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. 1998 Nov 1;512(Pt 3):883–892. doi: 10.1111/j.1469-7793.1998.883bd.x

Efferent projection from the preoptic area for the control of non-shivering thermogenesis in rats

Xiao-Ming Chen *, Takayoshi Hosono *, Tamae Yoda *, Yutaka Fukuda *, Kazuyuki Kanosue *
PMCID: PMC2231233  PMID: 9769429

Abstract

  1. To investigate the characteristics of efferent projections from the preoptic area for the control of non-shivering thermogenesis, we tested the effects of thermal or chemical stimulation, and transections of the preoptic area on the activity of interscapular brown adipose tissue in cold-acclimated and non-acclimated anaesthetized rats.

  2. Electrical stimulation of the ventromedial hypothalamic nucleus (VMH) elicited non-shivering thermogenesis in the brown adipose tissue (BAT); warming the preoptic area to 41.5 °C completely suppressed the thermogenic response.

  3. Injections of d,l-homocysteic acid (DLH; 0.5 mm, 0.3 μl) into the preoptic area also significantly attenuated BAT thermogenesis, whereas injections of control vehicle had no effect.

  4. Transections of the whole hypothalamus in the coronal plane at the level of the paraventricular nucleus induced rapid and large rises in BAT and rectal temperatures. This response was not blocked by pretreatment with indomethacin. The high rectal and BAT temperatures were sustained more than 1 h, till the end of the experiment. Bilateral knife cuts that included the medial forebrain bundle but not the paraventricular nuclei elicited similar rises in BAT and rectal temperatures. Medial knife cuts had no effect.

  5. These results suggest that warm-sensitive neurones in the preoptic area contribute a larger efferent signal for non-shivering thermogenesis than do cold-sensitive neurones, and that the preoptic area contributes a tonic inhibitory input to loci involved with non-shivering thermogenesis. This efferent inhibitory signal passes via lateral, but not medial, hypothalamic pathways.

Brown adipose tissue (BAT) is an important effector for non-shivering thermogenesis and is activated by the sympathetic nervous system (Flaim et al. 1976, 1977; Perkins et al. 1981; Saito et al. 1987) during cold exposure (Foster & Frydman, 1978) or during periods of increased energy intake (Flaim et al. 1976, 1977) in rodents. Non-shivering thermogenesis is influenced by various nuclei in the hypothalamus. The ventromedial nucleus (VMH) plays a key role, because electrical or chemical stimulation of the VMH in both anaesthetized and unanaesthetized rats activates BAT thermogenesis (Addae et al. 1986; Freeman & Wellman, 1987; Holt et al. 1987, 1988; Amir, 1990; Hugie et al. 1992), while local anaesthesia of the VMH suppresses the metabolic response to cooling of the preoptic area (Imai-Matsumura & Nakayama, 1987).

The preoptic area is a major central thermosensitive site for the regulation of body temperature. This nucleus contains both warm- and cold-sensitive neurones. It has long been believed that warm-sensitive neurones are important for the control of heat loss, while cold-sensitive neurones are important for the control of heat production (Bligh, 1973; Hori, 1991; Boulant, 1996). A recent study by Zhang et al. (1995) showed that warm-sensitive neurones in the preoptic area send efferent signals for shivering and skin vasodilatation. It is still an open question, however, whether efferent signals for non-shivering thermogenesis also originate from warm-sensitive neurones or from cold-sensitive neurones.

Our first aim was to examine this question. We tested the effect on non-shivering thermogenesis by injecting the excitatory amino acid d,l-homocysteic acid (DLH) into the preoptic area of anaesthetized rats. Since local preoptic cooling facilitates non-shivering thermogenesis (Imai-Matsumura et al. 1984; Imai-Matsumura & Nakayama, 1987), if DLH suppresses non-shivering thermogenesis the main contribution to its control would be inhibitory signals from warm-sensitive neurones. If, however, DLH enhanced non-shivering thermogenesis, the main contribution would be excitatory signals from cold-sensitive neurones.

