Neuroscience Letters 653 (2017) 71–77
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Involvement of serotonin in the ventral tegmental area in thermoregulation of freely moving rats Takayuki Ishiwata a,b,∗ , Hiroshi Hasegawa c , Benjamin N. Greenwood b a b c
Graduate School of Community & Human Services, Rikkyo University, 1-2-26 Kitano, Niiza, Saitama 352-8558, Japan Department of Psychology, College of Liberal Arts & Sciences, University of Colorado Denver, Denver, CO, USA Graduate School of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8521, Japan
h i g h l i g h t s • • • •
Thermoregulatory responses and neurotransmitters were measured in conscious rats. Heat exposure is associated with 5-HT release in the VTA. Increasing extracellular 5-HT in the VTA activated heat loss mechanisms. 5-HT in the VTA may mediate the heat loss component of thermoregulation.
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Article history: Received 10 April 2017 Received in revised form 11 May 2017 Accepted 16 May 2017 Available online 17 May 2017 Keywords: Thermoregulation Serotonin Ventral tegmental area Microdialysis Freely moving rat
a b s t r a c t We have recently reported that the serotonin (5-HT) projections from the midbrain’s raphe nuclei that contains 5-HT cell bodies may play a role both in heat production and in heat loss. The purpose of the present study was to clarify the involvement of 5-HT in the ventral tegmental area (VTA), where 5-HT is suggested to participate in thermoregulation, using the combined methods of telemetry, microdialysis, and high performance liquid chromatography, with a special emphasis on regulation of the body temperature (Tb ) in freely moving rats. First, we measured changes in Tb , tail skin temperature (Ttail ; an index of heat loss), heart rate (HR; an index of heat production), locomotor activity (Act), and levels of extracellular monoamines in the VTA during cold (5 ◦ C) or heat (35 ◦ C) exposure. Subsequently, we perfused citalopram (5-HT re-uptake inhibitor) into the VTA and measured the thermoregulatory parameters and monoamines release. Although Tb , Ttail , and HR changed during both exposures, signiﬁcant changes in extracellular level of 5-HT (138.7 ± 12.7% baseline, p < 0.01), but not dopamine (DA) or noradrenaline (NA) were noted in the VTA only during heat exposure. In addition, perfusion of citalopram into the VTA increased extracellular 5-HT levels (221.0 ± 52.2% baseline, p < 0.01), but not DA or NA, while Tb decreased from 37.4 ± 0.1 ◦ C to 36.8 ± 0.2 ◦ C (p < 0.001), Ttail increased from 26.3 ± 0.4 ◦ C to 28.4 ± 0.4 ◦ C (p < 0.001), and HR and Act remained unchanged. Our results suggest that the VTA is a key area for thermoregulation, and 5-HT, but not DA or NA, modulates the heat loss system through action in the VTA. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The central serotonin (5-HT) pathways are involved in many physiological functions such as sleep, sex, locomotion, food intake,
Abbreviations: 5-HT, serotonin; Act, locomotor activity; DA, dopamine; HPLC, high performance liquid chromatography; HR, heart rate; NA, noradrenaline; PO/AH, preoptic area and anterior hypothalamus; Tb , body temperature; Ttail , tail skin temperature; VTA, ventral tegmental area. ∗ Corresponding author at: Rikkyo University, 1-2-26 Kitano, Niiza, Saitama 3528558, Japan. E-mail address: [email protected]
(T. Ishiwata). http://dx.doi.org/10.1016/j.neulet.2017.05.030 0304-3940/© 2017 Elsevier B.V. All rights reserved.
