Functional Decortication Lowers Ventromedial Hypothalamic Activation Induced by Hippocampal Neostigmine Injection

M. Monda, A. Viggiano and V. De Luca

Dipartimento di Fisiologia Umana e Funzioni Biologiche Integrate ‘Filippo Bottazzi’, Seconda Università di Napoli, Via Costantinopoli 16, 80138 Napoli, Italia


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
This experiment tested the effects of cortical spreading depression (CSD) on the ventromedial hypothalamic activity and on the related thermogenesis, both stimulated by an injection of neostigmine in the hippocampus. The firing rate of the neurons of the ventromedial hypothalamus, and the temperature of the interscapular brown adipose tissue and of the colon (TIBAT 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 (5 x 10–7 mol) in the first and second groups or of saline in the third and fourth groups. The hippocampal injection was preceded by CSD in the first and third groups, while CSD was not induced in the second and fourth groups. The same procedure was carried out in the other four groups of six rats each and oxygen consumption was monitored. The results show an increase in the firing rate, TIBAT, TC and oxygen consumption after the neostigmine injection. CSD significantly reduces these enhancements. The findings demonstrate that: (i) the activation of ventromedial hypothalamic neurons are involved in the thermogenic changes due to the effects of a neostigmine injection into the hippocampus; and (ii) integrity of cerebral cortex is required for this activation of thermogenesis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Both neocortex and paleocortex are involved in body thermoregulation. Indeed, an electrical stimulation of the prefrontal cortex causes an increase in body temperature (De Luca et al., 1989Go), while a functional decortication induced by cortical spreading depression (CSD) reduced the hyperthermia due to pyrogens (Komaromi et al., 1994Go; Monda et al., 1994aGo,bGo). On the other hand, chemical stimulation of paleocortex produces an increase in heat production. Neostigmine injection into the hippocampus activates thermogenesis through an increase in the sympathetic activity (Iguchi et al., 1992Go) and in the conversion of tiroxina in triiodotironine (Monda et al., 1996Go). A recent report (Monda et al., 1999bGo) demonstrates that the neocortex influences the paraventricular activation induced by hippocampal neostigmine injection.

The ventromedial hypothalamus (VMH) influences non-shivering thermogenesis through a control on the activity of the interscapular brown adipose tissue (IBAT), the principal effector of non-shivering thermogenesis in the rodents (Cannon et al., 1998Go). Indeed, electrical stimulation of the VMH increases IBAT temperature (Thornhill and Halvorson, 1994Go), while a lesion of the VMH reduces the thermogenic activation induced by various stimuli (Monda et al., 1997Go; Dube et al., 1999Go).

The aim of this experiment was to evaluate: (i) the effects of an hippocampal injection of neostigmine on the firing rate of the neurons of the VMH and on the related thermogenesis; (ii) the modifications of these effects due to a functional decortication induced by CSD, in order to test the importance of integrity of the cortex in these phenomena.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Animals

We used male inbred Sprague–Dawley rats (n = 48), 3 months old and weighing 270–320 g. They were housed in pairs at controlled temperature (22 ± 1°C) and humidity (70%) with a 12:12 h light–dark cycle from 07.00 to 19.00 h. The experiments were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Apparatus

Extracellular single unit activities were recorded using a tungsten electrode (Clark Electromedical Instruments) with tip diameter of 10 µm and impedance of 12 M{Omega}. The tip of the microelectrode reached the VMH stereotaxically at the following coordinates: 1.0 mm lateral to the midline, 0.2 mm anterior to the bregma, 9.9 mm below the cranial theca (Pellegrino et al., 1979Go). The electrical pulses were amplified by a condenser-coupled amplifier and were filtered by band-path filters (NeuroLog System, Digitimer). The raw pulses were displayed on an oscilloscope (Tektronix) and sent to a window discriminator. Square waves from the discriminator were sent to an analog–digital converter (DAS System, Keithley) and stored on a personal 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 pen recorder (Vitatron). The threshold level of the event detector was fixed during the experiment at 3–4 times above background noise. Electrocortico-graphical activity and slow potential changes were recorded with two wick calomel cell electrodes applied to the parietal cortex. The reference electrode was placed on the nasal bone. The three electrodes were connected to a polygraph (Dynograph, Beckman). Thermocouples (Ellab) were used to monitor colonic and IBAT temperatures (TC and TIBAT) and the values were stored on a chart recorder. The cannula for drug injections was 0.4 mm longer than the guide cannula.

