c-Fos expression in the parabrachial nucleus following intraoral bitter stimulation in the rat with dietary-induced zinc deficiency

Accepted Manuscript Research report C-Fos Expression in the Parabrachial Nucleus Following Intraoral Bitter Stimulation in the Rat with Dietary-induced Zinc Deficiency Akiyo Kawano, Shiho Honma, Chizuko Inui-Yamamoto, Akira Ito, Hitoshi Niwa, Satoshi Wakisaka PII: DOI: Reference:

S0006-8993(17)30023-9 http://dx.doi.org/10.1016/j.brainres.2017.01.020 BRES 45256

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Brain Research

Received Date: Revised Date: Accepted Date:

4 August 2016 14 January 2017 16 January 2017

Please cite this article as: A. Kawano, S. Honma, C. Inui-Yamamoto, A. Ito, H. Niwa, S. Wakisaka, C-Fos Expression in the Parabrachial Nucleus Following Intraoral Bitter Stimulation in the Rat with Dietary-induced Zinc Deficiency, Brain Research (2017), doi: http://dx.doi.org/10.1016/j.brainres.2017.01.020

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To: Brain Research

C-Fos Expression in the Parabrachial Nucleus Following Intraoral Bitter Stimulation in the Rat with Dietaryietary-induced Zinc Deficiency

Akiyo Kawanoa, b*, Shiho Honmaa, Chizuko Inui-Yamamotoa, Akira Itoa, Hitoshi Niwab, Satoshi Wakisakaa aDepartment

of Oral Anatomy and Developmental Biology

Osaka University Graduate School of Dentistry and bDepartment

of Dental Anesthesiology

Osaka University Graduate School of Dentistry, Osaka, Japan

*Corresponding author: Akiyo Kawano Department of Dental Anesthesiology Osaka University Graduate School of Dentistry 1-8 Yamadaoka Suita, Osaka 565-0871, Japan. Tel: +81-6-6879-2972; Fax: +81-6-6879-2975 E-mail: [email protected]

Abstract Zinc deficiency causes various symptoms including taste disorders. In the present study, changes in expression of c-Fos immunoreactivity in neurons of the parabrachial nucleus (PBN), one of the relay nuclei for transmission of gustatory information, after bitter stimulation to the dorsal surface of the tongue were examined in zinc-deficient rats. Experimental zinc-deficient animals were created by feeding a low-zinc diet for 4 weeks, and showed the following symptoms of zinc deficiency: low body weight, low serum zinc content 1

and behavioral changes to avoid bitter stimulation. In normal control animals, intraoral application of 1 mM quinine caused increased numbers of c-Fos-immunoreactive (c-Fos-IR) neurons in the external lateral subnucleus and external medial subnucleus of the PBN (elPBN and emPBN, respectively) compared with application of distilled water. However, in the zinc-deficient animals, the numbers of c-Fos-IR neurons in the elPBN and emPBN did not differ significantly between application of quinine and distilled water. After feeding the zinc-deficient animals a normal diet for 4 weeks, the symptoms of zinc deficiency recovered, and the expression of c-Fos-IR neurons following intraoral bitter stimulation became identical to that in the normal control animals. The present results indicate that dietary zinc deficiency causes alterations to neuronal activities in the gustatory neural circuit, and that these neuronal alterations can be reversed by changing to a normal diet.

Key Words: Words zinc-deficiency, taste disorder, c-Fos, bitter, parabrachial nucleus

Highlights ● Rats fed a low-zinc diet showed symptoms of zinc deficiency. ● Bitter stimuli evoked increased c-Fos expression in elPBN and emPBN in normal rats. ● Increased c-Fos expression in elPBN and emPBN was reduced in zinc-deficient rats. ● c-Fos in elPBN and emPBN in rescued rat became almost identical to normal rats.

