Regional differences of cFos immunoreactive cells in the preoptic areas in hypothalamus associated with heat and cold responses in mice

Accepted Manuscript Title: Regional differences of cFos immunoreactive cells in the preoptic areas in hypothalamus associated with heat and cold responses in mice Authors: Yuki Uchida, Keisuke Onishi, Ken Tokizawa, Kei Nagashima PII: DOI: Reference:

S0304-3940(17)30962-X https://doi.org/10.1016/j.neulet.2017.11.053 NSL 33261

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

24-9-2017 21-11-2017 24-11-2017

Please cite this article as: Yuki Uchida, Keisuke Onishi, Ken Tokizawa, Kei Nagashima, Regional differences of cFos immunoreactive cells in the preoptic areas in hypothalamus associated with heat and cold responses in mice, Neuroscience Letters https://doi.org/10.1016/j.neulet.2017.11.053 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title: Regional differences of cFos immunoreactive cells in the preoptic areas in hypothalamus associated with heat and cold responses in mice

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Authors: Yuki Uchida a1 , Keisuke Onishia2 , Ken Tokizawa a3 and Kei

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Nagashima a,b

Institutions:

Body Temperature and Fluid Laboratory, Faculty of Human Sciences,

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Institute of Applied Brain Sciences, Waseda University, Saitama, Japan

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a

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Kei Nagashima, MBBS, DMSci,

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Corresponding author:

Body Temperature and Fluid Laboratory, Health and Welfare, Faculty of Human Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa,

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Saitama 359-1192, Japan

E-mail: [email protected]

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Tel. and Fax: INT+81-4-2947-6918 Present address: 1 Women's Environmental Science Laboratory,

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Department of Health Sciences, Faculty of Human Life and Environment, Nara Women's University, Nara, Japan, 2 Tokyo Fire Department, Tokyo, Japan, National Institute of Occupational Safety and Health, Kanagawa, Japan

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HIGHLIGHTS 1. The difference of cFos expression in the preoptic area in mice between the heat and cold was examined. 2. In the heat, all areas in the preoptic area were activated. 3. In the cold, the dorsal part of the medial preoptic area and the ventral part of

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the lateral preoptic were activated.

4. There were regional differences in the activated preoptic areas between the

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heat and cold.

Abstract

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cFos expression in the preoptic area (PO), which is thermoregulatory center

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increased by both heat and cold exposures; however, the regional difference is

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unknown yet. We aimed to determine if cFos expression in the PO was regionally different between heat and cold exposures. Mice were exposed to 27,

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10, or 38°C for 90 min, and body temperature (T b ) was measured. cFos-immunoreactive (cFos-IR) cells in the PO were counted by separating the

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PO into the ventral and dorsal parts in the rostral (bregma 0.38 mm), central (-0.10 mm), and caudal (-0.46 mm) planes. T b at 10°C remained unchanged;

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however, it increased at 38°C. Counts of cFos-IR cells in all areas were greater

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at 38°C than at 27°C. In the dorsal and ventral parts of the central and the dorsal part of caudal PO, counts of cFos-IR cells were greater at 10°C than at 27°C. In conclusion, the areas of increased cFos expression in the PO in the heat were different that in the cold in mice.

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Keywords: cFos, hypothalamus, cold, heat, preoptic area

1. Introduction

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The preoptic area (PO) is a neural area, from which many efferent neurons are projected [26] and afferent neurons are received for thermoregulation [27].

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Each nucleus in the PO relates to thermoregulation: The lateral preoptic area

(LPO) [9] , median preoptic area (MnPO) [27], medial preoptic nucleus (MPO)

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[6], anterior hypothalamic area (AH) [19] , and lateral hypothalamic area (LH)

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[9].

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The PO has many thermosensitive neurons [29]. Neurons which increase

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their steady discharge frequency in response to a higher or lower local

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temperature are termed “warm-sensitive neurons” (WSNs) or “cold-sensitive neurons” (CSNs), respectively [28]. WSNs are more abundant than CSNs in the PO [31]. CSNs can increase body temperature (T b ). The activation of WSNs

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induces heat loss by stimulating responses such as panting and sweating [5].

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Activation of WSNs in the PO by electric or glutamate stimulation suppressed shivering; however, activation of CSNs by anesthetic did not affect shivering.