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 2.5 l respiratory chamber and a CO2 trap (soda lime), by a peristaltic pump at a rate of 2 l/min. 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 for temperature and pressure and was expressed as ml min–1 (kg body wt)–0.75 (Kleiber, 1975Go).

Drug

Neostigmine was purchased from Sigma Chemical (St Louis, MO) and dissolved in a pyrogen-free saline solution (5 x 10–7 mol of neostigmine in 1 µl of saline).

Surgery

All animals were anesthetized with i.p. pentobarbital sodium (50 mg/kg body 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, 2.7 mm below the cranial theca (Pellegrino et al., 1979Go). 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 (5 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 (internal diameter 1.5 mm; volume 30 µl) 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.

Procedure

After recovery, six animals (first group) were anesthetized with i.p. urethane (1.2 g/kg body wt) and mounted in a stereotaxic instrument (Stoelting). The level of anesthesia was kept constant as evaluated by skeletal muscle relaxation, and eye and palpebral responses to stimuli. The content of the wells was removed and the parietal cortex was previously exposed before the experiment. TC was measured by inserting the thermocouple into the colon at 4 cm from the anus, while TIBAT was monitored by inserting the thermocouple in the left side of the estimated IBAT region. Firing rate, TIBAT and TC were recorded for 40 min before and for 40 min after injection of neostigmine (1 µl of 0.5 M solution) into the hippocampus. An application (Bures and Buresova, 1972Go) of KCl (10 µl of 2.0 M solution) to the frontal cerebral cortex (which causes several waves of CSD) preceded the neostigmine injection by 5 min. In addition, electrocorticographical activity and slow potential changes of the parietal cortex were recorded at the same time. The same variables were recorded in another six rats (second group), but the KCl solution applied to the cortex was substituted (Bures and Buresova, 1972Go) with 2.0 M NaCl solution (which has no effect on cortical function). The same procedure used with the first group was carried out with another six animals (third group), except that saline was injected into the hippocampus. The rats of the fourth group were used as controls: 2.0 M NaCl solution was applied to 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 other words, a servo system for controlling the animal's temperature was not used.

The same procedure of CSD induction and hippocampal injection was carried out in other four groups of urethane-anesthetized rats and O2 consumption was measured (see Table 1Go).


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Table 1 Experimental procedure carried out in each group of rats (n = 6)
 
Histology

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 in the first twenty-four animals. A stain (bromophenol blue) was injected in the hippocampus of all animals at the same volume used for drug administration to identify the location of the cannula. The rats were then injected with an overdose of pentobarbital (150 mg/kg body wt) and were perfused with 0.9% NaCl followed by 10% (v/v) formalin solution. The brain was removed and stored in formalin solution. After a few days, 50 µm coronal sections of the fixed brain were cut and stained with neutral red.

Statistical Analysis

The values are presented as means ± SE. Statistical analysis was performed using analysis of variance. Multiple comparisons were made using the Newman–Keuls post hoc test.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
The percentage changes in firing rate of the neurons of the VMH are shown in Figure 1Go. Neostigmine injection caused a rise that peaks at 10 min. This increase was reduced by CSD. The NaCl or KCl applied upon the cortex did not cause any modification in the control rats. Three-way ANOVA (drug x CSD x time, 2 x 2 x 9) with repeated measures on the last factor showed significant main effects of neostigmine [F(1,20) = 287.714, P < 0.01], of CSD [F(1,20) = 62.482, P < 0.01], of time [F(8,160) = 61.112, P < 0.01], as well as significant first-order interactions, neostigmine x CSD [F(1,20) = 63.038, P < 0.01], neostigmine x time [F(8,160) = 59.386, P < 0.01], CSD x time [F(8,160) = 11.823, P < 0.01], and second-order interaction (drug x CSD x time) [F(8,160) = 11.040, P < 0.01]. A Newman–Keuls post hoc test showed that the neostigmine group without CSD was different from other groups at 5–20 min. Differences were demonstrated between the neo-stigmine + CSD group and other groups at 5–15 min. There were no differences in the baseline absolute values of all groups, as reported in Table 2Go. Figure 2Go shows examples of the actual changes in firing rate.