Abbreviations c-Fos-IR: c-Fos-immunoreactive NTS: nucleus tractus solitarius PB: phosphate buffer PBN: parabrachial nucleus dlPBN: dorsal lateral subnucleus of PBN 2

elPBN: external lateral subnucleus of PBN emPBN: external medial subnucleus of PBN PBS: phosphate-buffered saline

1.I 1.Introduction Zinc is one of the essential minerals for humans and animals, and low-zinc conditions cause various abnormalities such as growth retardation, anorexia, epilation, skin parakeratosis, and impaired immune responses (Prasad et al., 1963; Henkin and Bradley, 1969; Mills et al., 1969; Hambidge et al., 1972; Prasad, 2013). Zinc deficiency also causes taste disorders such as hypogeusia (decreased taste acuity) (Henkin and Bradley, 1969). Zinc deficiency can be rapidly and easily created by dietary zinc restriction, and various studies using experimental zinc-deficient animals have been carried out to determine the relationships between zinc deficiency and taste disorders. As zinc contributes to cell proliferation and differentiation, many studies have shown that zinc deficiency causes morphological and functional abnormalities in the taste buds where rapid and continuous turnover of taste bud cells occurs (Kobayashi and Tomita, 1986; Naganuma et al., 1988; Ohki, 1990; Sekine et al., 2012; Ikeda et al., 2013). At the sites of nerve fibers in the taste buds, an electrophysiological study revealed decreased taste sensitivity in zinc-deficient animals (Goto et al., 2001). However, little is known about the neuronal activity in the central gustatory neural circuit of zinc-deficient animals. Immunohistochemistry of c-Fos has been used as an anatomical marker for activated neurons in the central nervous system. Previous studies revealed that gustatory stimulation evoked c-Fos immunoreactivity in different parts of the brain involved in the gustatory neural circuit, such as the nucleus tractus solitarius (NTS) (Houpt et al., 1994, 1996; Harrer and Travers, 1996; Travers et al., 1999; Travers, 2002), reticular formation (DiNardo and Travers, 1997), parabrachial nucleus (PBN) (Travers et al., 1999; Yamamoto et al., 1993, 1994; Yamamoto and Sawa, 2000; King et al., 2003), and amygdala (Yamamoto et al., 1997) under normal and experimental conditions. One of the advantages of c-Fos immunohistochemistry is 3

that it can reveal the topographic localization of neurons activated by different taste stimuli. Yamamoto and Sawa (2000) clearly showed increased numbers of c-Fos-immunoreactive (c-Fos-IR) neurons in the external lateral subnucleus and external medial subnucleus of the PBN (elPBN and emPBN, respectively) following intraoral stimulation by quinine, while intraoral infusion of sucrose evoked increased expression of c-Fos in the waist area and dorsal lateral subnucleus of the PBN (dlPBN). Thus, c-Fos immunohistochemistry in the gustatory neuronal circuit is a suitable method to analyze the alterations in neuronal activities following application of specific taste stimulations.

As zinc-deficient animals showed

decreased taste sensitivity at gustatory nerve fibers (Goto et al., 2001), alterations in neuronal activities in the gustatory neuronal circuit may have occurred in these animals. However, there have been no studies on changes in c-Fos expression in the gustatory neuronal circuit, such as neurons of the PBN, in experimental zinc-deficient animals. Thus, we applied c-Fos immunohistochemistry to investigate the changes in neural activity in the PBN in response to bitter stimulation in rats with dietary-induced zinc deficiency. Moreover, as zinc supplementation was reported to improve impaired taste sensation (Henkin and Bradley, 1969; Henkin et al., 1976; Heckmann et al., 2005), we also examined c-Fos expression in the PBN in rescued animals fed a normal diet after a low-zinc diet.

2. Results

2.1. General appearance The body weight of the zinc-deficient animals was significantly lower than that of the normal control animals (Fig 1). Careful observation revealed depilation and skin disorders around the eyes and limbs of the zinc-deficient animals. The body weight of the rescued animals increased gradually after switching from the low-zinc diet to the normal diet (Fig 1). The symptoms of depilation and skin disorders observed in the zinc-deficient animals recovered within 1 week after switching to the normal diet.