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Therefore, WSNs are more important than CSNs to control cold responses such as shivering and vasomotion in rats [44]. Thus, WSNs influence thermoregulatory responses in both hot and cold conditions. A number of previous studies showed that cFos expression [14] in the PO

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is increased by exposure to cold [2,8,21,24,25,37,43] or heat [1,8,11,13,21,24,33,37] conditions in rats and mice, suggesting that neural activity in the PO is increased by both parameters. Traditionally, increased

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cFos expression in response to heat conditions is considered to reflect activation of WSNs, which control heat loss. However, in cold condition,

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increased cFos expression in the PO may result from WSN or CSN control of heat production, but this is unknown.

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We defined that regional difference was the difference of cFos expression

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among the nuclei from rostral to caudal PO in the present study. The

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distribution of cFos expression during cold exposure differs between the rostral

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and caudal suprachiasmatic nucleus in hypothermic mice [42]. Thus, there may

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be regional differences of activated neurons in each nucleus in the PO related to thermoregulation. For example, injection of a GABA A receptor antagonist to the rostral and ventral sections of the caudal MnPO in anesthetized rats

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increased the temperature of the interscapular brown adipose tissue (iBAT) and

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T b [27]. Additionally, injection of a GABA A receptor antagonist to rostromedial PO (bregma 0–0.5 mm) or caudolateral PO (bregma -1–0 mm) in

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anesthetized rats increased or did not change tail sympathetic nerve activity [38]. However, electric stimulus and heating on the PO and AH induced vasodilation (bregma -0.26, -0.3 mm) [19] and saliva spreading (bregma -3.5–1 mm) [18]. To examine the relationship between these thermoregulatory

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functions in each areas in the PO and cFos expression in the PO, the PO was divided into dorsal and ventral parts in the rostral, central, and caudal parts in the present study. We hypothesized that cFos expression in the whole MnPO

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and rostromedial PO is not increased in response to cold, and that the distribution of cFos expression in the PO and AH would be consistent in

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response to heat.

In the present study, we examined this hypothesis by dividing the LPO,

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MnPO, MPO, AH and LH of mice into the rostral, central, and caudal, and the

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ventral to dorsal parts, and assessing them.

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2. Methods

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Animals. Male ICR mice (n=23; body weight, 34.9–40.4 g; age, 8 weeks; Takasugi, Saitama, Japan) were used in the present study. They were individually housed in a cage (12 cm × 20 cm × 10 cm) at an ambient

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temperature (T a ) of 27±0.5°C with in a 12:12-h light/dark cycle (lights on at

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07:00 hours; 300 lux). The Institutional Animal Care and Use Committee of Waseda University (Tokyo, Japan) approved the experimental procedures.

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Surgery. Following inhalation anesthesia with 2% sevoflurane, a

temperature sensor with a built-in data logger (Thermochron SL type, KN Laboratories, Osaka, Japan) was implanted in the abdominal cavity of mice for T b measurement. Mice were allowed to recover from the surgery for at least 1

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week. Experimental protocol. Mice were transferred to climatic chamber (Program Incubator IN602W, Yamato Scientific, Tokyo, Japan) and maintained

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at a Ta of 27°C. Mice were exposed to 27°C control (n=5) or 38°C heat (n=9) or 10°C cold (n=9) for 90 min (at 12:00–13:30 hours). T a of 10°C was set for cold

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condition to minify cold stress and induce cFos expression in the PO [21,25,43]. After the exposure, they were killed by cervical dislocation and perfused with

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4% paraformaldehyde. The brain was dissected out, post-fixed overnight with

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4% paraformaldehyde, and dehydrated in 20% sucrose for another two days.

