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J. Anim. Sci. 2005. 83:1845-1853
© 2005 American Society of Animal Science

ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Induced expression of c-fos in the diencephalon and pituitary gland of goats following transportation1

Y. Maejima*,{dagger},2, M. Aoyama*, A. Abe* and S. Sugita*

* Department of Animal Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan; and and {dagger} United Graduate School of Department of Animal Science, Tokyo University of Agriculture and Technology, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
To identify regions of the caprine diencephalone and pituitary gland related to transportation stress, the expression of c-fos protein was examined immunohistochemically as an indicator of neural activation. Ten castrated Shiba goats (Capra hircus), five transported and five controls, were used. Transported goats were trucked for 1 h and killed by transcardiac perfusion 1 h after the end of transportation. Control goats were housed in single pens killed in the same manner and at the same time as the transported goats. The diencephalon and the pituitary gland were removed after perfusion and used for immunostaining. Plasma cortisol concentrations during and after transportation also were investigated. During transportation, plasma cortisol concentrations increased (P < 0.05) compared with those in the controls. In the diencephalon, c-fos immunoreactive cells were detected in the subcallosa, the lateral septal area, the bed nucleus of stria terminalis (BNST), the preoptic hypothalamic area (POA), the suprachiasmatic nucleus (SCN), the supraoptic nucleus, the paraventricular hypothalamic nucleus parvocellular (PVNp), the paraventricular hypothalamic nucleus magnocellular (PVNm), the arcuate nucleus (ARC), the paraventricular thalamic nucleus, and the stria medullaris in both control and transported goats. The numbers of c-fos immunoreactive cells were increased (P < 0.05) by transportation in the PVNm, the PVNp, the BNST, the POA, the ARC, and the SCN (P < 0.10). In the anterior pituitary gland, the number of c-fos immunoreactive cells in transported goats was 4 to 30 times as much as in control goats; however, there were no differences in the intermediate and posterior lobes between control and transported goats. This study has identified regions in the caprine diencephalon and pituitary gland that show transport-induced increases in c-fos immunoreactive cells. In conclusion, the PVNm, the PVNp, the BNST, the POA, the SCN in the diencephalons, and the anterior lobe of pituitary gland may be involved in the stress responses of goats to transportation.

Key Words: C-fos • Diencephalon • Goat • Pituitary Gland • Stress • Transportation


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Literature Cited
 
Transportation has severe physiological effects on domestic animals, which result in serious economic losses to the livestock industry (Phillips et al., 1985Go; Kannan et al., 2000Go). Kannan et al. (2000)Go reported that 2.5 h of transportation led to approximately 10% weight loss in goats. In addition, dark-cutting meat, which can be induced by transport, results in a 10% or greater economic penalty (Hood and Tarrant, 1981Go). Transportation stimulates a remarkable activation of the hypothalamo-pituitary-adrenal (HPA) axis and the sympathetic nervous system; such activation is a well-known stress response in both goats (Nwe et al., 1996Go; Aoyama et al., 2003Go) and sheep (Parrott et al., 1994Go). A decrease in transportation stress is essential for both animal welfare and economic reasons, and considerable attention has been given recently to ways to decrease transportation-induced problems (Broom, 2003Go).

Transportation stress results from vehicle motion, noise, and vibration (Parrott et al., 1994Go). When an animal is exposed to a stressor, information is sent via the cerebral limbic system to the hypothalamus, the initial point of the HPA axis; this information is then sent from the hypothalamus to the pituitary gland. It is known that several nuclei in the diencephalon are involved with the stress reaction (Pacak et al., 1995Go). Expression of the c-fos protein has been used as an indicator of neural activity; therefore, c-fos expression provides an approach for identifying regions related to stress response (Sagar et al., 1988Go; Dragunow and Faull, 1989Go; Vellucci and Parrott, 1994Go). In addition, different stimuli result in different distributions of c-fos immunoreactive cells in the diencephalon (Briski and Gillen, 2001Go) and pituitary gland (Kononen et al., 1992Go). Thus, we may be able to characterize a stressor from the distribution of c-fos immunoreactive cells. In the present study, we evaluated the effects of transportation on expression of c-fos immunoreactive cells in the caprine diencephalon and pituitary gland.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Literature Cited
 
The experiment was conducted from October through December 2001, and from March through May 2002. The temperature was measured at 0900 on each day of the experiment. The range of temperatures during the experimental period was 4 to 15°C.