2.2. Serum electrolytes 4

The serum zinc content in the zinc-deficient animals was 36.1 ± 23.3 mg/dl, and significantly lower than that in the age-matched control animals (129.8 ± 13.5 mg/dl). There were no apparent differences in the contents of other electrolytes (Na, Cl, K, Ca, Mg) between the zinc-deficient animals and the age-matched control animals (Table 1). In the rescued animals, the serum zinc content was 151.6 ± 24.0 mg/dl, and showed no significant difference from that in the age-matched control animals (136.6 ± 12.3 mg/dl) (Table 1).

2.3. Two-bottle preference test The results of the two-bottle preference test are shown in Table 2. Although the total volume (mean ± SEM) of 0.01 mM quinine consumption in48 h was comparable between the zinc-deficient animals (9.5 ± 1.8 ml) and the age-matched control animals (9.8 ± 2.4 ml), the consumption volume per 100 g body weight in the zinc-deficient animals (11.6 ± 2.6 ml) was significantly greater than that in the age-matched control animals (3.8 ± 1.0 ml). The total amount of distilled water consumption of the zinc-deficient animals was 30.1 ± 3.2 ml, and significantly smaller than that in the age-matched control animals (75.5 ± 6.1 ml). However,when total volume of distilled water consumption was calculated as the consumption volume per 100 g body weight, there was no significant difference between the zinc-deficient animals (35.9 ± 2.2 ml) and the age-matched control animals (29.9 ± 3.4 ml) . The preference rate (mean ± SEM) for 0.01 mM quinine in the zinc-deficient animals and age-matched control animals was 23.9 ± 3.9% and 10.7 ± 2.5%, respectively, with a significant difference (Table 2). The amount of distilled water and 0.01 mM quinine consumption in the rescued animals was smaller than that in the age-matched control animals, but no significant difference was noted. The consumption volume per 100 g body weight of distilled water and 0.01mM quinine in the rescued animals was comparable to that in the age-matched control animals. The preference rate in the rescued animals was 6.0±1.6%, and almost identical to that in the age-matched control animals (6.5 ± 2.6%) (Table 2).

2.4. c-Fos immunoreactivity in the PBN 5

Although c-Fos-IR neurons were detected in all subnuclei of the PBN after intraoral application of solution, we focused on the expression of c-Fos-IR neurons in the elPBN, emPBN and dlPBN in the present study. Intraoral application of distilled water or 1 mM quinine induced the appearance of c-Fos-IR neurons in the elPBN, emPBN (Fig. 2), and dlPBN (Fig. 3) in all experimental animals. In the elPBN, application of 1 mM quinine evoked an increased number of c-Fos-IR neurons compared with application of distilled water in the control animals (Figs. 2A, B) and rescued animals (Fig. 2E, F), but not in the zinc-deficient animals (Fig. 2C, D). Intraoral application of 1 mM quinine also induced the appearance of c-Fos-IR neurons in the dlPBN, but the changes were similar to those after application of distilled water in the control animals (Fig. 3A, B), zinc-deficient animals (Fig. 3C, D), and rescued animals (Fig. 3E, F). The results of the quantitative analysis are presented in Figure 4. In the control animals, the total number (mean ± SEM) of c-Fos-IR neurons in three consecutive sections of the elPBN and emPBN from the same animal was 292.7 ± 18.8 and 17.3 ± 2.9, respectively (Fig. 4A, B). Following intraoral application of 1 mM quinine, significantly increased numbers of c-Fos-IR neurons (428.7 ± 23.3 in the elPBN and 41.0 ± 4.1 in the emPBN) were detected (Fig. 4A, B). In the zinc-deficient animals, application of distilled water induced approximately 276.0 ± 27.9 and 17.3 ± 2.9 c-Fos-IR neurons in the elPBN and emPBN, respectively. Following application of 1 mM quinine, approximately 278.3 ± 8.0 and 18.3 ± 3.2 c-Fos-IR neurons were observed in the elPBN and emPBN, respectively, showing no significant difference compared with application of distilled water (Fig. 5A, B). In the rescued animals, the number of c-Fos-IR neurons in the elPBN and emPBN following application of distilled water was 280.2 ± 16.4 and 15.5 ± 1.4, respectively, showing no significant differences compared with both the control animals and the zinc-deficient animals. Following application of 1 mM quinine, significantly increased numbers of c-Fos-IR neurons were detected in the elPBN (429.2 ± 20.4) and emPBN (45.3 ± 6.8), and the numbers were almost identical to those observed in the control animals following application of bitter stimulation (Fig. 4A, B). The number of c-Fos-IR neurons in the dlPBN following intraoral application of 6