Our second aim was to determine whether the efferent signals for non-shivering thermogenesis pass through the medial forebrain bundle or through the periventricular pathway (Conrad & Pfaff, 1976a, b), by making selective hypothalamic transections.

METHODS

Animals and surgery

Thirty adult male Wistar rats (300–500 g) were used in this study. They were reared with free access to food and water at a room temperature of 5°C and a 12 : 12 h on : off lighting schedule (08.00–20.00 h on) for at least 3 weeks prior to the experiment and were taken out to a room of neutral temperature (22°C) just before experiments. All the experiments were done in the period of 10.00–18.00 h. The experiment was approved by the Animal Care Committee of Osaka University Medical School.

Rats were anaesthetized with urethane (1.4 g kg−1, i.p.). Additional doses of urethane were given as needed to keep the depth of anaesthesia at or below the first plane of stage 3 (surgical anaesthesia) (Lumb & Jones, 1984). Each rat was mounted in a stereotaxic apparatus according to the co-ordinate system of Paxinos & Watson (1986). A small incision was made about 5 cm posterior to the scapulae. A thermocouple probe was inserted through the incision and placed under the BAT for measuring BAT temperature (Tbat). A small hole was drilled at 1.0 mm right from the mid-line and 0.0 mm posterior to the bregma to insert an electrode-thermocouple assembly (Kanosue et al. 1991) 9.5 mm below the skull surface. This assembly consisted of an insulated stainless steel tube (0.4 mm i.d., 0.6 mm o.d.) with a bared, sharp tip for thermal stimulation and a Cu-Co thermocouple glued inside for monitoring local hypothalamic temperature (Thy). Tissue around the electrode is not damaged if Thy is not raised above 42°C (Kanosue et al. 1994). For drug injections into the preoptic area, a guide cannula (0.5 mm o.d.) was implanted in the right brain at the same anterio-posterior and lateral location as the electrode- thermocouple assembly but to a depth of 9.0 mm. A stainless steel electrode (0.3 mm diameter) for monopolar stimulation was implanted into the right VMH (0.6 mm from mid-line, 2.6 mm posterior to bregma, and 9.5–10.3 mm below the skull surface). Various assemblies were fixed to the skull with dental cement.

Experimental procedures

After surgery each animal was put into a climatic chamber (30 cm × 40 cm × 80 cm). A thermocouple for measuring rectal temperature (Tre) was inserted 6 cm beyond the anal sphincter and fixed to the tail with surgical tape. Tre and Tbat were monitored continuously. For preoptic warming, radio frequency current (500 kHz; Lesion Generator RF-4; Radionics, Burlington, MA, USA) was passed between the tip of the electrode-thermocouple assembly and a subcutaneous needle electrode in the back. For electrical stimulation of the VMH electric current (0.075 mA or 0.2 mA; 33 Hz; 0.5 ms) was passed (SEN-3201, Nihon Kohden, Tokyo, Japan) for 2 min between the electrode used as cathode and another subcutaneous needle electrode in the back. Tre was maintained between 36.0 and 37.5°C by manipulating air temperature between 23 and 28°C.

Thermal and chemical stimulation study

To test the effects of thermal stimulation of the preoptic area, electrical stimulation of the VMH was confirmed to produce Tbat increase. When Tbat returned to a steady state, the preoptic area was warmed to keep Thy at 41.5°C and the same VMH stimulation was applied. Finally, after preoptic warming was stopped, VMH stimulation was applied again. Seven rats were used in this portion of the study.