pain modulation, mood, stress, and thermoregulation . The two main sources of forebrain 5-HT innervation are the dorsal raphe nucleus (DRN) and median raphe nucleus (MRN) . These raphe nuclei send serotonergic ﬁbers to a variety of forebrain areas, with the olfactory bulb, hypothalamus, septal area, thalamus, caudateputamen, hippocampal region, amygdala, and cerebral cortex as the main targets . We have recently reported that inhibition of neuronal activity by perfusion of the voltage-gated sodium channel blocker tetrodotoxin (TTX) into the MRN or DRN using reverse microdialysis induced a considerable decrease in body temperature (Tb ) with an increase of heat loss and no change of heat production in freely moving rats. These ﬁndings suggest that the 5-HT
T. Ishiwata et al. / Neuroscience Letters 653 (2017) 71–77
projections from the midbrain’s raphe nuclei may play a role in thermoregulation . In the thermoregulatory system, several studies have focused on the relationship between 5-HT and Tb regulation especially in the preoptic area and anterior hypothalamus (PO/AH), which are the crucial loci for the maintenance of Tb . For example, Feldberg and Myers , in pioneer experiments, reported that microinjection of 5-HT into the PO/AH induced a rise of Tb . However, other 5-HT studies in the PO/AH using the same agent failed to reproduce the same rise in Tb . For example, Cox et al.  reported a Tb decrease after 5-HT microinjection into the PO/AH. Clark and Lipton  reviewed different results of Tb response after microinjection of 5-HT into the PO/AH and concluded that the role of 5-HT in the PO/AH in thermoregulation is equivocal. The reasons for the differences between studies regarding the effects of 5-HT in thermoregulation could be due limitation in the use of bolus injections and large injection volumes by microinjection, or conditions such as the use of anesthesia. It is well known that anesthesia impairs thermoregulation, and more speciﬁcally decreases Tb . Therefore, it is important to investigate the roles of brain nuclei involved in thermoregulation in conscious animals without any external factors affecting baseline thermoregulatory parameters. The microdialysis technique provides a solution to this experimental problem . With this method, we have already reported, in freely moving rats, that both cold and heat exposure caused no signiﬁcant changes in extracellular levels of 5-HT or its metabolite in the PO/AH. Similarly, perfusion of 5-HT agents into the PO/AH did not affect Tb , low-intensity exercise did not alter 5-HT release in the PO/AH, and increased extracellular 5-HT elicited by perfusion of the selective reuptake inhibitor (SSRI) citalopram into the PO/AH had no effect on thermoregulation during low-intensity exercise in a warm environment . These data suggest that 5-HT in the PO/AH may not mediate acute changes in thermoregulation. Investigating the roles of nuclei beyond the PO/AH in thermoregulation, Nagashima et al.  and Romanovsky  both implicate the ventral tegmental area (VTA) as an important area for thermoregulation, especially in the control of cutaneous vasomotor tone. Zhang et al.  have found cutaneous’vasoconstrictor neurons in the VTA and showed that cutaneous vasodilatory response to warming the PO is inhibited by electrical and pharmacological activation of neurons in the VTA. Although there are many reports of a relationship between 5-HT and vasomotor regulation in the hypothalamus or medullary raphe regions , the role of 5-HT in the VTA in regulating Tb remains unclear. The aim of the present study was to clarify the involvement of 5-HT in the VTA using the combined methods of telemetry, microdialysis, and high performance liquid chromatography (HPLC), with a special emphasis on the regulation of Tb in freely moving rats. Given that little is known regarding the pattern of 5-HT release in the VTA during active thermoregulation, we measured monoamine release, Tb , tail skin temperature (Ttail ) as an index of heat loss [4,17], heart rate (HR) as an index of heat production [4,18] and locomotor activity (Act) during exposure to cold (5 ◦ C) and heat (35 ◦ C). We also simultaneously measured Tb , Ttail , HR, Act, and extracellular monoamine levels in the VTA during perfusion of citalopram into the VTA.