distilled water and 1 mM quinine in the control animals was 68.7 ± 6.1 and 69.5 ± 20.1, respectively, showing no significant difference. In the zinc-deficient animals, the corresponding numbers were 61.0 ± 8.2 and 48.5 ± 8.6, respectively, with no significant difference. In the rescued animals, approximately 52.3 ± 7.5 and 57.8 ± 3.7 neurons showed c-Fos immunoreactivity following application of distilled water and 1 mM quinine, respectively (Fig. 4C).

3. Discussion Various experimental animal models for zinc deficiency have been created and analyzed. Dietary zinc restriction has been widely used to create zinc-deficient animals because zinc deficiency can be easily and rapidly produced. In rodents, diets containing less than 10 mg/kg of zinc induce marginal or mild zinc deprivation. It was reported that the zinc content in a 5–7 mg/kg diet was associated with moderate zinc deprivation, while a zinc content of less than 1–2 mg/kg was associated with severe zinc deprivation (Golub et al., 1995). In the present study, we fed the experimental animals with a diet containing 1.2 mg/kg of zinc from postnatal day 21, and confirmed that their serum zinc contents were significantly low. Thus, we consider that our experimental animals were classified as marginal zinc deficiency. The experimental animals showed typical symptoms of zinc deficiency, such as growth retardation and dermatological disorders. Several lines of clinical evidence have shown that zinc supplementation normalizes the serum zinc content and improves taste disturbance (Henkin and Bradley, 1969; Henkin et al., 1976; Heckmann et al., 2005). In the present study, the serum zinc content of the rescued animals recovered to the normal level after switching the zinc-deficient diet to the normal diet. The growth rate of the rescued animals also recovered, and the typical symptoms of zinc deficiency, such as dermatological disorders, were rescued. Therefore, the present animal model can reflect the taste disorder observed in humans. In the present study, behavioral changes were examined by the two-bottle preference test, and all experimental animals tended to avoid drinking 0.01 mM quinine (preference rates of all experimental groups were less than 50%). We found that the preference rate of the 7

zinc-deficient animals was significantly higher than that of the age-matched control animals. It is known that PBN taste neurons receive both ascending inputs from taste receptors and descending inputs from forebrain regions. Thus, it is possible that the perception of bitter stimulation can be modified at the cortical level Volume of water consumption is known to be closely related to body weight in adult animals. Therefore, we calculated the consumption volume per 100 g body weight, and found that the consumption volume per 100 g body weight was increased for 0.01 mM quinine in the zinc-deficient animals. This finding is accordance with a previous study showing increased intake of quinine in zinc-deficient animals (Catalonotto and Lacy, 1977). It is interesting that the consumption volume per 100 g body weight for total liquids (0.01 mM quinine and distilled water) in the zinc-deficient animals was significantly higher than that in the age-matched control animals. Clinically, increased intake of liquid (polydipsia) is not the major symptom of zinc-deficient patients. We cannot explain why polydipsia occurred in our zinc-deficient animals. One possible explanation is that the changes in balance of serum electrolytes as observed in the present study may have affected the balance of body fluids, resulting in polydipsia. Further experiments are required to elucidate the mechanism of the polydipsia caused by zinc-deficiency. In the present study, we found that intraoral bitter stimulation induced increased numbers of c-Fos-IR neurons in the elPBN and emPBN, but not in the dlPBN, in the normal control animals. These findings were consistent with those in a previous report (Yamamoto and Sawa, 2000). In the zinc-deficient animals, we observed significantly decreased numbers of c-Fos-IR neurons in the elPBN and emPBN. It is possible that the low zinc levels specifically affected the neuronal activity in the gustatory neuronal circuit. However, this speculation is unlikely, because experimental dietary zinc-deficient animals were reported to show various neurological disorders such as learning and memory deficits, and altered emotionality (for review, see Hagmeyer et al., 2015). Thus, we think that the low zinc contents may have affected the neuronal activity in various neuronal circuits, rather than being specific for the activity in the gustatory neuronal circuit. 8

