Decreased GABAergic signaling, fewer parvalbumin-, somatostatin- and calretinin-positive neurons in brain of a rat model of simulated transport stress
Introduction
With the rapid development of animal husbandry in China, the transport of livestock has steadily increased. During transportation, animals may be subjected to various harsh conditions such as bumps, crowding, high or freezing temperatures, hunger, noise, and others. These can cause various symptoms of physical and mental discomfort, including changes in blood composition, behavior, and the endocrine system; as well as psychological conditions, such as depression-like and anxiety-like behavioral responses and even death (Warriss and Brown, 1994). Animals are stimulated by various stressors, which usually result in increased weight loss, mortality, decreased meat quality and even affect the male reproductive system, resulting in sub-fertility or infertility and significant economic losses (Kadim et al., 2009; Minka and Ayo, 2007; Arun et al., 2018). Therefore, transportation stress directly affects animal welfare, and understanding it is crucial to successfully improving transportation conditions for animals, assuring the quality of meat products, and enhancing animal husbandry practices.
γ-aminobutyric acid (GABA), is widely distributed in the CNS and a major inhibitory neurotransmitter in the mammalian CNS (Li and Xu, 2008). GABA can be regulated by multiple subtype transporter proteins such as GAT1 and plays a significant role in regulating sleep and cognition (Klausberger and Somogyi, 2008). There are many psychological problems when GABA contents decrease (Buddhala et al., 2009), such as tension, anxiety, insomnia, and depression (Petroff, 2002), suggesting that GABA is closely related to stress. GAT1 is a crucial GABA transporter in the nervous system, which has the function of reabsorbing GABA (Radian et al., 1990). GAD is a rate-limiting enzyme in the synthesis of GABA, GAD expression is closely related to GABAergic activity. Increased expression of GAD has been considered as a sign of GABAergic hyperactivity (Lindefors, 1993) while decreased expression is associated with reduced GABA synthesis (Mason et al., 2001). Moreover, GABAergic interneurons such as PV, SOM and CR play crucial roles in neuroprotection (Liu et al., 2004; Wu et al., 2004), learning, memory, circadian rhythm, immunity, anxiety and depression (Calza et al., 1993; Dawson and Dawson, 1996).
A study result has shown that the dorsal striatum and medial PFC plays essential roles in automatic and controlled processing of a future conditioned stimulus (Pérez-Díaz et al., 2017). The PFC is related to cognitive function, including learning and memory (Euston et al., 2012; Good, 2002). The striatum plays an important role in movement control (Kravitz and KreitzerStriatal, 2012). The striatum can be divided into the dorsal lateral (DL), dorsal medial (DM), ventral lateral (VL), and ventral medial (VM) striatum regions. Both the PFC activity and the response to a pleasant stimulus from the medial frontal cortex of patients with depression were demonstrated to be low (Mitterschiffthaler et al., 2003). The striatum and PFC contain many GABAergic neurons (Zhu et al., 2017). The striatum widely receives excitatory afferents from the cerebral cortex, thalamus and midbrain (Reiner et al., 1998), and projects GABAergic neurons to the reticular part of the substantia nigra, the medial part of the globus pallidus and the lateral part of the globus pallidus. GABAergic neurons are the main neurons in the striatum. In the striatum, 95% of the neurons are medium-sized spinous process neurons (Medium spiny neurons, MSN), and MSN are GABAergic inhibitory neurons; 5% are large non-spinous cholinergic neurons and medium-sized non-spinous GABA neurons, including intermediate neurons such as SOM, CR and PV positive neurons (Tepper et al., 2004). Stress can cause neuroendocrine dysfunction dominated by the HPA axis, which leads to a series of neuropsychiatric symptoms. Glutamatergic neurons in PFC, hippocampus and GABAergic neurons in medial amygdaloid nucleus and lateral septum project to hypothalamic paraventricular nucleus (PVN) to regulate the activity of HPA axis. The hypothalamic–pituitary–adrenal (HPA) axis regulates stress through negative feedback of the GABAergic signal from the ventral hippocampus to the hypothalamic paraventricular nucleus (Cullinan et al., 1993).
In the current study, we investigated the effect of transport stress on behavior and GABAergic neurons in the striatum and PFC of rats based on a rat model of simulated transport stess (STS). Serum concentrations of corticosterone (CORT) and norepinephrine (NE) were compared. The changes of the expression of GABAergic interneurons in PFC and striatum of rats were analyzed to further explore the function of GABAergic neurons in stress responses.
Section snippets
Animals
Thirty-six adult male Sprague-Dawley rats weighing 200 to 230 g were obtained from Peking University Laboratory Animal Center (SCXK, Beijing, China, 2012–0001) (Wan et al., 2014; Wang et al., 2019).
The rats were routinely housed for 7 days to adapt to the environment. The rats were housed in cages (54 × 39 × 20 cm) under standard laboratory conditions: room temperature (25 ± 2 °C), 12 h light/dark cycle, 60% ± 5% humidity, and free access to water and food. Lights were on and off at 8:00 am and
Responses and serum concentrations change of CORT and NE after STS in rats
The behavioral scores of rats in each group were analyzed by one-way ANOVA, and the difference was extremely significant (df = 2, f = 363.909, P = 0.001). Compared with the control group, the rats in the STS groups exhibited significantly lower scores of the OFT in TS1d (P = 0.001) and TS7d (P = 0.001) (Fig. 2A). However, the total scores of the OFT in the TS7d group were significantly higher than the TS1d group (P = 0.001). The serum concentrations of CORT in each group were analyzed by
Discussion
This study aimed to explore the effect of stress on behavior, HPA axis, GABA transmitters and the distribution of GABAergic interneurons in the PFC and striatum by a rat model of simulated transport stress (STS). We successfully induced the simulated transport stress model of rats, and made the rats show anxiety-like behavior. The results showed that STS may affect the HPA axis to increase GABA neurotransmitters, thus resisting excitotoxicity and regulating stress response. STS significantly
Conclusions
In conclusion, rats exhibited anxiety-like behavior after STS which then altered the serum concentrations of CORT and NE of rats. Our findings demonstrated that STS can reduce PV-, CR-, and SOM-positive cells, decrease GABAergic signaling in the PFC and striatum, increase excitotoxicity and decrease inhibition function of brain, thus aggravating brain injury. TS7d may increase expression of GABA neurotransmitters through regulation of HPA axis, thus resisting excitotoxicity and regulating
Author contributions
The study was conceived and designed by Y Ma. All data were collected by W Zhang and S Wang. Statistical analyses were conducted by S Wang, Z Deng and J Wang. The manuscript was drafted by S Wang, Z Deng, J Wang and Y Ma. Administrative, technical, and material supports were provided by Y Ma, F Liu and J Xu.
Declaration of Competing Interest
All authors, Shujing Wang, Ziteng Deng, Jia Wang, Wenjun Zhang, Fenghua Liu, Jianqin Xu, Yunfei Ma, agree to the publication of this manuscript and declare that they have no conflicts of interest.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (no. 31772686, no. 31502025) and the Ministry of Agriculture, Public Service Sector Agriculture Research Projects (no. 201403051-07). This manuscript was edited by Wallace Academic Editing.
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These authors contributed equally to this work.