2. Materials and methods 2.1. Animals Male Wistar rats (CLEA Japan, Inc. Tokyo, Japan; 300–360 g body weight) were housed individually in plastic cages. Home cages were kept within temperature chambers (BIOTRON, LPH200, Nihon Medical and Chemical Instrument, Osaka, Japan) [4,11],
under controlled conditions of ambient temperature (23 ◦ C), relative humidity (50%), and 12:12 h light/dark cycle (lights on at 0700 h). Rats had access to food and water ad libitum except during the experiments. All experiments were carried out according to the Guiding Principles for the Care and Use of Animals in the Field of Physiological Science of the Physiological Society of Japan. Experiments were approved by the committee for safety and ethics in research and experiments related to Rikkyo University Life Sciences (No. LS09004A). 2.2. Thermoregulatory parameters Following intraperitoneal (i.p.) injection of Somnopentyl anesthesia (pentobarbital sodium, 50 mg/kg), telemetry devices (TA11CTA-F40; Data Sciences International, USA) were implanted into the peritoneal cavity, in order to continuously monitor Tb , HR, and Act in freely moving rats . Ttail was measured on the dorsal surface of the skin about 10 mm from the base of the tail using a mini temperature data logger (TSDL-HT1, Techno Science, Tokyo, Japan), which was covered with a plastic cladding for protection. All experiments were performed in the home cage. The ambient temperature was set by the temperature chambers at 23 ◦ C (normal environment). Either cold exposure (5 ◦ C) or heat exposure (35 ◦ C) was used for 2 h to activate thermoregulation in the rats. 2.3. Microdialysis At least 7 days were allowed between the insertion of the telemetry sensors and preparation for microdialysis. Three days prior to the experiments, the microdialysis probe (0.22 mm outer diameter, 1.0 mm long cellulose dialyzing membrane with a molecular weight cut-off of 50,000; CX-I-12-01, Eicom, Kyoto, Japan) was stereotaxically placed in the left lateral VTA (coordinates from bregma: AP −5.2 mm; L +0.5 mm; D −8.3 mm from dura) . The microdialysis probes were left in place until the start of the experiment. After conﬁrmation of a clear circadian rhythm of Tb , HR, and Act (2 days after surgery), microdialysis perfusion (1 L/min) commenced at 0900 h over 7 h using a microinjection pump (ESP-64, Eicom, Kyoto, Japan). We used a coiled Teﬂon tube (CT-20, Eicom, Kyoto, Japan) to prevent the pulling and tangling of the microdialysis tubes. A citalopram solution (1 M) was perfused for 60 min between 1200 and 1330 h into the VTA to inhibit 5-HT reuptake, as previously reported . Citalopram is an SSRI that results in an increase in extracellular levels of 5-HT, but not DA or NA . We have already reported that a citalopram concentration as low as 1 M is sufﬁcient to increase 5-HT release without increasing DA or NA levels . Ringer’s solution (147 mM NaCl, 4 mM KCl, and 2.3 mM CaCl2 ; pH 6.0) was continuously perfused except during the periods requiring modiﬁcation of the perfusate. 2.4. HPLC Concentration of NA, DA, and 5-HT from a single sample of brain microdialysate were measured using on-line HPLC (HTEC500, Eicom, Kyoto, Japan). The HTEC-500 is all-in-one with a small footprint and includes an online degasser, an amperometric electrochemical detector, a pump, a column temperature controller, and a manual injector. Peaks in a sample were identiﬁed by matching the retention times of peaks with those of authentic standards (PowerChrom, eDAQ Pty Ltd, Denistone East, Australia). We used an EICOMPAK CAX column (SC-5ODS, 200 × 2.0 mm inside diameter [i.d.], Eicom) and measured the concentration of NA, DA, and 5-HT in the same run. A 20-L aliquot of the sample was injected directly into the HPLC by using an auto-injector (EAS-20S, Eicom) from the microdialysis tube. The mobile phase solution contained 70% 0.1 M/L acetate ammonium buffer (pH 6.0) including 50 mg/L
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Fig. 1. Schematic representation of the locations of the microdialysis probe on a coronal sections and a representative photomicrograph depicting probe placement in the VTA. Each vertical bar (䊏: thermal exposure study, 䊐: citalopram study) indicates the length of a microdialysis probe membrane implanted into the VTA. fr, fasciculus retroﬂexus; MM, medial mammillary nucleus; ml, medial lemniscus; scp, superior cerebellar peduncle; SNR, substantia nigra; VTA, ventral tegmental area.
EDTA-2Na and 0.05 M/L sodium sulfate, 30% methanol. The ﬂow rate of the mobile phase was 0.25 mL/min. The graphite electrode (WE-3G, Eicom) was set at a potential of 450 mV, relative to an Ag/AgCl reference electrode. Analysis of one sample took about 15 min and the obtained sensitivity is as low as 100 fg/injection.
The probes were located from bregma −5.20 mm to −5.8 mm in the VTA.