The decreased numbers of c-Fos-IR neurons in the elPBN and emPBN, where neurons were activated by bitter stimulation in the normal control animals, indicated that two possible types of taste disorders occurred in the zinc-deficient animals. One possibility is that the zinc-deficient animals recognized quinine as another taste, for example, sweet. It is known that animals prefer sweet substances. However, our behavioral analysis using the two-bottle preference test showed avoidance of quinine in the zinc-deficient animals. In addition, the numbers of c-Fos-IR neurons in the dlPBN, where neurons were reported to be activated by intraoral administration of sweet stimuli and water (Yamamoto and Sawa, 2000), did not differ significantly between the normal control animals and the zinc-deficient animals. Thus, we believe that the zinc-deficient animals, at least, did not recognize quinine (bitter stimulus) as sweet. Another possibility is that our experimental animals recognized quinine as non-taste. It is well-known that zinc deficiency causes hypogeusia (decreased taste acuity). Zinc-deficient rats were reported to increase their intake of supra-taste-threshold concentrations of sodium chloride, hydrochloric acid, quinine sulfate, and sucrose (Catalanotto and Lacy, 1977). We believe that the increased intake of normally-avoided quinine, as shown by the two-bottle preference test, in the present study arose through hypogeusia caused by the zinc deficiency. This speculation is supported by the findings in our zinc-deficient animals that the numbers of c-Fos-IR neurons in the elPBN and emPBN following bitter stimulation were almost identical to those following intraoral administration of distilled water. In addition to changes in the gustatory neuronal circuit in the central nervous system, it is possible that the taste disorder caused by low dietary zinc might have arisen through functional abnormalities of the taste buds, as the peripheral taste receptors. The first step for gustatory transmission is contact of chemical substances with the taste buds via taste pores. It was reported that zinc deficiency causes parakeratosis and hyperkeratosis, thereby blocking the contact between chemical substances and taste pores (Catalanotto and Nanda, 1977; Naganuma et al., 1988). An ultrastructural study revealed decreased numbers of microvilli at taste pores (Kobayashi and Tomita, 1986). Furthermore, the number of taste 9

cells expressing bitter taste receptor genes was decreased in zinc-deficient rats (Sekine et al., 2012; Ikeda et al., 2013). The lifespan of taste bud cells was reported to be about 10 days, and continuous turnover of taste bud cell occurs (Beidler and Smallman, 1965; Farbman, 1980). As many genes involved in cell differentiation are zinc-dependent, low zinc caused prolonged turnover of taste bud cells (Ohki, 1990). Taken together, we speculate that the taste disorder induced by dietary zinc deficiency in our experimental animals was mostly caused by functional abnormalities of the taste buds. In addition to ascending inputs from peripheral taste receptors to the PBN, descending inputs from forebrain regions to the PBN were reported to have a significant impact on taste responses in the PBN (Lundy and Norgren, 2004; Li et al., 2005; Baez-Santiago et al., 2016). Thus, feedback from forebrain regions to the PBN may also contribute to changes in c-Fos expression in the PBN. Clinical supplementation of zinc can occasionally relieve taste disorders. Many experimental studies using dietary-induced zinc-deficient animals have revealed that alterations to taste bud morphology caused by low zinc were recovered by zinc supplementation (Hamano et al., 2006; Kinomoto et al., 2010). In the present study, we created rescued animals by switching the low-zinc diet to the normal diet, and found that their preference rate against bitter stimulation became almost identical to that of the age-matched control animals. In addition, the numbers of c-Fos-IR neurons in the elPBN and emPBN following intraoral bitter stimulation in the rescued animals became almost identical to those in the age-matched normal control animals. These findings indicate that the symptoms of zinc deficiency were recovered by feeding the normal diet. The present study has clearly demonstrated the behavioral and anatomical changes against bitter stimulation in zinc-deficient rats. In the present study, we created our experimental zinc-deficient animals by feeding a low-zinc diet from postnatal day 21. However, it is known that zinc deficiency in adult life can have specific and distinct effects (for review, see Hagmeyer et al., 2015). For example, Keller et al. (2001) showed that the effect of low dietary zinc on short-term memory deficiency was obvious in rats aged less than 62 days upon starting zinc restriction. Our preliminary study showed that feeding 7-week-old animals 10