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Figure 1. Means ± SE of changes in the firing rate of the neurons of the ventromedial hypothalamus. The neostigmine or saline was injected at time 0. The KCl or NaCl was applied to the cortex at time –5.

 

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Table 2 Absolute values ± SE of firing rate (spikes/5 s) at time of neostigmine injection
 


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Figure 2. Firing rate changes in a non-decorticated (A) or decorticated (B) rat receiving neostigmine and in a non-decorticated (C) or decorticated (D) rat receiving saline. The arrow indicates the hippocampal injection of neostigmine or saline.

 
Figure 3Go illustrates the TIBAT changes. Neostigmine injection caused a rise that peaked at 15 min in the rats without CSD. This increase was reduced by CSD. In the control rats, the NaCl or KCl applied to the cortex did not cause any modification. Three-way ANOVA (drug x CSD x time, 2 x 2 x 9) with repeated measures on the last factor showed significant main effects of neostigmine [F(1,20) = 7.586, P < 0.05 ], of CSD [F(1,20) = 6.303, P < 0.05], and of time [F(8,160) = 4.434, P < 0.01], as well as significant first-order interactions, neostigmine x CSD [F(1,20) = 4.406, P < 0.05], and neostigmine x time [F(8,160) = 3.673, P < 0.01]. A Newman–Keuls post hoc test showed that the neostigmine group without CSD was different from other groups at 10–40 min. Differences were demonstrated between the neostigmine + CSD group and other groups at 10 and 15 min.



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Figure 3. Means ± SE of changes in interscapular brown adipose tissue temperature. The neostigmine or saline was injected at time 0. The KCl or NaCl was applied to the cortex at time –5.

 
Figure 4Go shows 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 (drug x CSD x time, 2 x 2 x 9) with repeated measures on the last factor showed significant main effects of neostigmine [F(1,20) = 15.635, P < 0.01], of time [F(8,160) = 4.644, P < 0.01 ], as well as significant first-order interactions, neostigmine x time [F(8,160) = 3.651, P < 0.01]. A Newman–Keuls post hoc test showed that the neostigmine group without CSD was different from other groups at 15–30 min. Differences were demonstrated between the neostigmine + CSD group and other groups at 15 and 20 min.



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Figure 4. Means ± SE of changes in colonic temperature. The neostigmine or saline was injected at time 0. The KCl or NaCl was applied to the cortex at time –5.

 
Figure 5Go illustrates the changes in O2 consumption. Neostigmine injection caused an increase that was reduced by CSD. The NaCl or KCl applied upon the cortex did not cause any modification in the control rats. Three-way ANOVA (drug x CSD x time, 2 x 2 x 2) with repeated measures on the last factor showed significant main effects of neostigmine [F(1,20) = 45.738, P < 0.01], of time [F(1,20) = 79.101, P < 0.01], as well as significant first-order interactions, neostigmine x time [F(1,20) = 84.426, P < 0.01]. A Newman–Keuls post hoc test showed that the neostigmine group without CSD was different from other groups. Differences were demonstrated between the neostigmine + CSD group and other groups.



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Figure 5. Means ± SE of changes in oxygen consumption before (basal) and after neostigmine or saline injections.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
To our knowledge, these findings are the first to demonstrate that: (i) hippocampal cholinergic stimulation induces an increase in unit activity of ventromedial hypothalamic neurons; and (ii) CSD is able to reduce this increase.

