417. Y. Uemura, A. Iguchi, A. Yatomi, H. Miura, A. Honmura, M. Yanase, N. Sakamoto, Involvement of the hippocampus in central nervous system-mediated glucoregulation in rats, Endocrinology 124 Ž1989. 2449–2455. H. Yoshimatsu, M. Egawa, G.A. Bray, Sympathetic nerve activity after discrete hypothalamic injections of L-glutamate, Brain Res. 601 Ž1993. 121–128.
Research report
Cortical spreading depression reduces paraventricular activation induced by hippocampal neostigmine injection M. Monda ) , A. Viggiano, A. Sullo, V. De Luca Dipartimento di Fisiologia Umana e Funzioni Biologiche Integrate ‘Filippo Bottazzi’, Seconda UniÕersita` di Napoli, Via Costantinopoli 16, 80138 Napoli, Italy Accepted 9 February 1999
Abstract The firing rate of the neurons of the hypothalamic paraventricular nucleus, the temperatures of the interscapular brown adipose tissue and of the colon ŽT IBAT and Tc . were monitored in 24 urethane-anesthetized male Sprague–Dawley rats divided into four groups. These variables were measured before and after hippocampal injection of neostigmine Ž5 = 10y7 mol. in the 1st and 2nd groups or of saline in the 3rd and 4th groups. The hippocampal injection was preceded by cortical spreading depression in the 1st and 3rd groups, while the cortical depression was not induced in the 2nd and 4th groups. The results show an increase of firing rate, T IBAT and Tc after neostigmine injection in the rats without cortical depression. Cortical spreading depression significantly reduces these enhancements. These findings demonstrate that: Ž1. the paraventricular nucleus plays a significant role in the hyperthermia induced by neostigmine injection into the hippocampus; and Ž2. the cerebral cortex is involved in the control of the paraventricular activity. q 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Cortical spreading depression; Hippocampus; Hyperthermia; Paraventricular nucleus; Unit activity; Rat
1. Introduction The paraventricular nucleus of the hypothalamus ŽPVN. serves as the crossroads of integrative physiology. This discrete hypothalamic area receives neural, humoral, and endocrine input concerning the state of the cardiovascular, endocrine, and immune systems, as well as fluid, electrolyte and energy balances w7,11,29x. Integration of afferent inputs results in efferent neural or hormonal regulation of specific organ systems w2x. The PVN, like the anterior w10,27x and posterior w17x hypothalamus, plays a role in the control of body temperature. The variety of physiological variables influenced by the PVN indicates that this cell population is functionally complex. Several studies have shown that neurons of the PVN project to a number of areas causally involved in the regulation of autonomic functions w30x, and stimulation of the PVN activates neuronal outflow to a number of brain stem nuclei w14,23x and peripheral organs w32x. In rodents, a major tissue for heat production related to non-shivering thermogenesis is inter-
) Corresponding [email protected]
author.
Fax:
q 39-81-566-5820;
E-mail:
scapular brown adipose tissue ŽIBAT. w9x. IBAT is known to be under the peripheral neurogenic control of the sympathetic nervous system w13x. Stimulation of the PVN by local glutamate microinjections results in an increase in IBAT temperature in urethane-anaesthetized rats w1x, associated with increased electrical activity in the sympathetic fibers innervating this tissue w32x. In contrast, the firing rate of nerve filaments terminating in IBAT was reduced after acute lesions of the rat PVN, indicative of reduced PVN-mediated sympathetic activity w24x. 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 cold-sensitive units in the anterior hypothalamic area w12x. We have demonstrated the involvement of the neocortex in the control of heat production. In fact, functional decortication induced by cortical spreading depression ŽCSD. reduces thermogenesis in overfed rats w6x and blocks the hyperthermic reaction induced by an intracerebroventricular injection of prostaglandin E 1 w19x. Excitatory neurotransmitters increase in the frontal cortex during prostaglandin E 1 hyperthermia w21x. Stimulation of the
0006-8993r99r$ - see front matter q 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 2 2 7 - 5
120
M. Monda et al.r Brain Research 824 (1999) 119–124
frontal cortex raises IBAT temperature w5x, and this increase is blocked by a lesion of the septal nuclei w16x. This last point suggests that the limbic pathway is involved in the thermogenesis. As a component of the limbic system, the hippocampus has been linked to the mechanisms of emotion w3x, and hippocampal cholinergic neurons have been reported to be stimulated by some forms of stress w8x. The hippocampus also affects autonomic system activity. Uemura et al. w31x have shown an involvement of the hippocampus in central nervous system-mediated glucoregulation. We have recently demonstrated that neostigmine injection into the hippocampus activates thermogenesis by an increase in sympathetic activity and T4-to-T3 conversion w18x. The aim of this experiment was to evaluate: Ž1. the effects of hippocampal injection of neostigmine on the firing rate of the paraventricular neurons, and Ž2. the role of the neocortex in these phenomena.