Animals
The 10 healthy, castrated Shiba goats (Capra hircus) used in the present study were obtained from the experimental station of the University of Tokyo. They ranged in age from 1 to 4 yr and in BW from 15 to 40 kg. The animals were taken to the research farm of the agricultural faculty of Utsunomiya University. Each experimental animal was housed in a single pen (2.5 x 2.5 m) for 1 wk before the experiment to allow adaptation to the new environment. The single pen was bedded with straw. The goats were castrated at least 1 mo before the experiment. They were fed daily with alfalfa hay ad libitum and 100 g of pelleted diet (ZC, Oriental Yeast Co., Ltd., Tokyo, Japan; major ingredients were 35% carbohydrate and 17% protein), and water was available ad libitum.

Transportation and Sampling Schedule
The animals were divided into five pairs. Each pair was made up of one control goat and one animal that was subjected to transportation stress. Each pair was made considering age and BW. Thus, the age and BW of control and transported goat in each pair were similar. A jugular venous catheter was inserted into each animal on the day before the experiment. To avoid isolation stress, the experimental goat was accompanied during transportation by another goat that was not used in the experiment.

During transportation, the goats were driven in a truck around the research farm for 1 h (0900 to 1000), and the animals were kept in individual cages while on the truck. The length of the transportation course was 6.4 km. The average and maximum speeds of the truck were 26 and 60 km/h, respectively. The control goats were housed in their single pens during the transportation period. Food and water withheld from the control goats during the time when the other goats were transported.

In the experimental animals, 2.5- to 3.0-mL blood samples were collected before the start of transportation (0900), during transportation (0915, 0930, 0945, 1000), and 1 h after transportation (1100). In the control animals, blood samples were collected at the same times as the animals undergoing transportation.

Plasma Cortisol Assay
Heparin was added to blood samples to a final concentration of 10 IU/mL. Blood samples were centrifuged immediately at 1,400 x g for 15 min at 4°C and the plasma was stored at –20°C until use. Radioimmunoassay was used for the analysis of plasma cortisol concentrations (DPC cortisol kit, Diagnostic Products Corp., Los Angeles, CA).

Tissue and Fixation
At 1100, animals were deeply anesthetized with pentobarbital (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan; 500 to 1,000 mg of pentobarbital sodium per animal). The animals were then perfused transcardially with 6 L of ringer solution (0.9% NaCl, 0.042% KCl, 0.025% CaCl2 in distilled water, wt/vol) and 6 L of Zanboni’s fixative (0.1 M phosphate buffer, pH 7.4, containing 2.0% paraformaldehyde and 0.2% picric acid, wt/vol). Each brain was removed and cut into several blocks. These blocks were fixed overnight at 4°C and then submersed in 30% (wt/vol) sucrose in Zanboni’s fixative for 3 d. Serial 40-µm coronal sections were cut using a freezing microtome from blocks that included the diencephalon. Sections of 22 to 34 mm also were cut along the interaural line based on the stereotaxic atlas of the Shiba goat brain (Zuccolilli et al., 1995Go). The pituitary glands of 6 of the 10 goats (three control and three transported goats) were removed and treated in the same manner as the diencephalon. Sagittal sections (40 µm) were cut using a freezing microtome from the regions of the pituitary gland containing the anterior lobe (AL), the intermediate lobe (IL), and the posterior lobe (PL). The diencephalon and pituitary gland sections were stored at –20°C in cryoprotectant solution (Watson et al., 1986Go).