Although the involvement of the VMH in responses to cholinergic stimulation of the hippocampus has been hypothesized by other authors, the present findings support this hypothesis with direct proof. Indeed, an administration of atropine (Iguchi et al., 1991Go) or muscimol (Ozawa et al., 1993Go) into the VMH sup-presses hyperglycemia induced by hippocampal administration of neostigmine, suggesting that both cholinergic and GABAergic neurons have inhibitory effects on the sympathetic activation (Iguchi et al., 1992Go) that induces hyperglycemia. The above-mentioned researches have been carried out with the injection of a drug, but these experimental procedures did not present direct information about the changes in concentration of neurotransmitters, like microdialysis, for example, which could make it. Our experiment presents a direct electrophysiological evidence of neuronal activation. The enhanced firing rate precedes the rise in TIBAT and TC, indicating that an activation of ventromedial hypothalamic neurons induces an increase in IBAT activity. The rise in body temperature and O2 consumption corroborates other evidence showing that experimental manipulations of the VMH induce a modification of the sympathetic activity of nerves to IBAT (Monda et al., 1997Go) and of metabolic variables (Vilberg and Keesey, 1990Go; Monda et al., 1993Go), but this experiment is the first to demonstrate directly an involvement of ventromedial hypothalamic neurons in the thermogenic and metabolic changes induced by cholinergic stimulation of the hippocampus.

CSD reduces the increase in VMH activity, indicating cortical control on the hippocampus and/or VMH. The effects of CSD on the activity of other hypothalamic areas are already known. Functional decortication blocks the hyperthermic response in rats with a lesion of lateral hypothalamus (De Luca et al., 1987), while the posterior hypothalamic activity is reduced by CSD in rats with intracerebroventricular injection of a pyrogen, e.g. PGE1 (Monda et al., 1994aGo,bGo). However, the present experiment emphasizes the importance of neocortex in the response of VMH neurons to stimulus induced in the paleocortex, in that the cholinergic hippocampal influence on VMH is not exerted completely unless the neocortex is intact. Evidence showing connections from the prefrontal cortex to the limbic system and the hypothalamic areas provides anatomical support to the thermogenic responses tested in this experiment (Gaykema et al., 1991Go; Prince 1999Go).

This involvement of the neocortex in a paleocortical– hypothalamic interaction assumes strong relevance, considering that VMH controls not only the sympathetic activity and TIBAT, but also food intake (Bray, 1984Go; Sakata et al., 1997Go), which is strictly related to sympathetic tone (Bray, 1991Go; Monda et al., 1999aGo). This indicates that the neocortex controls a mechanism that could be involved in the regulation of body weight. Indeed, body weight is the result of food intake and energy expenditure (Keesey and Powley, 1986Go). Although food intake has not been measured, this experiment underlines the role that the neocortex plays in the increase of energy expenditure induced by hippocampal stimulation. O2 consumption and body temperature are expressions of energy expenditure, so that a modification of these variables could induce a change in energy intake and/or body weight.

CSD alone does not modify the firing rate of the VMH in the control rats, and this confirms previous data showing a phasic influence of the cerebral cortex on subcortical structures (De Luca et al., 1987aGo,bGo; Monda and Pittman, 1993Go). In other words, CSD modifies the thermogenic and metabolic response only during hippocampal stimulation, suggesting that the cerebral cortex does not influence body temperature and O2 consumption under basal conditions.

The rise in TIBAT that precedes the increase in TC indicates an activation of heat production unrelated to shivering (Himmis-Hagen 1984Go). Thus, hippocampal cholinergic stimulation induces hyperthermia through an activation of IBAT. This indicates that the VMH controls IBAT activity in this experimental model of hippocampal stimulation, as has been reported for the paraventricular hypothalamic nucleus (Monda et al., 1999bGo).

In conclusion, the neuronal activity of the VMH is stimulated by the hippocampal injection of neostigmine, and functional decortication of the neocortex causes a reduction of this stimulation.

Although our attention has been focused exclusively on the VMH, we do not exclude the possibility that other hypothalamic areas could be involved in the control of these thermogenic responses, as result of a fine interplay between different hypothalamic and brain stem cells. In perspective, the role of the other hypothalamic nuclei could be the object of further experiments.


    Notes
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
This research has been supported by the Italian National Research Council.

Address correspondence to Dr Marcellino Monda, MD, Dipartimento di Fisiologia Umana, Via Costantinopoli 16, 80138 Napoli, Italy. Email: marcellino.monda{at}unina2.it.


    References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
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