Fig. 2 shows the changes in thermoregulatory parameters (A) and monoamine levels in the VTA (B) under hot (35 ◦ C) ambient temperature. Exposure to heat induced a signiﬁcant increase in Tb (F(23, 138) = 24.714, p < 0.001) with an immediate increase in Ttail (F(23, 138) = 92.987, p < 0.0001) and decrease in HR (F(23, 138) = 3.796, p < 0.0001). The maximum Tb and Ttail were 38.9 ± 0.2 ◦ C (at 175 min) and 37.6 ± 0.2 ◦ C (at 185 min), respectively, while the minimum HR was 296.4 ± 19.0 beats/min (at 185 min). Although Act increased transiently during heat exposure (F(23, 138) = 1.855, p = 0.016), post-hoc test showed no signiﬁcant differences compared with values prior to heat exposure (2.1 ± 0.8 counts/min at 55 min). Moreover, NA and DA levels did not change during heat exposure compared with baseline levels (Fig. 2B). On the other hand, 5-HT concentration increased from baseline levels during heat exposure (F(11, 66) = 2.977, p = 0.003). Post-hoc tests revealed a signiﬁcant increase above baseline levels in 5HT concentration between 90 min (138.7 ± 12.7%) and 130 min (119.7 ± 9.3%). The mean basal concentrations of extracellular 5HT, DA, and NA in the VTA were 0.30 ± 0.05, 0.46 ± 0.14, and 0.48 ± 0.08 pg/20 L sample, respectively.
Citalopram (Tocris, St. Louis, MO, USA) was freshly dissolved in Ringer’s solution on the day of each experiment. 2.6. Histological examination At the end of each experiment, rats were sacriﬁced using an overdose of somnopentyl (120 mg/kg, i.p.). The placement of the probe was veriﬁed in coronal sections stained with bromophenol blue by using a digital microscope (WM401, 3R System Corporation, Japan). 2.7. Data collection and statistical analysis Tb was measured every 1 min and averaged every 10 min. Microdialysates were collected every 20 min. Sufﬁcient time was taken until change of substances was stabilized before thermal or pharmacological manipulation. Three samples taken at 1 h before modiﬁcation of the perfusate or thermal exposure were considered as the baseline value and corresponded to 100%. Statistically signiﬁcant differences were evaluated by using repeated measures analysis of variance (ANOVA) followed by Fisher’s least signiﬁcant difference post-hoc test. All statistical analyses were performed ® ® using IBM SPSS Statistics (Version 23.0 for Mac; IBM Corp., Chicago, IL). Values are expressed as the mean ± standard error of the mean (SEM). A P < 0.05 was regarded as statistically signiﬁcant. 3. Results A total of 12 rats were used in this study, including each 7 rats for cold or heat exposure studies and each 5 rats for citalopram study. Fig. 1 is a schematic representation of the length of the probe membrane within a coronal brain sections and a representative photomicrograph depicting probe placement in the VTA.
3.1. Heat loss mechanisms during heat exposure is associated with 5-HT release in the VTA
3.2. Exposure to cold increased heat production without altering extracellular monoamines in VTA Fig. 3 depicts the changes in thermoregulatory parameters (A) and monoamine levels in the VTA (B) under cold (5 ◦ C) ambient temperature. Exposure to cold temperatures induced a signiﬁcant increase in Tb (F(23, 138) = 9.630, p < 0.0001) and HR (F(23, 138) = 17.673, p < 0.0001) with an immediate decrease in Ttail (F(23, 138) = 277.480, p < 0.0001). The maximum Tb and HR were 38.0 ± 0.1 ◦ C (at 115 min) and 510.9 ± 11.1 beats/min (at 95 min), respectively, while minimum Ttail was 10.3 ± 0.3 ◦ C (at 165 min). Although Act also increased transiently during cold exposure (F(23, 138) = 1.644, p = 0.042), post-hoc test showed no signiﬁcant differences compared with values prior to cold exposure (0.9 ± 0.6
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Fig. 2. Changes in thermoregulatory parameters (A: body temperature (䊉), tail skin temperature (䊏), heart rate (), locomotor activity ()), and extracellular monoamine levels (B: NA (), DA (䊐), 5-HT (♦)) in the VTA under hot ambient temperature (35 ◦ C). The average concentration of three microdialysis samples before heat exposure was considered as the baseline value and corresponded to 100%. Microdialysis samples were expressed relative to the baseline value. Values are expressed as mean ± SEM (n = 7). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with baseline value at 55 min.