C-fos Immunohistochemical Staining
Sections at 200-µm intervals from the series cut through the diencephalon and 20 to 30 pituitary gland sections were stained using a peroxidase–antiperoxidase immunohistochemical method (Sternberger, 1979Go). A polyclonal antibody against the family of human c-fos proteins (sc-52, Santa Cruz Biotechnology Inc., Santa Cruz, CA) was used as the primary antibody. Briefly, anti-c-fos was diluted 1:5000 in PBS (pH 7.4) containing 2% normal goat serum, 2% BSA, and 3% Triton-X. The sections were incubated with the anti-c-fos for 48 h at 4°C. The sections were then incubated with goat anti-rabbit IgG (ICN Pharmaceuticals, Inc., Costa Mesa, CA) diluted 1:500 in PBS containing 2% goat serum. The sections were incubated with rabbit peroxidase–antiper-oxidase complex (ICN Pharmaceuticals Inc.) diluted 1:500 in PBS containing 2% goat serum. Immunoreactions were visualized by incubation in Tris•HCl buffer containing 0.02% 3',3'-diaminobenzidine (Dojin Laboratories, Kumamoto, Japan), 0.3% ammonium nickel (II) sulfate hexahydrate, and 0.015% hydrogen peroxidase for 10 min. To avoid variability of staining, sections from one control and one transported goat in each pair were immunostained simultaneously.

Nissl Staining
Forty-micrometer sections of the diencephalon and the pituitary gland, adjacent to those used for c-fos immunohistochemical staining, were stained by cresyl violet.

Quantification of c-fos Immunoreactivity
The number of c-fos immunoreactive cells in each section of diencephalon was counted under a light microscope (Labophot-2, Nikon, Tokyo, Japan). After the sections were stained with cresyl violet, topographical identification of the nuclei and the areas on the sections of the diencephalons were determined with the stereotaxic atlas of the Shiba goat (Zuccolilli et al., 1995Go).

Five sections were selected at random from each pituitary gland. The c-fos immunoreactive cells in the pituitary gland sections were quantified by counting the number of immunostained cells per unit area (0.0064 mm2). The AL was divided into six areas: zona tuberalis, caudal, fore ventral, hind ventral, hind dorsal, and medial regions (Figure 5AGo). Mean density of c-fos immunoreactive cells in each area of five sections from each goat was obtained.



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  		Figure 5. Schematic diagram of the caprine pituitary gland and c-fos immunoreactive cells in the pituitary gland. Panel A shows the schema of a sagittal section of the caprine pituitary gland. The anterior lobe is divided into six areas: the zona tuberalis (ZT); caudal (C); fore ventral (FV); hind ventral (HV); hind dorsal (HD); and medial (M). The posterior lobe and intermediate lobe are indicated by PL and IL, respectively. Panel B shows the number of c-fos immunoreactive cells per unit area (0.0064 mm2) in the caprine pituitary gland. Each bar represents the mean ± SEM. Because the number of samples was small (n = 3), statistical comparison between means was not performed.

 
Specificity of Anti-c-fos
To confirm the specificity of the anti-c-fos for c-fos, some sections were incubated with primary antibody that had been preabsorbed with a synthetic c-fos peptide (13.4 nmol/mL, PP10-100UG, Calbiochem, Darmstadt, Germany).

Statistical Analyses
The result of cortisol concentration and the number of c-fos immunoreactive cells in the diencephalons and pituitary gland are represented as mean ± SEM. The effect of transportation and time trends on plasma cortisol concentration on the number of c-fos immunoreactive cells in the diencephalons and pituitary gland was analyzed by ANOVA for repeated measures, followed by Tukey’s test. Standard errors of the mean were generated from the ANOVA.

An Excel spreadsheet (Microsoft Corp., Redmond, WA) was used to analyze the data, as described by Yoshida (1998)Go. To analyze plasma cortisol concentrations, the model used to analyze the data contained the term for time x treatment x pair (three-way ANOVA). To analyze the expression of c-fos, the model used to analyze the data contained term for treatment x pair (two-way ANOVA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Literature Cited
 
Plasma Cortisol Concentrations
Plasma cortisol concentrations increased after transportation and were greater (P < 0.05) than those of the control goats (Figure 1Go). The mean peak value observed in transported goats (64 ng/mL at 1000) was nine times greater than the corresponding mean from control goats.