2. Materials and methods 2.1. Animals We used male inbred Sprague–Dawley rats Ž N s 24., 3 months old and weighing 270–300 g. These were housed in pairs at controlled temperature Ž22 " 18C. and humidity Ž70%. with a 12:12 h light–dark cycle from 0700 to 1900 h. The experiments conformed with the European Convention for the Protection of Vertebrate Animals used for Experimental and Scientific Purpose ŽCouncil of Europe No. 123, Strasbourg 1985..
2.2. Apparatus Extracellular single unit activities were recorded using a tungsten electrode ŽClark Electromedical Instruments. with tip diameter of 10 mm and impedance of 12 M V. The tip of the microelectrode reached the PVN stereotaxically at the following coordinates: 0.6 mm anterior to the bregma, 0.4 mm lateral to the midline and 7.7–7.9 mm from the surface w22x. The electrical pulses were amplified by a condenser-coupled amplifier and were filtered by band-path filters ŽNeuroLog System, Digitimer.. The raw pulses were displayed on an oscilloscope ŽTektronix. and sent to a window discriminator. Square waves from the discriminator were sent to an analog–digital converter ŽDAS system, Keithley. and stored on a computer ŽPersonal Computer AT, IBM. every 5 s. Furthermore, a rate meter with a reset time of 5 s was used to observe the time course of the unit activity recorded by a pen recorder ŽVitatron.. The threshold level of the event detector was fixed during the experiment at 3–4 times above background noise. Electrocorticographic activity and slow potential changes were recorded with two wick calomel cell electrodes applied on the parietal cortex. The reference electrode was placed on the nasal bone. The three electrodes were connected to a polygraph ŽDynograph, Beckman.. Thermocouples ŽEllab. were used to monitor colonic and IBAT temperatures and the values were stored on a chart recorder. The cannula for drug injections was 0.4 mm longer than the guide cannula. 2.3. Drug Neostigmine was purchased from Sigma ŽSt. Louis, MO. and dissolved in a pyrogen-free saline solution Ž5 = 10y7 mol of neostigmine in 1 ml of saline..
Fig. 1. Means" S.E. of changes in the firing rate of the neurons of the PVN. The neostigmine or saline was injected at time 0. The KCl or NaCl was applied on the cortex at time y5.
M. Monda et al.r Brain Research 824 (1999) 119–124 Table 1 Absolute values"S.E. of firing rate Žspikesr5 s. at time of neostigmine injection Group
Firing rate
1st
2nd
3rd
4th
21.43"3.78
21.76"5.15
22.25"6.11
19.42"4.23
121
4th group were used as controls: 2.0 M NaCl solution was applied on the cerebral cortex and saline was injected into the hippocampus. The baseline values of Tc from all animals used were maintained constant by a heating pad. The electrical energy supplied to the pad was not altered during the experimental period. In the other words, a servo system for controlling the animal’s temperature was not used.