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Immunohistochemistry. Coronal sections of 40-µm thickness were

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obtained using a cryostat (CM1510S, Leica, Wetzlar, Germany). Sections were

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rinsed with phosphate-buffered saline (PBS) and incubated in 0.3% hydrogen peroxide and 0.3% Triton X-100 in PBS for 30 min. Section were incubated with a rabbit anti-cFos polyclonal IgG primary antibody (1:15,000 dilution;

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Calbiochem, Merk, Tokyo, Japan) diluted in PBS with 0.3% Triton X for 12 h,

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followed by incubation with a biotinylated donkey anti-rabbit IgG secondary antibody (1:1,000 dilution; Jackson ImmunoResearch Laboratories, West Grove,

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PA) for 90 min at RT. After reaction with the avidin-biotin complex (1:1,000 dilution; Vectastain Elite ABC standard kit, Vector Laboratories, Burlingame, CA) for another 90 min. The sections were stained with 5% diaminobenzidine-tetrahydrochloride (Sigma, St. Louis, MO) and 0.3%

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hydrogen peroxide in PBS. Sections were mounted onto gelatin-coated glass slides, each of which was coverslipped. cFos-immunoreactive (cFos-IR) cells were counted in three different

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levels from the rostral to the caudal aspect of the section (0.38, -0.10, and -0.46 mm from the bregma, based on the atlas of Paxinos and Franklin [34]; defined

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as rostral, central and caudal, respectively). Digital images of the MPO, MnPO, and LPO in the rostral section, the MPO and LPO in the central section, and the

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MPO, AH and LH in the caudal section were taken in three subsequent slices

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(Digital Camera HC 2500 3CCD, FUJIFILM, Tokyo, Japan; ECLIPSE E600,

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Nikon, Tokyo, Japan; Adobe Photoshop, Adobe System, San Jose, CA). Each

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digital image of nucleus was divided to two parts (dorsal and ventral,

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respectively). The cells were counted and calculated the mean of three subsequent sections, which was divided by the counted area (density). Statistics. T b was averaged every 5 min. The baseline was defined as the

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30-min mean before the exposure. Differences between groups were assessed

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by two-way ANOVA without repeated measures with R language (R version 2.6.0, The R Foundation for Statistical Computing). Tukey-Kramer's test was

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used to identify significant differences at specific time points of T b and among the groups of the number of cFos-IR cells. Data are presented as the mean ± standard error. The null hypothesis was rejected at the level of P<0.05.

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3. Results T b during the exposure at 27, 10, and 38°C is shown in Fig. 1. No significant differences were observed in T b between 27 and 10°C. T b at 38°C

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was higher than the baseline level at 40–90 min, and greater than that at 27°C and 10°C at 25–90 min.

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Fig. 2 shows photo images of the rostral, central, and caudal PO obtained from one mouse exposed to T a of 27°C, 10°C or 38°C for 90 min with the

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into two parts (dorsal and ventral, respectively).

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schemas modified from the brain atlas [34]. Each image of nucleus was divided

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Figs. 3A and B show counts of cFos-IR cells in the PO. The cFos-IR cells

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were not observed in all areas at 27°C. In all areas, counts of cFos-IR cells at

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38°C were greater than at 27°C. Counts of cFos-IR cells at 38°C were greater than at 10°C except in the AH and LH in the caudal area. Only in the caudal MPO, counts of cFos-IR cells at 10°C were greater than at 27°C (Fig. 3A).

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After dividing the areas into ventral and dorsal parts, counts of cFos-IR cells

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were greater at 38°C than at 27 or 10°C. Only in the ventral part of central LPO and the dorsal part of the central and caudal MPO, counts of cFos-IR cells were

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greater at 10°C than at 27°C (Fig. 3B). Fig. 3C illustrates the densities of cFos-IR cells in the ventral and dorsal

parts of the PO. In all areas, the densities of cFos-IR cells were greater at 38°C than at 27 or 10°C. Only in the ventral part of the rostral LPO, and the dorsal

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part of the caudal AH and MPO, the densities of cFos-IR cells were greater at 10°C than at 27°C. In mice at 10°C, the density of cFos-IR cells in the dorsal part of the rostral MnPO was greater than in the other parts (the ventral and

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dorsal parts of the rostral and central LPO, the ventral part of the central MPO, and the ventral and dorsal parts of the caudal LH). In mice at 38°C, the density

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of cFos-IR cells in the ventral part of the rostral MnPO was greater than in the other parts (the ventral part of the rostral and central LPO and the ventral and

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dorsal parts of the caudal LH). In addition, the density of cFos-IR cells in the

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dorsal part of the rostral MnPO was greater than that in the ventral part of the

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rostral LPO.