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  		Figure 1. Cortisol concentrations during and after transportation in goats. Each value represents the mean ± SEM, with the SEM generated from ANOVA. The horizontal bar from 0900 to 1000 in the figure indicates the period of transportation. Control goats were housed in their pens as usual and not transported. Letters indicate that values differ from the control at the same time (a; P < 0.05) or compared with the value at 0900 to other time points within treatment (b; P < 0.05).

 
Distribution of the c-fos Immunoreactive Cells in the Diencephalon
None of sections that was incubated with the antibody preabsorbed with a-fos peptide showed immunoreactions. This observation confirms that the antibody used in the present study was specific for c-fos. The c-fos immunoreactive cells of both control and transported goats were located in the area subcallosa, the lateral septal area (LSA), the bed nucleus of stria terminalis (BNST), the preoptic hypothalamic area (POA), the suprachiasmatic nucleus (SCN), the supraoptic nucleus (SON), the paraventricular hypothalamic nucleus parvocellular (PVNp), the paraventricular hypothalamic nucleus magnocellular (PVNm), the arcuate nucleus (ARC), the paraventricular thalamic nucleus and the stria medullaris (Figure 2Go). The number of c-fos immunoreactive cells in the transported goats was greater (P < 0.05) than those in control goats in several regions (Figure 3Go): the PVNp (Figure 4Go), the PVNm (Figure 4Go), the BNST (Figure 3Go), and the POA (Figure 3Go). A similar tendency also was noted in the SCN (P = 0.082) and the ARC (P = 0.052; Figure 3Go).



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  		Figure 2. Schematic diagram illustrating sequential coronal section of the caprine hypothalamus from transported goats showing the distribution of c-fos immunoreactive cells. Panels A, B, C, D, and E are diagrams of sections taken 32, 30, 29, 26, and 23 mm, respectively, from the caprine interaural line. A black dot in any panel shows the presence of approximately 100 c-fos immunoreactive cells. Scale bar = 3 mm. ARC = arcuate nucleus, AS = area subcallosa, BNST = bed nucleus of stria terminalis, LSA = lateral septal area, POA = preoptic area, PVNm = paraventricular hypothalamic nucleus magnocellular, PVNp = paraventricular hypothalamic nucleus parvocellular, PVT = paraventricular thalamic nucleus, SCN = suprachiasmatic nucleus, SM = stria medullaris, and SON = supraoptic nucleus.

 


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  		Figure 3. The number of c-fos immunoreactive cells in caprine diencephalons of control and transported goats. Each bar represents the mean ± SEM. Symbols indicate the comparison for differences between value ({dagger}P < 0.10; *P < 0.05). ARC = arcuate nucleus, AS = area subcallosa, BNST = bed nucleus of stria terminalis, LSA = lateral septal area, POA = preoptic area, PVNm = paraventricular hypothalamic nucleus magnocellular, PVNp = paraventricular hypothalamic nucleus parvocellular, PVT = paraventricular thalamic nucleus, SCN = suprachiasmatic nucleus, SM = stria medullaris, and SON = supraoptic nucleus.

 


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  		Figure 4. Photomicrographs showing c-fos immunoreactive cells in the paraventricular hypothalamic nucleus (PVN) of the caprine diencephalon of a control goat (A) and transported goat (B). Magnocellular and parvocellular areas are indicated in the photomicrographs by M and P, respectively. Horizontal scale bar = 100 µm.

 
Distribution of the c-fos Immunoreactive Cells in the Pituitary Gland
In control goats, there were few c-fos immunoreactive cells in the pituitary gland (Figures 5BGo and 6AGo). The densities of c-fos immunoreactive cells in the six regions of the AL in transported goats ranged from approximately 4 to 30 times greater than those in control goats (Figures 5BGo and 6BGo). In contrast to the AL, the density of c-fos immunoreactive cells in the IL of transported goats was only 1.5 times greater than control goats (Figure 5BGo). In the PL, the densities of c-fos immunoreactive cells in transported goats showed the same value with controls (Figure 5BGo).