Fig. 3. Changes in thermoregulatory parameters (A: body temperature (䊉), tail skin temperature (䊏), heart rate (), locomotor activity ()), and extracellular monoamine levels (B: NA ((), DA ((䊐), 5-HT ((♦)) in the VTA under cold ambient temperature (5 ◦ C). The average concentration of three microdialysis samples before cold exposure was considered the baseline value and corresponded to 100%. Microdialysis samples were expressed relative to the baseline value. Values are expressed as mean ± SEM (n = 7). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with baseline value at 55 min.
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Fig. 4. Changes in thermoregulatory parameters (A: body temperature (䊉), tail skin temperature (䊏), heart rate (), locomotor activity ()), and extracellular monoamine levels (B: NA ((), DA ((䊐), 5-HT ((♦)) elicited by perfusion of the VTA with a dialysate containing 1 M citalopram under normal temperature (23 ◦ C). The average concentration of three microdialysis samples before heat exposure was taken as the baseline and was deﬁned as 100%. Microdialysis samples were expressed relative to the baseline value. Values are expressed as mean ± SEM (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with baseline value at 55 min.
counts/min at 55 min). No changes were observed in monoamine levels during cold exposure compared with baseline levels (Fig. 3B). However, the variability in extracellular 5-HT did seem to increase after cold exposure compared with other monoamines. The mean basal concentrations of extracellular 5-HT, DA, and NA in the VTA were 0.28 ± 0.04, 0.46 ± 0.18, and 0.42 ± 0.05 pg/20 L sample, respectively.
3.3. Increasing extracellular 5-HT in the VTA with intra-VTA citalopram activated heat loss mechanisms As shown in Fig. 4, perfusion of citalopram into the VTA under normal ambient temperature induced a Tb decrease (F(17,68) = 9.630, p < 0.0001), facilitated heat loss (F(17,68) = 14.909, p < 0.0001), but did not alter heat production. Tb decreased immediately after perfusion of citalopram, and was signiﬁcantly different from 75 min and thereafter. Ttail gradually increased and was signiﬁcantly different from 105 min and thereafter. HR and Act were not altered by citalopram perfusion in the VTA. The minimum Tb and maximum Ttail were 36.8 ± 0.2 ◦ C (at 85 min) and 29.2 ± 0.2 ◦ C (at 175 min), respectively. 5-HT concentration but not DA or NA increased from baseline levels during heat exposure (F(8.32) = 4.592, p = 0.001) (Fig. 4B). Post-hoc tests revealed a signiﬁcant increase above baseline levels in 5-HT concentration between 70 min (177.2 ± 31.6%) and 150 min (141.2 ± 13.3%), and maximum concentration was 221.0 ± 52.2% (90 min). The mean basal concentrations of extracellular 5-HT, DA, and NA in the VTA were 0.30 ± 0.03, 0.40 ± 0.04, and 0.38 ± 0.03 pg/20 L sample, respectively.
4. Discussion In the present study, we demonstrated that signiﬁcant changes in the extracellular level of 5-HT, but not levels of DA or NA, in the VTA were noted only during exposure to heat. In addition, perfusion of the VTA with the SSRI citalopram in freely moving rats increased extracellular levels of 5-HT, but not DA or NA, in combination with decreased Tb , increased Ttail , and unaltered HR and Act. These results indicate that the VTA is a key area for thermoregulation and that 5-HT in the VTA may mainly mediate heat loss. Nagashima et al.  and Romanovsky  investigated the neuronal circuitries involved in thermoregulation and revealed that the VTA is one important area implicated in thermoregulation; particularly cutaneous vasomotor tone. Zhang et al.  reported that the cutaneous vasodilatory response to warming the PO is inhibited by electrical and pharmacological activation of neurons in the VTA. In addition, blocking downstream signaling from the VTA by transection of the area caudal to the VTA elicits cutaneous vasodilatation. In contrast, blocking signal inputs upstream of the VTA by transection of the area rostral to the VTA suppresses the cutaneous vasodilatory response to warming the PO. Serotonergic innervation of the rat VTA examined by light and electron microscopic radioautography have shown that 5-HT innervates both dopaminergic and non-dopaminergic neurons in the VTA . In addition, a trans-synaptic retrograde tracing study using pseudorabies virus into the tail artery revealed infection in the VTA . Despite the many reports suggesting a relationship between 5-HT and vasomotor regulation in the hypothalamus or medullary raphe regions , the role of 5-HT in Tb regulation via the VTA remains unclear. In the present study, we focused on the role of 5-HT in the VTA using the combined methods of telemetry, microdialysis, and HPLC, with a special emphasis on thermoregulation in freely moving rats.