Six rats were used to test the effects of d,l-homocysteic acid (DLH) injection into the preoptic area. An injection cannula (0.3 mm o.d.) for drug application was inserted into the guide cannula so that its tip protruded 0.5 mm beyond the tip of the guide cannula. A polyethylene tube connected the injection cannula to a 5 μl microsyringe (Hamilton, Reno, NV, USA). As in the experiment of thermal stimulation, electrical stimulation of the VMH was first confirmed to produce Tbat increase. Then, DLH (0.5 mm; pH 7.4; osmolality 290 mosmol (kg H2O)−1; 0.3 μl in phosphate-buffered saline) was injected into the preoptic area through the injection cannula. The 0.5 mm concentration was chosen because in preliminary experiments a 0.1 mm solution was found to have small, inconsistent effects. The solution was made just before each experiment, and pH and osmolality were adjusted by adding sodium hydoxide and sodium chloride. Two minutes after the DLH injection, VMH electrical stimulation was done again. The effect of vehicle control injection was also tested in six other rats. Procedures were the same as the DLH experiment except that phosphate-buffered saline (0.5 mm, pH 7.4; osmolality 290 mosmol (kg H2O)−1) was injected instead of DLH.

At the end of experiments, 0.3 μl of Pontamine Sky Blue in sodium acetate was injected through the injection cannula to mark the position of the cannula tip, and DC current (1 mA; 0.5 s) was passed through the VMH electrode to mark the position of the electrode tip.

Additionally, we tested the effect of preoptic area DLH injections on Tbat change by VMH stimulation in eight non-acclimated rats, which were reared in the same way as the cold-acclimated rats except that room temperature was kept at 22°C.

Knife-cut study

Eleven rats were used in this series of experiments. To monitor the effect of knife cuts on shivering, an electromyogram (EMG) was recorded from needle electrodes in the m. quadriceps femoris. The signal was amplified (gain 2000 from 10 Hz to 1 kHz). Small holes for inserting a microknife were opened bilaterally 1.5 mm (medial cut) or 3.5 mm (complete or lateral cut) from the mid-line and 1.3–1.8 mm posterior to bregma. The microknife consisted of a thin stainless steel tube containing a wire blade (Blatteis et al. 1982). First, the Tbat response was confirmed to be produced by electrical stimulation of the VMH. Then transection was made on the left side by lowering the microknife with the blade sheathed, extending the blade 1.5 mm (medial cut), 3.0 mm (lateral cut), or 4.0 mm (complete cut), and then lowering and raising the knife 4 mm. After the transection on the left side, transection on the right side was also conducted using the same procedure. In another three rats, indomethacin (15 mg kg−1 in sesame oil) was injected subcutaneously. Twenty minutes after the injection, complete transection was made as noted. Additionally, we tested the effect of complete cut in three non-acclimated rats.

At the end of each experiment, the anaesthetized rat was killed by perfusing first with saline and then 10 % formalin solution. The brain was removed and 40 μm brain sections were made. They were stained with Toluidine Blue, and the positions of the electrode- thermocouple assembly, cannula and electrode, and the extent of knife cut were verified.

Data are expressed as means ± s.e.m., and the significance of difference was evaluated with ANOVA for repeated measures.

RESULTS

In the present study, Tre was not regulated with a heating pad, because of the effects of induced changes in skin temperature on thermoregulatory responses. We dealt with the effects of anaesthetic level and other unknown factors by analysing only data obtained from rats of normothermic Tre, that is, 36.0–37.5°C.

Thermal and chemical stimulations

In seven cold-acclimated rats used for the experiments of preoptic thermal stimulation, the tip of the thermode was located adjacent to the base of the brain in the medial and lateral preoptic area (Fig. 1A), and the tip of the electrode was located in, and slightly lateral to, the VMH (Fig. 1B). Electrical stimulation of the VMH produced a rise in Tbat, while warming the preoptic area suppressed this rise in Tbat. Figure 2 shows an example of this effect of preoptic warming. In the initial condition (without preoptic warming) electrical stimulation of the VMH produced a 1.0°C rise in Tbat which peaked 4 min after cessation of the electrical stimulation. The rise in Tbat was followed by a slight rise in Tre. The effect of the rise in Tbat elicited with the same electrical stimulation was completely abolished by warming the preoptic area to 41.5°C. It reappeared at nearly full strength when VMH stimulation was reapplied, after the preoptic warming was stopped. The inhibitory effect of the rise in Tbat by preoptic warming to 41.5°C was obtained in all seven rats (Fig. 3). There was no significant difference between the Tbat response before and after preoptic warming (Fig. 3).