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  		Figure 6. Photomicrographs showing c-fos immunorective cells in the zona tuberalis (ZT) of the caprine anterior pituitary gland. Panels A and B show the pituitary gland of control and transported goats, respectively. Horizontal scale bar = 100 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
The expression of c-fos protein, a product of immediate-early gene c-fos, has been used as an indicator of neuronal activity. A number of topographical studies on the response of the central nervous system have been reported by investigating c-fos expression (Sagar et al., 1988Go; Kononen et al., 1992Go; Vellucci and Parrott, 1994Go).

The PVN can be divided into the parvocellular in the medial part (PVNp) and the magnocellular in the lateral part (PVNm). The PVNp contains a significant number of corticotrophin releasing hormone (CRH) neurons. Cells in the PVNm contain arginine vasopressin (AVP) as well as CRH neurons (Kikusui et al., 1997Go). Corticotrophin releasing hormone and AVP play critical roles in the activation of the HPA axis in various species of animal, including ruminants (Redekopp et al., 1985Go). The activation of the HPA axis by transportation is confirmed in the present study by increased plasma cortisol concentrations with transportation. Cells found to express c-fos in the present study can be CRH and AVP neurons. It has been shown that c-fos-immunoreactive cells can be induced in the PVN by transport simulation and isolation stress in sheep (Vellucci and Parrott, 1994Go).

A large number of AVP neurons also are present in the caprine SON (Kikusui et al., 1997Go). Arginine vasopressin in the SON contributes to the regulation of the HPA axis (Aguilera and Rabadan-Diehl, 2000Go). It is known that osmotic stress induces c-fos immunoreactive cells in the SON of rats (Sagar et al., 1998); however, transportation did not increase the number of c-fos immunoreactive cells in the present study. Similarly, restraint and immobilization stress in rats (Briski and Gillen, 2001Go) and transport simulation in pigs (Parrott and Vellucci, 1994Go) and sheep (Vellucci and Parrott, 1994Go) did not induce c-fos immunoreactive cells in the SON. Therefore, it is likely that AVP neurons in the SON are not involved in the HPA axis response to transportation stress in goats.

Generally, stress stimuli are sent to the hypothalamus via the cortex and the limbic systems via neurotransmitter (Pacak et al., 1995Go). In rats, the hypothalamus is connected to the limbic sites via two neuronal pathways: the stria terminalis pathway and the ventral amygdalo-fugal pathway (Pacak et al., 1995Go). There was no significant difference between control and transported goats in the population of c-fos immunoreactive cells of the LSA. The LSA is included in the ventral amygdalofugal pathway. The BNST and the POA, in which the number expressing c-fos immunoreactive cells was significantly increased by transportation, are included in the stria terminalis pathway. If goats have neuronal pathways similar to rats, then our results suggest that the stria terminalis pathway, but not the ventral amygdalofugal pathway, may be involved in transportation-induced activation of the HPA axis in goats.

A significant increase in the number of c-fos immunoreactive cells was seen in the BNST, which is part of the stria terminalis pathway. The BNST projects to the PVN in rats (Sawchenko and Swanson, 1983Go). Changes in glucocorticoid secretion (Dunn, 1987Go) and stress-like behavior (Casada and Dafny, 1991Go) occur in rats after the stimulation of the BNST. In addition, lesions of the BNST in rats induce a decrease in CRH mRNA in the PVN (Herman et al., 1994Go). If the phenomena described above also occur in goats, the BNST may be activated to regulate the response of the HPA axis to transportation stress.

The number of c-fos immunoreactive cells in the POA was increased by transportation. The projection from the median POA is distributed uniformly over that part of PVNm in which AVP-containing cells are concentrated in rats (Sawchenko and Swanson, 1983Go). Thus, the cells in the caprine POA also may contribute to the response of the HPA axis through AVP secretion to transportation.

The number of c-fos immunoreactive cells tended to increase in the SCN (P = 0.082) and ARC (P = 0.052) after transportation. Immobilization stress also induces c-fos-immunoreactive cells in the SCN of rats (Briski and Gillen, 2001Go). Although there is no anatomical connection between the PVN and the SCN (Buiji et al., 1993), the neurons in the SCN send endocrine information to the neighboring nuclei of the PVN (Buijs et al., 1993Go). The bilateral lesion of the SCN in rats induces loss of ACTH (Cascio et al., 1987Go) and of corticosterone circadian rhythm (Moore and Eichler, 1972Go). Previous reports indicate that the SCN may be involved indirectly in the HPA response to stress.