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First, since little is known about the pattern of monoamine releases in the VTA during active thermoregulation, we measured their release during cold (5 ◦ C) and heat (35 ◦ C) exposure. Tb increased persistently under both conditions. The Tb increase during cold exposure was likely due to the facilitation of heat production and inhibition of heat loss, since a decrease in Ttail and increase in HR during cold exposure were observed. On the other hand, the Tb increase during heat exposure likely reﬂected a failure to maintain thermoregulation capacity, based on the signiﬁcant rise in Ttail and decrease in HR. Interestingly, signiﬁcant changes in extracellular levels of 5-HT, but not DA or NA, in the VTA were noted only during heat exposure. Apart from a single report suggesting that chronic cold stress reduces the spontaneous activity of DA neurons in the VTA , to our knowledge, no study has examined the release of monoamines in the VTA of freely moving rats during exposure to cold or heat by using microdialysis. We also manipulated the extracellular 5-HT levels in the VTA by perfusing the SSRI citalopram using microdialysis, while simultaneously measuring of Tb , Ttail , HR, and Act. Perfusion of citalopram into the VTA increased extracellular 5-HT, but not DA or NA, while decreasing Tb , incresing Ttail , and leaving HR and Act unchanged. These results indicate that 5-HT in the VTA mainly mediates the heat loss components of thermoregulation. Using microdialysis, previous studies have reported that citalopram selectively inhibits 5-HT re-uptake in the nerve terminals, and thus increases extracellular 5-HT levels. For example, we have already reported that local perfusion citalopram (1 M) into the PO/AH only increased extracellular 5-HT, but not DA or NA, during acute low-intensity exercise in a warm environment . Many studies have reported a 3-fold increase in extracellular 5-HT levels in the ventral hippocampus  and striatum , and a 4-fold increase in the DRN and MRN  following the addition of citalopram (1 M) to the perfusion medium. On the other hand, Romero et al.  reported a 5-fold increase in extracellular 5-HT level in the DRN and a 4-fold increase in the ventral hippocampus after perfusion of 1 M citalopram. In the present study, the increase in extracellular 5-HT level was comparatively smaller, possibly reﬂecting differences in density of 5-HT neuron terminals in the different brain regions, or variable length of the microdialysis probe membrane between studies. Both gamma-aminobutyric acid ([GABA]ergic) and glutaminergic modulation of thermoregulatory circuits have been well discussed, but there is little information on the role of 5-HT in midbrain regions [5,16]. It is noteworthy that our results suggest signiﬁcant changes in extracellular 5-HT in the VTA, combined with a decreased Tb and increased Ttail in the absence of HR changes after perfusion of citalopram into the VTA. In addition, extracellular 5HT levels increased only during heat exposure. Two explanations for this effect stand out. First, as a population of VTA neurons are thought to be vasoconstrictive , 5-HT could be contributing an inhibitory inﬂuence over the activity of VTA neurons, perhaps through 5-HT2C receptor-mediated activation of GABA neurons . Second, dopaminergic neurons in the VTA project their ﬁbers to the forebrain, and number of studies have argued that DA in the forebrain is involved in thermoregulation . For example, Zheng and Hasegawa  recently reported that central dopaminergic neurotransmission plays an important role in thermoregulation and performance during endurance exercise. Thus, 5-HT in the VTA may also modulate this dopaminergic pathway. However, despite the small size of the VTA, we used a 1-mm long microdialysis probe. Therefore, potential contribution of regions surrounding the VTA in the current results cannot be ruled out. Additionally, although understanding the projections of speciﬁc temperature-sensitive 5-HT neurons could help identify the key targets mediating the thermoregulatory effects of 5-HT, these regions remain elusive. In addition to the VTA, potential candidates could be the dorsomedial hypothalamus or periaqueductal gray [13,14,16]. Further research