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4. Discussion

The present study showed that heat exposure activated all areas in the PO in mice; however, the areas activated by cold exposure were the dorsal part of

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the central and caudal MPO and the ventral part of the central LPO. There were

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regional differences in the activated PO areas between heat and cold exposures. T b at 38°C was greater than that at 27°C (Fig. 1). This result coincides with

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previous studies that T b increased during various heat exposures: 34°C in mice [41] and 32–40°C in rats [3,11,21,32]. Consistent with previous studies that T b did not change during cold exposures: 10°C and 20°C [12,39-41] in mice and 10°C in rats [21], 10°C exposure did not affect T b (Fig. 1). Thus, a T a of 10°C

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may be a mild cold stimulus, and a T a of 38°C may be a strong heat stimulus for mice. Heat exposure increased cFos-IR cells in all parts in the brain regions

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assessed (Fig. 3); this supported our hypothesis. In rats, heat exposure increased cFos-IR cells in the MPO [8,11,24,37], MnPO [8,11,33,37,43] and

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LPO [8,37]. In mice, heat exposure increased cFos-IR cells in the MPO [1], MnPO [1,13], LPO, AH, and LH [13]. These studies are consistent with the

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results of present study. Local heating and electronic stimulation of the PO

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increased vasodilatation of the tail [19] and saliva-spreading behavior [18] in

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rats. WSNs exist in the PO in rats [7,10,15,17,20,23], mice [22,35,36], and cats

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[30]. The percentage of WSNs in the PO was 30 [10,20], 35 [16], and 48% [23]

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in rats; however, it remains unknown in mice. Few previous studies showed a regional difference of WSNs from rostral to caudal parts of the PO in rats; the percentage of WSNs was 49–63% in the rostral area [10] and 15% in the caudal

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area [7]. Thus, WSNs may be distributed in the widespread PO. Although we

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did not investigate the relationship between WSNs and cFos-IR cells in heat conditions, the activated neurons may reflect WSNs to control heat dissipation

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in a heat environment. Cold exposure increased cFos-IR cells only in the dorsal part of the central

(bregma -0.10 mm) and caudal MPO (bregma -0.46 mm) and the ventral part of the central LPO; this also supported our hypothesis. Previous studies also

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showed that cFos-IR cells in the MPO [2,8,24] and LPO [8] of rats were increased by cold, though they did not divide each nucleus into different parts. Unlike our results, cFos-IR cells in the MnPO [8] and LH [2] were increased by

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cold (4°C for 2 or 3h) in rats. Even though the same cold exposure protocol (10°C for 1.5h) was used as the present study, cFos-IR cells in the MPO and

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LPO (bregma -0.14 mm) did not increase in mice [1]. These differences may result from variations in exposure T a , time and the distance from bregma. In

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addition to the presence of WSNs, CSNs exist in the PO; the percentage was 10

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[20] and 4% [10] in rats. The percentage was 50% in the caudal PO in rats [10].

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Hence, CSNs are distributed in the PO with regional differences from the

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rostral to caudal areas, but the ratio may be small. A local application of GABA

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to the PO decreased T b [4]. CSNs in the PO were excited by GABA A antagonists, suggesting that GABA directly inhibits CSNs [17]. Thus, CSNs may partly affect thermoregulatory responses to increase T b . The increased

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cFos-IR cells in specific parts of the MPO and LPO in response to cold may

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reflect CSN and WSN activit y to control heat production in the cold. The other possible interpretation is that these neurons may detect cold signals from the

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skin, which results in the disinhibition of neurons in the MnPO [27] and rostromedial PO [38], which projects to the dorsomedial hypothalamic nucleus and raphe to induce the iBAT thermogenesis and tail vasoconstriction. The density of cFos-IR cells in the dorsal and ventral parts of the rostral

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MnPO that were increased by hot conditions (Figs. 3A and B) was greater than that in the other areas (Fig. 3C). MnPO neurons activated in response to heat stimuli project to the rostral periaqueductal gray matter, which results in

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vasodilation in the tail [43]. Thus, the high density of cFos-IR cells in the MnPO may relate to heat dissipation in hot conditions. However, the density of

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cFos-IR cells in the dorsal part of the rostral MnPO was greater than that in the other areas in cold exposure (Fig. 3C). Cold exposure did not affect cFos-IR

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cells in the rostral MnPO (Fig. 3B). Thus, the greater density of cFos-IR cells

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in the dorsal part of the rostral MnPO might not affect thermoregulatory

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responses in the cold.