The ARC contains a considerable amount of ß-endorphin derived from proopiomelanocortin (Ellacott and Cone, 2004Go), neuropeptide Y (NPY), neurotensine, and dopamine (Chronwall, 1985Go). In addition, 8% of the neurons that synthesize proopiomelanocortin and 20% that synthesize NPY project into the PVN in rats (Baker and Herkenham, 1995Go). Both {alpha}-melanophore-stimulating hormone and NPY stimulate the CRH neuron in the PVN and regulate the HPA axis in rats (Bell et al., 2000Go). If the caprine ARC has a similar neuronal structure to that of the rat, then the ARC in goats is likely to be involved in the HPA axis responses to transportation.

Interestingly, when rats were stressed by exposure to cats, c-fos immunoreactive cells also were expressed in the dorsomedial part of the ventromedial hypothalamic nucleus (VMH) (Canteras et al., 1997Go). However, novel environmental stress does not induce c-fos expression in the rat VMH (Briski and Gillen, 2001Go). There were no c-fos immunoreactive cells in the VMH of either control or transported goats in the present study. The VMH, along with the mammillary nucleus and the hypothalamic nucleus, forms the medial hypothalamic defensive system to environmental threats (Canteras, 2002Go). As far as the rat is concerned, exposure to a predator, such as a cat, constitutes a sufficient environmental threat as to induce c-fos immunoreactive cell expression in the VMH. The lack of c-fos immunoreactive cells in the VMH of goats suggests that transportation stress does not induce the medial hypothalamic defensive system.

In the pituitary gland, the number of c-fos immunoreactive cells in the AL of transported goats was greater than that of controls. The c-fos immunoreactive cells can be induced in the AL of the rat pituitary gland by noxious thermal stimulation (Autelitano, 1998Go) and by novel environmental stress (Handa et al., 1993Go). Although the type of cells that expressed c-fos immunoreactivity in the present study was not confirmed, Pan et al. (1996)Go reported that most c-fos immunoreactive cells were corticotrophs that contained ACTH and ß-endorphin in rats following electroacupuncture and noxious thermal stimulation. In addition, all of the c-fos-expressing cells contained ACTH in rats after immobilization stress (Kononen et al., 1992Go). On the basis of these reports, it is possible that c-fos immunoreactive cells of the caprine pituitary gland include corticotrophs. The transportation-induced increase in cortisol in the present study supports this hypothesis.

In addition to the corticotrophs, some of c-fos immunoreactive cells in the pituitary glands of the transported goats can be regarded as mammotrophs that secrete prolactin. Plasma prolactin concentrations are increased by transportation in sheep (Vellucci and Parrott, 1994Go). Thus, in this study, mammotrophs also may have expressed a positive immunoreaction for c-fos.

Transportation did not cause an increase in c-fos immunoreactive cells in the IL. In the rat, noxious thermal stress did not induce c-fos immunoreactive cells in the IL (Autelitano, 1998Go) whereas, restraint stress for 4 h induced c-fos immunoreactive cells (Kononen et al., 1992Go). The cause of these differences in the induction of c-fos immunoreactive cells in the IL is not clear, but it may be related to the nature of the stressors, species differences, or both.

In conclusion, this study identified the regions involved in transportation stress in the caprine diencephalone and pituitary gland. Our results indicate that transportation may induce physical stress rather than psychological stress in goats.


   Footnotes
 
1 H. Okamura of the National Institute of Agrobiological Resource is thanked for providing the c-fos peptide for the absorption test. We are grateful to J. Watanabe of Tokyo University of Agriculture and Technology for technical help. This work was partially supported by the Grant Aid for the Young Scientists in Utsunomiya University 2002. Back

2 Correspondence: 350 Minemachi 321-8505 (phone: +81-28-649-5438; fax: +81-28-649-5436; e-mail: maejimayuko{at}hotmail.com).

Received for publication January 23, 2005. Accepted for publication April 29, 2005.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 


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