is required to identify the afferent and efferent 5-HT pathways involved in thermoregulation. 5. Conclusion Our results suggest that the VTA is a key area for the thermoregulatory system and that 5-HT, but not DA or NA, in the VTA may speciﬁcally be involved with the heat loss component of thermoregulation. Conﬂicts of interest Authors of the present study declare no conﬂict of interest. Funding The present study was partly supported by JSPS KAKENHI Grant Number JP21700596 (T.I). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2017.05. 030. References  B.L. Jacobs, E.C. Azmitia, Structure and function of the brain serotonin system, Physiol. Rev. 72 (1992) 165–229.  A.P. Descarries, A. Beaudet, Organization of ascending serotonin systems in the adult rat brain. A radioautographic study after intraventricular administration of [3 H]5-hydroxytryptamine, Neuroscience 6 (1981) 115–138.  I. Pollak Dorocic, D. Fürth, Y. Xuan, Y. Johansson, L. Pozzi, G. Silberberg, M. Carlén, K. Meletis, A whole-brain atlas of inputs to serotonergic neurons of the dorsal and median raphe nuclei, Neuron 83 (2014) 663–678.  T. Ishiwata, A. Oshimoto, T. Saito, Y. Kotani, S. Nomoto, Y. Aihara, H. Hasegawa, B.N. Greenwood, Possible mechanisms of hyporthermia after inhibition of the median or dorsal raphe nucleus of freely moving rats, Neuroreport 27 (2016) 1287–1292.  S.F. Morrison, K. Nakamura, Central neural pathways for thermoregulation, Front. Biosci. 16 (2011) 74–104.  W. Feldberg, R.D. Myers, A new concept of temperature regulation by amines in the hypothalamus, Nature 200 (1963) 1325.  B. Cox, A. Davis, V. Juxon, T.F. Lee, D. Martin, A role for an indoleamine other than 5-hydroxytryptamine in the hypothalamic thermoregulatory pathways of the rat, J. Physiol. (London) 337 (1983) 441–450.  W.G. Clark, J.M. Lipton, Changes in body temperature after administration of adrenergic and serotonergic agents and related drugs including antidepressants: II, Neurosci. Biobehav. Rev. 10 (1986) 153–220.  B. Redfors, Y. Shao, E. Omerovic, Inﬂuence of anesthetic agent, depth of anesthesia and body temperature on cardiovascular functional parameters in the rat, Lab. Anim. 41 (2014) 6–14.  B.H.C. Westerink, Brain microdialysis and its application for the study of animal behavior, Behav. Brain. Res. 70 (1995) 103–124.  T. Ishiwata, T. Saito, H. Hasegawa, T. Yazawa, M. Otokawa, Y. Aihara, Changes of body temperature and extracellular serotonin level in the preoptic area and anterior hypothalamus after thermal or serotonergic pharmacological stimulation of freely moving rats, Life Sci. 75 (2004) 2665–2675.  S. Takatsu, T. Ishiwata, R. Meeusen, S. Sophie, H. Hasegawa, Serotonin release in the preoptic area and anterior hypothalamus is not involved in thermoregulation during low-intensity exercise in a warm environment, Neurosci. Lett. 482 (2010) 7–11.  K. Nagashima, S. Nakai, M. Tanaka, K. Kanosue, Neuronal circuitries involved in thermoregulation, Auton. Neurosci. 85 (2000) 18–25.  A.A. Romanovsky, Thermoregulation: some concepts have changed. Functional architecture of the thermoregulatory system, Am. J. Physiol. 292 (2007) R37–R46.  Y.H. Zhang, T. Hosono, M. Yanase-Fujiwara, X.M. Chen, K. Kanosue, Effect of midbrain stimulations on thermoregulatory vasomotor responses in rats, J. Physiol. 503 (1997) 177–186.  Y. Ootsuka, M. Tanaka, Control of cutaneous blood ﬂow by central nervous system, Temperature (Austin) 2 (2015) 392–405.  C.J. Gordon, Thermal biology of the laboratory rat, Physiol. Behav. 47 (1990) 963–991.  J.B. Chambers, T.D. Williams, A. Nakamura, R.P. Henderson, J.M. Overton, M.E. Rashotte, Cardiovascular and metabolic responses of hypertensive and
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