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In conclusion, the present study showed that neural activation in the PO of

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mice was regionally different between heat and cold exposures. However, a relationship between the regional difference and CSNs and WSNs is unknown

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yet, and needs to be clarified.

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Acknowledgements

The present research was partially supported by the Ministry of Education,

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Culture, Sports, Science, and Technology; Grant-in-Aids for Scientific Research (B), No. 20390066; Grant-in-Aid for Research Activity, No. 24800047; Grant-in-Aid for challenging Exploratory Research, No. 16K13055; MEXT. KIBANKEISEI (2010); the Strategic Research Platforms for Private

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University; and the Hayashi Memorial Foundation for Female Natural Scientists.

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[40] K. Tokizawa, Y. Uchida, K. Nagashima, Thermoregulation in the cold changes depending on the time of day and feeding condition: physiological and anatomical analyses of involved circadian mechanisms, Neuroscience

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164(3) (2009) 1377-1386.

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[41] P. Trayhurn, W.P. James, Thermoregulation and non-shivering thermogenesis in the genetically obese (ob/ob) mouse, Pflugers Arch

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373(2) (1978) 189-193.

[42] Y. Uchida, K. Tokizawa, K. Nagashima, Characteristics of activated neurons in the suprachiasmatic nucleus when mice become hypothermic during fasting and cold exposure, Neurosci Lett 579(2014) 177-182.

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[43] K. Yoshida, M. Konishi, K. Nagashima, C.B. Saper, K. Kanosue, Fos activation in hypothalamic neurons during cold or warm exposure: projections to periaqueductal gray matter, Neuroscience 133(4) (2005)

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1039-1046. [44] Y.H. Zhang, M. Yanase-Fujiwara, T. Hosono, K. Kanosue, Warm and cold

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signals from the preoptic area: which contribute more to the control of

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shivering in rats? J Physiol 485 ( Pt 1)(Pt 1) (1995) 195-202.

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Figure Legends

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Figure 1. Body core temperature (T b ) during a 90-min exposure at 27, 10, and

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38°C. Data are presented as the mean ± standard error. Significant difference

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between 38°C and 10°C (*), 27°C and 10°C (#), and 27°C and 38°C (†) exposures, P<0.05. Ta, ambient temperature.

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Figure 2. Representative images of the preoptic area. Images were obtained

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from one mouse exposed to a T a of 38°C or 10°C for 90 min, and those of the rostral, central, and caudal sections (2Aa-c). Each image of nucleus was

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divided to two parts: d and v, respectively. The left schemas were modified from the stereotaxic atlas of Paxinos and Franklin [34]. Magnified images of the ventral part of MPO in the rostral, central, and caudal areas after exposure to a T a of 27°C, 10°C, or 38°C are shown in 2Ba-i. Dark staining indicates cFos

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immunoreactive cells. MPO, medial preoptic nucleus; MnPO, median preoptic nucleus; LPO; lateral preoptic area; AH, anterior hypothalamus; LH, lateral hypothalamic area; 3V, third ventricle; d, dorsal; v, ventral; T a, ambient

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temperature.

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Figure 3. Total counts of cFos-IR cells in the preoptic area (A), counts (B), and density (C) of cFos-IR cells in the d and v parts of the preoptic area after

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exposure to 38°C and 10°C. Data are expressed as the mean ± standard error.

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Significant difference between 10°C and 38°C (*), 27°C and 10°C (#), 27°C

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and 38°C (†) exposures, the v rostral MnPO and v rostral LPO (a), the v rostral

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MnPO and v central LPO (b), the v rostral MnPO and v caudal LH (c), the v

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rostral MnPO and d caudal LH (d), the d rostral MnPO and v rostral LPO (e), the d rostral MnPO and d rostral LPO (f), the d rostral MnPO and v central LPO (g), the d rostral MnPO and d central LPO (h), the d rostral MnPO and v central

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MPO (i), the d rostral MnPO and v caudal LH (j), and the d rostral MnPO and d

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caudal LH (k), P<0.05. MPO, medial preoptic nucleus; MnPO, median preoptic nucleus; LPO, lateral preoptic area; AH, anterior hypothalamus; LH, lateral

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hypothalamic area; cFos-IR, cFos immunoreactive; d, dorsal; v, ventral.

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