2.4. Surgery
2.6. Histology
All animals were anesthetized with intraperitoneal pentobarbital sodium Ž50 mgrkg b.wt., and a 20-gauge stainless guide cannula was positioned stereotaxically above the dorsal hippocampus at the following coordinates: 1.5 mm lateral to the midline, 2.0 mm posterior to the bregma, 2.2 mm from the surface w22x. The guide cannula was secured to the skull by screws and dental cement. A stylet was inserted into the guide tube and removed only during drug administration. In addition, two symmetrical craniotomies Ž5 mm in diameter. were performed in the frontal bone. The centers of the trephine openings were located at 3 mm anterior to the bregma and at 3 mm lateral to midline. Two polyethylene wells Žwith internal diameter of 1.5 mm and volume of 30 ml. were placed upon the frontal cortex in the animals. These wells were filled with saline solution and capped until the experiment began. Rats were given 7–10 days to recover from surgery as judged by recovery of preoperative body weight.
At the end of the experiment, a 2 mA cathodal current was passed through the recording electrode for 40 s to produce an electrolytical lesion for identifying the locus of registration. A stain Žbromophenol blue. was injected in the hippocampus at the same volume used for drug administration to identify the location of the cannula. The rats
2.5. Procedure After recovery, 6 animals Ž1st group. were anesthetized with urethane Ž1.2 grkg b.w. i.p.. and mounted in a stereotaxic instrument ŽStoelting.. The level of anesthesia was kept constant as evaluated by skeletal muscle relaxation, eye and palpebral responses to stimuli w28x. The content of wells was removed and the parietal cortex was previously exposed before the experiment. 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 estimated region. Firing rate, TIBAT and Tc were recorded for 30 min before and for 40 min after injection of neostigmine Ž1 ml of 0.5 M solution. into the hippocampus. An application of KCl Ž10 ml of 2.0 M solution. upon the frontal cerebral cortex Žwhich causes several waves of CSD. w4x preceded the neostigmine injection by 5 min. In addition, electrocorticographic activity and slow potential changes of the parietal cortex were recorded at the same time. The same variables were recorded in another 6 rats Ž2nd group., but the KCl solution applied to the cortex was substituted with 2.0 M NaCl solution Žwhich has no effect on cortical function. w4x. The same procedure used with the 1st group was carried out with another 6 animals Ž3rd group. except than saline was injected into the hippocampus. The rats of the
Fig. 2. Firing rate changes in a non-decorticated Žpanel A. or decorticated Žpanel B. rat receiving neostigmine and in a non-decorticated Žpanel C. or decorticated Žpanel D. rat receiving saline. The arrow indicates the hippocampal injection of neostigmine or saline.
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Fig. 3. Means " S.E. of changes in interscapular brown adipose tissue temperature. The neostigmine or saline was injected at time 0. The KCl or NaCl was applied on the cortex at time y5.
were then injected with an overdose of pentobarbital Ž150 mgrkg b.wt. and were perfused with 0.9% NaCl followed by 10% Žvolrvol. formalin solution. 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 neutral red.
3. Results Fig. 1 shows the percentage changes in firing rate of the neurons of the PVN. Neostigmine injection caused a rise
that peaks at 10 min in the rats. This increase was reduced by CSD. In the control rats, the NaCl or KCl applied upon the cortex did not cause any modification. Three-way ANOVA Ždrug = CSD = days, 2 = 2 = 9. with repeated measures on the last factor showed significant main effects of neostigmine w F Ž1,20. s 115.305, p - 0.01x, of CSD w F Ž1,20. s 15.256, p - 0.01x, of days w F Ž8,160. s 60.961, p - 0.01x, as well as significant first order interactions, neostigmine = CSD w F Ž1,20. s 15.185, p - 0.01x, neostigmine= days w F Ž8,160. s 59.821, p - 0.01x, CSD = time w F Ž8,160. s 6.586, p - 0.01x, and second order interaction Ždrug = CSD = days. w F Ž8,160. s 6.303,
Fig. 4. Means" S.E. of changes in colonic temperature. The neostigmine or saline was injected at time 0. The KCl or NaCl was applied on the cortex at time y5.