Figure 1. Locations of the tips of thermode assembly (A) and locations of electrode tips (B).

Figure 1

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The filled circles indicate the locations of the thermode tip (A) and the electrode tip (B). AC, anterior commissure; OX, optic chiasm; ic, internal capsule; f, fornix; mfb, medial forebrain bundle; ml, medial lemniscus; mt, mammillothalamic tract; opt, optic tract; DM, dorsomedial hypothalamic nuclei; 3V, 3rd ventricle; VMH, ventromedial hypothalamic nuclei; TS, triangular septal nuclei; MPO, medial preoptic nuclei.

Figure 2. Changes in brown adipose tissue thermogenic response following electrical stimulation of the ventromedial hypothalamic nucleus before, during and after warming of the preoptic area.

Figure 2

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A, temperature of interscapular brown adipose tissue (Tbat), rectal temperature (Tre), and hypothalamic temperature (Thy); the filled bars indicate electrical stimulation (ES) of the VMH: 0.075 mA, 33 Hz, 0.5 ms. B, the filled circle indicates the location of the thermode tip (left), and the electrode tip (right). Abbreviations as in Fig. 1.

Figure 3. Increases in brown adipose tissue temperature in response to VMH electric stimulation before, during and after preoptic (PO) warming to 41.5 °C.

Figure 3

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Electrical stimulation (ES) of the VMH (0.075 mA, 33 Hz, 0.5 ms) was applied for 2 min before (•), during (▪), and after (○) preoptic warming. Values are means and vertical bars are ± s.e.m. (n = 7).

The effect of DLH injection was tested in six cold-acclimated rats, and control injection of phosphate-buffered saline (PBS) was tested in six separate cold-acclimated rats. All injections were made in the medial and lateral preoptic areas (Fig. 4A). The tips of the electrodes were distributed throughout the VMH and in adjacent areas (Fig. 4B). In two cases where the electrode was in the dorsomedial hypothalamus, electrical stimulation produced changes in Tbat identical to those following VMH stimulation. The data from these animals are included in the following analysis.

Figure 4. Locations of cannula tips for drug microinjection (A) and electrode tips for electrical stimulation (B).

Figure 4

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The filled circles indicate the locations of cannula and electrode tips in the DLH-injected group and the open triangles indicate those in the control group. Abbreviations as in Fig. 1.

Injection of 0.5 mm DLH suppressed, but did not abolish, the Tbat rise following VMH stimulation (Fig. 5A). A rise in Tbat solely in response to DLH was never observed. Figure 6A shows the changes in Tbat after VMH stimulation alone or after injection of DLH into the preoptic area. The initial peak increase in Tbat was 0.41 ± 0.04°C. This response was significantly suppressed after DLH injection. Injection of control vehicle into the preoptic area had no effect on the rise in Tbat in response to VMH stimulation (Figs 5B and 6B). There was no significant difference in the initial Tbat responses to VMH stimulation between the DLH and PBS groups. Mean Tre just before VMH stimulation was 36.6 ± 0.2°C, which also did not differ between DLH and PBS groups.

Figure 5. Effects of injection of DLH (A) or control vehicle (B) into the preoptic area on the rise in Tbat and Tre by electrical stimulation of the VMH in separate cold-acclimated rats.

Figure 5

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A and B, the open bars indicate application of DLH (0.5 mm, 0.3 μl) or control vehicle (phosphate-buffered saline, PBS) and the filled bars indicate electrical stimulation (ES, 0.075 mA, 33 Hz, 0.5 ms). C, the filled circles indicate the location of DLH injection and electrical stimulation in the DLH-injected rat and the open circles indicate the location of PBS injection and electrical stimulation in the control rat. Abbreviations as in previous figures.

Figure 6. Changes in brown adipose tissue temperature in response to VMH electrical stimulation before and after the injection of DLH (A) or control vehicle (B) into the preoptic area.

Figure 6

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Values are means and vertical bars are ± s.e.m. (n = 6). Asterisks indicate significant difference from the value before drug injection: *P < 0.05; **P < 0.01.