with the low-zinc diet for 4 weeks did not cause apparent growth retardation compared with the present experimental animals fed the low-zinc diet from 3 weeks after birth (unpublished observations). Thus, further analyses are needed to determine whether changes in c-Fos expression in the PBN also occur in rats with induced zinc deficiency during adulthood. In addition, further studies are required to investigate whether similar behavioral and anatomical alterations occur against other taste stimuli and whether changes in c-Fos expression are also observed in other relay nuclei for gustatory information such as NTS..

4. Experimental Procedures The present study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. All experimental procedures were reviewed and approved by the Intramural Animal Use and Care Committee at Osaka University Graduate School of Dentistry (Permit Number 24-09).

4.1. Experimental animals A total of 24 male Sprague-Dawley rats at 3 weeks of age were purchased from Nihon Dobutsu (Osaka, Japan). The animals were individually housed in polycarbonate cages and maintained on a 12-h/12-h light/dark cycle. Experimental animals were created by feeding a low-zinc diet (1.2 mg Zn/kg) for 4 weeks (zinc-deficient animals; n = 6). Some of the zinc-deficient animals were then fed a normal diet (52.8 mg Zn/kg) for an additional 4 weeks to rescue the zinc deficiency (rescued animals; n = 6). As controls, age-matched animals were fed the normal diet for 4 or 8 weeks (normal control animals; n = 6 each). The body weights of the animals were measured every week.

4.2. Two-bottle preference test To examine the changes in taste preference, a 48-h two-bottle preference test was performed. Briefly, after water deprivation for 9 h, two bottles, one containing 0.01 mM quinine hydrochloride (A) and the other containing distilled water (B), were placed in each cage at 11

18:00, and the animals were allowed free access to both bottles. To avoid any influence of side preference and/or recognition, the left/right positions of the bottles were alternated after 24 h. Consumption over 48 h was measured. Since volume of water consumption is closely related to body weight in adult animals, the consumption volume per 100 g body weight was calculated. The preference rate was calculated as follows: preference rate (%) = volume of bottle A consumption / volume of bottle A and bottle B consumption × 100.

4.3. Bitter stimulation Application of bitter stimulation was performed at least 12 h after completion of the two-bottle preference test. Under anesthesia with chloral hydrate (400 mg/kg, intraperitoneal; supplemented as necessary), either 1 mM quinine hydrochloride dissolved in distilled water or distilled water only was applied to the dorsal surface of the tongue. Approximately 40 µl of solution was applied every 3 min for 30 min, and the animals were euthanized at 90 min after the final stimulation.

4.4. Measurement of serum zinc concentration Blood samples were collect prior to perfusion fixation, and centrifuged at 3,000 rpm for 5 min. The serum was separated and stored at −20°C until measurements of the serum levels of zinc and other electrolytes (Na, K, Cl, Ca, Mg) using the Nitro PAPS method and ion-selective electrodes (FALCO, Kyoto, Japan).