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p - 0.01x. Newman–Keuls post hoc test showed that the neostigmine group without CSD and the neostigmineq CSD were different from other groups at 10 and 15 min. Differences were demonstrated between the neostigmineq CSD group and other groups at 10 and 15 min. There were no differences in the baseline absolute values of all groups, as reported in Table 1. Examples of the changes in firing rate are shown in Fig. 2. Fig. 3 illustrates the T IBAT changes. Neostigmine injection caused a rise that peaked at 20 min in the rats without CSD. This increase was reduced by CSD. In the control rats, the NaCl or KCl applied upon the cortex did not cause any modification. Three-way ANOVA Ždrug = CSD = days, 2 = 2 = 9. with repeated measures on the last factor showed significant main effects of neostigmine w F Ž1,20. s 16.543, p - 0.01x, of CSD w F Ž1,20. s 5.533, p - 0.01x, and of days w F Ž8,160. s 4.778, p - 0.01x, and as well as significant first order interactions, neostigmine = CSD w F Ž1,20. s 4.654, p - 0.01x, and neostigmine= days w F Ž8,160. s 4.476, p - 0.01x. The second order interaction was not significant. Newman–Keuls post hoc test showed that the neostigmine group without CSD was different from other groups at 10 to 40 min. Differences were demonstrated between the neostigmineq CSD group and other groups at 15 and 20 min. Fig. 4 illustrates the Tc changes. Neostigmine injection caused a rise that peaks at 20 min. This increase was reduced by CSD. In the control rats, the NaCl or KCl applied upon the cortex did not cause any modification. Three-way ANOVA Ždrug = CSD = days, 2 = 2 = 9. with repeated measures on the last factor showed significant main effects of neostigmine w F Ž1,20. s 20.697, p - 0.01x, of CSD w F Ž1,20. s 5.983, p - 0.01x, of days w F Ž8,160. s 5.864, p - 0.01x, as well as significant first order interactions, neostigmine= CSD w F Ž1,20. s 5.368, p - 0.01x, and neostigmine= days w F Ž8,160. s 4.846, p - 0.01x. The second order interaction was not significant. Newman– Keuls post hoc test showed that the neostigmine group without CSD was different from other groups at 15 to 40 min. Differences were demonstrated between the neostigmine q CSD group and other groups at 15 and 20 min.
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we showed that the PVN is involved in the sympathetic activation induced by a cortical stimulation. Indeed, procaine injection into the PVN reduces the sympathetic and thermogenic changes due to stimulation of frontal cortex w20x. The present experiment is the first to show that a cholinergic hippocampal stimulation induces an increase in the firing rate of the paraventricular neurons. The modification of the firing rate precedes temperature changes. This suggests the hippocampus may control body temperature through the PVN. Furthermore, these findings indicate that the cerebral cortex is involved in these complex phenomena. In fact, CSD reduces the increase in the paraventricular activity, suggesting a cortical control on the hippocampus andror PVN. CSD does not modify the firing rate of the PVN in the control rats, and this confirms previous data showing a phasic influence of the cerebral cortex on subcortical structures w6,15x. In other words, CSD modifies the thermogenic response only during the hippocampal stimulation, suggesting that the cerebral cortex does not influence body temperature under basal conditions. Shibata et al. w25x indicate that in urethane anesthetized rats firing rate of warm-sensitive and cold sensitive neurons in preoptic arearanterior hypothalamus is decreased and increased, respectively, during invasion of a CSD wave into frontal cortex. Furthermore, CSD activates in non-anesthetized rats behavioral thermoregulation and metabolic heat production, increasing colonic temperature w26x. The difference of our results can be due to procedural factors ŽPVN vs. preoptic arearanterior hypothalamus, several vs. single CSD waves, etc... In conclusion, the neurons of the PVN are stimulated by the hippocampal injection of neostigmine and functional decortication induced by CSD reduces this stimulation.
Acknowledgements The Italian National Research Council.
References 4. Discussion The findings of the present experiment emphasize: Ž1. the role of the PVN in the thermogenic activation induced by hippocampal cholinergic stimulation; and Ž2. the effects of CSD on the neuronal activity of the PVN. Uemura et al. w31x have shown that a neostigmine injection in the hippocampus produces hyperglycemia by epinephrine secretion. This demonstrates that the hippocampus is involved in the control of metabolic variables. We demonstrated that hippocampal cholinergic stimulation by neostigmine injection increases the firing rate of sympathetic nerves to IBAT and the temperature of this tissue w18x. Furthermore,
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