In eight non-acclimated rats VMH stimulation alone also produced an increase in Tbat, the peak of which was 0.43 ± 0.07°C. DLH injection into the preoptic area significantly suppressed this response.

Knife cuts

In two cold-acclimated rats, the knife cut totally transected the hypothalamus at the level of the paraventricular nucleus (complete cut, Fig. 7A). In these rats the knife cut resulted in a rise in Tbat and Tre. Figure 8 shows an example. In this case the first unilateral knife cut on the right side produced a slight increase in Tbat. This was certainly activation of the BAT, because Tbat became higher than Tre after the knife cut. The subsequent knife cut on the contralateral side produced a steeper rise in Tbat and Tre. As shown in the figure, Tbat reached 40.0°C in 37 min, and Tre reached 39.7°C. These high temperatures were maintained for 2 h till the experiment was halted. The EMG did not show any activity either before the knife cut or after the cut when BAT was strongly activated.

Figure 7. Extent of knife cuts.

Figure 7

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The extent of transection in complete (A, n = 2), lateral (B, n = 3) and medial cuts (C, n = 3). LV, lateral ventricle; sm, stria medullaris thalami; PV, paraventricular nuclei; LH, lateral hypothalamic area. Other abbreviations as in previous figures. Different forms of hatching and shading indicate the extent of transection in different animals.

Figure 8. Effects of complete cuts on brown adipose tissue thermogenesis.

Figure 8

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L and R indicate knife cuts applied on the left and right sides, respectively. In the inset, the hatched areas indicate the extent of the knife cut. Abbreviations as in previous figures.

In three cold-acclimated rats, the knife cut was made bilaterally through the medial forebrain bundle and its adjacent region (lateral cut, Fig. 7B). In this preparation the paraventricular nucleus was left intact. In another three cold-acclimated rats, a 0.5 mm strip on both sides along the third ventricle, which included the paraventricular nucleus, was transected (medial cut, Fig. 7C).

Lateral and medial cuts have contrasting effects on BAT thermogenesis. Figure 9 shows an example of the lateral cut. First, a lateral knife cut was made on the left side. This unilateral cut did not have notable effect on the change in Tbat. However, as soon as a cut was made on the other side, Tbat and, with a delay, Tre also began to increase. Increases in Tbat and Tre were also observed in the other two rats that received lateral cuts.

Figure 9. Effects of lateral cuts on brown adipose tissue thermogenesis.

Figure 9

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L and R indicate knife cuts applied on the left and right sides, respectively. In the inset, the hatched areas indicate the extent of the knife cut. Abbreviations as in previous figures.

In contrast to the lateral cut, a medial cut had no notable effect on Tbat. Figure 10A shows an example. Bilateral medial cuts did not influence Tbat. In this case the Tbat response to VMH stimulation was 1.0°C, which is far greater than the usual response. Thus, the lack of increase in Tbat after a medial cut was not due to a general lack of responsiveness by BAT. Warming of the preoptic area still had an inhibitory effect on the rise in Tbat in response to VMH stimulation even after the knife cut (Fig. 10B).

Figure 10. Lack of effect of medial cuts on brown adipose tissue thermogenesis.

Figure 10

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A, L and R indicate knife cuts applied on the left and right sides, respectively. B, effect of preoptic warming was tested after the knife cut. C, the filled circle indicates the location of the thermode tip (left), and the electrode tip (right). The hatched areas indicate the extent of the knife cut (middle). Abbreviations and symbols as in previous figures.

Indomethacin application had no effect on the increase in Tbat and Tre in another three rats, which received complete cuts (Fig. 11).

Figure 11. Lack of effect of indomethacin injection (s.c. 15 mg kg−1) on brown adipose tissue thermogenesis induced by complete knife cuts.

Figure 11

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L and R indicate knife cuts applied on the left and right sides, respectively. In the left inset, the hatched areas indicate the extent of the knife cut. In the right inset, the filled circle indicates the location of the electrode tip. Abbreviations as in previous figures.

Complete cuts were also applied in three non-acclimated rats. A sustained rise in Tbat and Tre followed the knife cuts, as observed in cold-acclimated rats.