4.5. Tissue preparation and immunohistochemistry Immediately after collection of blood samples, animals were transcardially perfused with 0.02 M phosphate-buffered saline (PBS; pH 7.2), followed by a mixture of 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.2). The brain containing the PBN was carefully removed, and immersed in 4% paraformaldehyde in 0.1 M PB for at least 48 h. After soaking in 20% sucrose in PBS overnight for cryoprotection, the specimens were sectioned at 40-µm thickness using a freezing microtome, collected into cold PBS, and treated 12

as free-floating sections for immunohistochemistry. For immunohistochemical staining, the sections were rinsed in PBS several times, incubated in PBS containing 0.3% H2O2 for 30 min to inactivate endogenous peroxidase activity, and incubated in PBS containing 3% bovine serum albumin (Sigma, St. Louis, MN) and 1% normal swine serum (DAKO, Copenhagen, Denmark) for 30 min to reduce non-specific binding. The sections were then incubated with a rabbit anti-c-Fos polyclonal antibody (1:2000; Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 16–18 h at room temperature. After several rinses with PBS, the sections were incubated with biotinylated swine anti-rabbit IgG (1:500; DAKO) and subsequently with ABC complex (Vector, Burlingame, CA) for 90 min each at room temperature. Peroxidase activity was visualized by incubation with 0.04% 3, 3′-diaminobenzidine in 0.05 M Tris-HCl-buffered saline (pH 7.5) containing 0.003% H2O2 with nickel ammonium sulfate (0.08–0.1%) enhancement. Sections were mounted onto gelatin-subbed glass slides, lightly counterstained with hematoxylin, dehydrated through an ascending series of ethanol, cleared with Lemosol (Wako, Osaka, Japan), and cover-slipped with Permount (Fisher Scientific, Fair Lawn, NJ). The sections were observed under a light microscope (BX50; Olympus, Tokyo, Japan).

4.6. Quantitative analysis of c-Fos-IR neurons As c-Fos-IR neurons induced by intraoral application of quinine increased in number in the elPBN and emPBN, and intraoral application of distilled water evoked an increased number of c-Fos-IR neurons in the dlPBN (Yamamoto and Sawa, 2000), we counted the numbers of c-Fos-IR neurons in the elPBN, emPBN and dlPBN. For counting of c-Fos IR neurons in the elPBN and emPBN, we selected three consecutive sections at 0.25 mm caudal to the contact of the inferior colliculus with the pons in each experimental group (three sections per animal; n = 6 per group).

For counting in the dlPBN, three consecutive sections at 0.5 mm caudal to

the same contact was selected (three sections per animal; n = 6 per group). The precise locations of c-Fos-IR neurons were plotted by camera lucida at ×40 magnification by an observer blinded to the condition of the animals, and the numbers of c-Fos-IR neurons in the 13

elPBN, emPBN and dlPBN were counted on one side (at least two animals per right or left side). The borders of the subnuclei of the PBN were determined according to the previous report (Fulwiler and Saper, 1984) and the stereotaxic atlas by Paxinos and Watson (2007). Data was presented as mean ± SEM per animal, and statistical significance was assessed by an unpaired Student’s t-test using Office Excel 2010 (Microsoft, Redmond, WA). Values of p < 0.05 were considered to indicate significant differences.

Acknowledgements: Acknowledgements This study was supported by the Japan Society for the Promotion of Science (JSPS) (# 258619370 to AI and #15K11007 to SW).

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Figure Legends

Fig 1. Temporal changes in the body weight of the normal control rats (closed circles), zinc-deficient rats (open triangles), and rescued rats (open circles). The body weight of the zinc-deficient rats is significantly lower than that of the normal control rats. The body weight of the rescued rats increases gradually after switching the low-zinc diet to the normal diet. *p < 0.05, versus the normal control rats. BW: body weight

Fig 2. Camera lucida drawings of c-Fos-IR neurons in the elPBN and emPBN at 0.25 mm caudal to the contact of the inferior colliculus with the pons following intraoral application of distilled water (DW) (A, C, E) or 1 mM quinine (QHCl) (B, D, F) in the normal control rats (A, B), zinc-deficient rats (C. D) and rescued rats (E, F). Each dot represents one c-Fos-IR neuron. BC: brachium conjunctivum; el: elPBN; em: emPBN. Scale bar: 500 µm.