DISCUSSION

The present study confirmed the excitatory effect of electrical stimulation of the VMH on BAT thermogenesis and added the finding that the BAT thermogenic response is inhibited by preoptic warming. The importance of the VMH to non-shivering thermogenesis initiated by preoptic cooling has already been reported (Imai-Matsumura et al. 1984; Imai-Matsumura & Nakayama, 1987). Efferent signals from the preoptic area clearly convey information to the areas that initiate the BAT thermogenic response. Is this information in the form of excitatory signals from cold-sensitive neurones, or inhibitory signals from warm-sensitive neurones? Local application of DLH into the preoptic area, which activates cell bodies but does not activate neural fibre passing through the area, suppressed BAT thermogenesis by VMH stimulation just as preoptic warming did. Additionally, DLH injection by itself never produced a rise in Tbat. Therefore, we propose that preoptic efferent signals for BAT thermogenesis are mainly via inhibitory outputs from warm-sensitive neurones.

This does not mean that cold-sensitive neurones play no role in the control of non-shivering thermogenesis. While preoptic warming completely suppressed the BAT thermogenesis by VMH stimulation (Fig. 3), application of DLH did so only partially (Fig. 6A). This might be because warming affected larger areas than did the drug application. Warming activated warm-sensitive neurones and inhibited cold-sensitive neurones, which would work co-operatively for suppressing BAT thermogenesis. However, in the case of DLH application warm- and cold-sensitive neurones are both activated. Hence warm-sensitive neurones suppress and cold-sensitive neurones activate BAT thermogenesis. The partial suppression of BAT thermogenesis by DLH application thus may mean that cold-sensitive neurones contribute to some extent to the control of BAT thermogenesis. However, the fact that DLH application suppressed BAT thermogenesis means that the contribution of cold-sensitive neurones is, at most, minor, compared with that of warm-sensitive neurones.

If non-shivering thermogenesis is under the control of inhibitory signals from the preoptic area, it should be activated when these signals are blocked. Indeed, BAT thermogenesis was strongly activated by a coronal transection just caudal to the preoptic area. The BAT thermogenesis was not secondary to the rise in Tre, because the rise in Tbat preceded the rise in Tre, and because shivering did not occur. This response corresponds well to the finding by Brück & Wünnenberg (1970) that lesion of the preoptic area produced a rise in oxygen consumption and Tbat in unanaesthetized animals. However, the observations that BAT thermogenesis is activated by the transection and that warm-sensitive neurones are mainly responsible for the response do not necessarily mean that the warm-sensitive neurones are inhibitory. They may be excitatory neurones which activate inhibitory systems in the lower brain stem (Shibata et al. 1987, 1996).

It has recently been postulated that fever is mediated by prostaglandin E2 (PGE2), which is produced in the blood vessels of the preoptic area (Cao et al. 1995). The destruction of these blood vessels by knife cuts could have induced the production of PGE2 and thus caused a fever. If this were the case, then the rise in Tbat and Tre would not necessarily be due to the blockade of efferent preoptic inhibitory signals. However, pretreatment with the antipyretic indomethacin did not block the rise in Tbat and Tre. Further, if the rise had been due to the non-specific destruction of blood vessels, it should have been elicited by any knife cut. However, while the lateral cut was as effective as the complete cut in producing the rise in Tbat and Tre, the medial cut had no effect at all. The rise in Tbat and Tre was not, therefore, fever, and was most probably produced by the transection of the efferent fibres which convey tonic, inhibitory inputs to the areas controlling BAT thermogenesis under normothermic conditions.

The effectiveness of the lateral cut indicates that efferent signals from the preoptic area pass through this area. The most likely pathway is via the medial forebrain bundle, which contains efferent fibres from the preoptic area (Conrad & Pfaff, 1976a, b). The ineffectiveness of medial cuts indicates that the periventricular pathway, another group of efferent fibres from the preoptic area, contributes little to the control of non-shivering thermogenesis.

Our findings are in substantial agreement with the results of Imai-Matsumura & Nakayama (1987). In their study, a thermode was placed slightly anterior to the preoptic area and was used to stimulate BAT thermogenesis by decreasing Thy. The effects of bilateral injections of lidocaine (lignocaine), applied to the anterior, ventromedial and lateral hypothalamus, on cooling-induced BAT thermogenesis were then studied. In the anterior hypothalamus, lidocaine injections produced an increase in BAT thermogenesis. We feel that this result was due to the removal of tonic inhibition from the warm-sensitive neurones of the preoptic area or anterior hypothalamus. The injection cannulae were near the border of the preoptic area and anterior hypothalamus, and these injections would have anaesthetized many temperature-sensitive neurones. In agreement with our work and many earlier studies, lidocaine injections in the VMH decreased on-going BAT thermogenesis.

The results of lidocaine injections into the lateral hypothalamus are difficult to reconcile with our findings. Imai-Matsumura & Nakayama (1987) observed no change in on-going BAT thermogenesis following such injections, while we found lateral pathways to be necessary for transmission of signals from the preoptic area. The above authors concluded that medially running fibres carried information from the preoptic area, while our results implicate lateral fibres in the medial forebrain bundle. Since our coronal knife cuts (lateral to the paraventricular nuclei) were anterior to the lidocaine injections (lateral to the VMH), it is conceivable that the descending fibres travel medially just after passing the paraventricular nuclei. More likely, the lateral fibres course in a straight line, and future experimentation will be required to clarify the discrepancy.

In a recent study, we showed that efferent signals from warm-sensitive neurones contribute more than those from cold-sensitive neurones not only to skin vasodilatation but also to shivering (Zhang et al. 1995). It is also suggested that thermally induced salivary secretion in rats is controlled by signals from warm-sensitive neurones, because warming and electrical stimulation both produce salivary secretion (Kanosue et al. 1990). In rats, therefore, warm-sensitive neurones send efferent signals for all autonomic thermoregulatory effectors; that is, salivation, vasomotion, shivering and non-shivering thermogenesis. They send excitatory signals for heat loss (vasomotion and salivation), and inhibitory signals for heat production (shivering and non-shivering thermogenesis). Additionally, different warm-sensitive neurones mediate the responses of salivary secretion, vasomotion and shivering (Kanosue et al. 1998). We do not yet know whether neurones for non-shivering thermogenesis are also different from those for other effectors. If so, the network for thermoregulation in the preoptic area seems much simpler than previously thought, because there is one dominant type of thermosensitive neurone (the warm-sensitive neurone) and there are separate channels for different effector controls (Kanosue et al. 1998).

Recently, Thornhill's group showed that electrical stimulation of the medial preoptic area produced a rise in Tbat, while stimulation of the lateral preoptic area produced increased shivering activity (Thornhill & Halvorson, 1994; Thornhill et al. 1994). The injection sites in the present study include both medial and lateral prepotic areas, but we did not observe any excitation of BAT following DLH injection. The difference between the studies might be due to the fact that electrical stimulation activates both cell bodies and passing fibres, while DLH activates only cell bodies. In a former study, however, we applied electrical stimulation to the preoptic area but never observed facilitation of shivering activity (Zhang et al. 1995).

The two main findings of the present study were obtained in both cold-acclimated rats and non-acclimated rats: (1) DLH injection into the preoptic area suppresses BAT thermogenesis, and (2) coronal transection applied caudal to the preoptic area produces an increase in BAT thermogenesis. Therefore, the preoptic mechanism controlling BAT thermogenesis is similar for normal and cold-acclimated animals.

In conclusion, warm-sensitive neurones in the preoptic area contribute a larger efferent signal for non-shivering thermogenesis than do cold-sensitive neurones. The efferent signal passes through the lateral part of the hypothalamus.

Acknowledgments

We thank Professor L. I. Crawshaw for his critical reading of and comments on this manuscript. This study was supported in part by a Grant-in-Aids for Scientific Research from the Ministry of Education, Science and Culture of Japan (Grants No. 9470016 and No. 04454143).

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