Fig 3. Camera lucida drawings of c-Fos-IR neurons in the dlPBN at 0.5 mm caudal to the contact of the inferior colliculus with the pons following intraoral application of distilled water (DW) (A, C, E) or 1 mM quinine (QHCl) (B, D, F) in the normal control rats (A, B), zinc-deficient rats (C. D) and rescued rats (E, F). Each dot represents one c-Fos-IR neuron. BC: brachium conjunctivum; dl: dlPBN; el: elPBN. Scale bar: 500 µm.

Fig 4. Numbers of c-Fos-IR neurons in the elPBN (A), emPBN (B), and dlPBN (C) in the normal control rats, zinc-deficient rats, and rescued rats following intraoral administration of distilled water (open columns) or 1 mM quinine (closed columns). *p < 0.05.

20

BW (g)

500

Normal Zn deficiency Rescued

400

* *

300

*

200

* * *

100 0

*

3

4

5

6

7

8

9

10

11

Number of c-Fos neurons (mean + SEM)

external lateral PBN (elPBN) 500

*

*

**

400

external medial PBN (emPBN) 60

*

*

*

*

dorsal lateral PBN (dlPBN) 90

45 60

300 30 200 30 15

100 0

A

Normal Zn deficiency Rescued

0

B

Normal Zn deficiency Rescued

0

C

Normal Zn deficiency Rescued

Table 1. Biological analyses of serum samples 7 weeks

11 weeks

Normal

Zn deficiency

Normal

Rescued

Zn (mg/dl)

129.8 ± 13.5

36.1 ± 23.3*

136.3 ± 12.3

151.6 ± 24.0

Na (mEq/l)

134.3 ± 3.8

136.4 ± 6.1

136.3 ± 2.4

136.2 ± 3.3

Cl (mEq/l)

97.1 ± 4.0

95.9 ± 3.1

96.5 ± 1.7

96.4 ± 3.0

K (mEq/l)

7.2 ± 1.3

6.2 ± 0.4

6.6 ± 0.5

6.4 ± 0.5

Ca (mEq/dl)

10.0 ± 0.4

9.5 ± 1.4

10.6 ± 0.6

10.3 ± 0.5

Mg (mEq/dl)

2.3 ± 0.5

1.8 ± 0.2

2.9 ± 0.4

2.9 ± 0.2

Data are presented as means ± SD. *p < 0.05, the serum zinc content is significantly lower in the zinc-deficient rats compared with that in the normal control rats.

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Table 2. Consumption volumes in the two-bottle preference test 7 weeks

11 weeks

Normal

Zn deficiency

Normal

Rescued

0.01mM quinine (ml)

9.8 ± 2.4 (3.3 ± 0.8)

9.5 ± 1.8 (13.8 ± 2.6*)

6.0 ± 2.0 (1.3 ± 0.5)

3.8 ± 0.9 (1.1 ± 0.4)

Distilled water (ml)

75.5 ± 6.1 (25.6 ± 2.0))

31.7 ± 3.5* (43.7 ± 4.5*)

89.0 ± 11.0 (19.0 ± 2.7))

68.2 ± 13.6 (20.7 ± 4.3)

Total volume (ml)

85.3 ± 7.7 (28.9 ± 2.5)

41.2 ± 3.3* (57.5 ± 4.9*)

95.0 ± 11.3 (20.3 ± 2.4)

71.9 ± 13.5 (21.9 ± 3.9)

Preference Rate

10.7 ± 2.5

23.9 ± 3.9*

6.5 ± 2.6

6.0 ± 1.6

All data are presented as means ± SEM. The data in parentheses show the consumption volume per 100 g body weight.

* p < 0.05, significant difference between the normal control animals and the zinc deficient animals, and between the normal control animals and the rescued animals (Student’s t-test).

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