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Journal of Animal Reproduction and Biotechnology 2023; 38(2): 77-83

Published online June 30, 2023

https://doi.org/10.12750/JARB.38.2.77

Copyright © The Korean Society of Animal Reproduction and Biotechnology.

Expression of the serotonin 1A receptor in the horse brain

Yeonju Choi1 and Minjung Yoon2,*

1Department of Animal Science and Biotechnology, Kyungpook National University, Sangju 37224, Korea
2Department of Horse, Companion and Wild Animal Science, Kyungpook National University, Sangju 37224, Korea

Correspondence to: Minjung Yoon
E-mail: mjyoonemail@gmail.com

Received: April 25, 2023; Revised: June 14, 2023; Accepted: June 14, 2023

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Serotonin receptors can be divided into seven different families with various subtypes. The serotonin 1A (5-HT1A) receptor is one of the most abundant subtypes in animal brains. The expression of 5-HT1A receptors in the brain has been reported in various animals but has not been studied in horses. The 5-HT1A receptor functions related to emotions and behaviors, thus it is important to understand the functional effects and distribution of 5-HT1A receptors in horses to better understand horse behavior and its associated mechanism.
Methods: Brain samples from seven different regions, which were the frontal, central, and posterior cerebral cortices, cerebellar cortex and medulla, thalamus, and hypothalamus, were collected from six horses. Western blot analysis was performed to validate the cross-reactivity of rabbit anti-5-HT1A receptor antibody in horse samples. Immunofluorescence was performed to evaluate the localization of 5-HT1A receptors in the brains.
Results: The protein bands of 5-HT1A receptor appeared at approximately 50 kDa in the frontal, central, and posterior cerebral cortices, cerebellar cortex, thalamus, and hypothalamus. In contrast, no band was observed in the cerebellar medulla. Immunofluorescence analysis showed that the cytoplasm of neurons in the cerebral cortices, thalamus, and hypothalamus were immunostained for 5-HT1A receptors. In the cerebellar cortex, 5-HT1A was localized in the cytoplasm of Purkinje cells.
Conclusions: In conclusion, the study suggests that 5-HT and 5-HT1A receptor systems may play important roles in the central nervous system of horses, based on the widespread distribution of the receptors in the horse brain.

Keywords: brain, horse, receptor, serotonin

Serotonin is a biogenic monoamine present in a variety of organisms. It functions as a neurotransmitter in animals and found in a variety of sites in the central and peripheral nervous system (Berger et al., 2009). It has been implicated in various physiological and behavioral processes.

Serotonergic signaling appears to play a key role in the generation and modification of various cognitive and behavioral functions such as sleep, mood, pain, addiction, locomotion, sexual activity, depression, anxiety, alcohol abuse, aggression, and learning. Novel roles for serotonin in heart disease, asthma, and phagocytosis have been recently reported.

The serotonin pathway plays a crucial role in brain development processes such as neurogenesis and axonal branching during various stages of development. Disruptions in serotonergic system have been implicated in the etiology of mental disorders such as schizophrenia, migraine, depression, suicidal behavior, infantile autism, eating disorders, and obsessive-compulsive disorder.

Several documents have reported that 5-HT influences animal behaviors such as social interaction, aggressiveness, and anxiety (Bacqué-Cazenave et al., 2020). Thus, investigation of the serotonergic system is important for understanding animal behavior and psychology.

5-HT receptors are groups of G protein-coupled receptors and are divided into seven families with at least 14 subtypes. 5-HT receptors are widely distributed and mediate the action of 5-HT on brain functions (Hannon and Hoyer, 2008; Mosienko et al., 2015). These receptor subtypes vary in their roles in controlling the serotonergic system and in their regional expression. Among these subtypes, the 5-HT1A receptor has been studied the most and is one of the most abundant and widely distributed 5-HT receptors in the brain (Albert and Vahid-Ansari, 2019).

The 5-HT1A receptor functions related to emotions and behaviors, such as decreasing aggression (de Boer and Koolhaas, 2005) and increasing sociality (Thompson et al., 2007), have been described well in several studies. Our previous study showed that 5-HT was negatively correlated with dominance in horses and positively correlated with their trainability (Kim et al., 2021). This suggests that 5-HT1A receptor activation may be directly related to the temperament of horses. Thus, to better understand horse behavior and its associated mechanisms, it is important to understand the functional effects and distribution of 5-HT1A receptors in the brain.

The existence of 5-HT1A receptors has been well described in various animals, including humans (Ito et al., 1999), monkeys (de Almeida and Mengod, 2008), and rodents (Bonnin et al., 2006). However, the precise regional patterns of 5-HT1A receptors have not been reported in horses. Therefore, the present study was designed to immunohistochemically localize 5-HT1A receptors in the horse brain.

Sample preparation

Brain tissues were collected from six Thoroughbred horses, including three mares and three geldings (1-2 years old). The horses were sacrificed at a local slaughterhouse in Jeju, South Korea. The brains were removed from the skulls and transported immediately to the laboratory in a 4℃ ice box. Samples were obtained from seven different regions of the brain, categorized as frontal, central, and posterior cerebral cortices; cerebellar cortex; cerebellar medulla; thalamus; and hypothalamus (Fig. 1). For the immunofluorescence assay, each tissue sample was cut into pieces of approximately 1 cm3 and immersed in 4% paraformaldehyde for 24 h at room temperature for tissue fixation. Following dehydration using a series of ethanol concentrations (25%, 50%, 70%, 80%, 90%, and 100%), tissues were embedded in paraffin. In addition, tissues (1 mg of each piece) were snap-frozen in liquid nitrogen and stored at -80℃ for western blot analysis.

Figure 1. The right lateral side of the horse brain. Frontal cerebral cortex (a); central cerebral cortex (b); posterior cerebral lobe (c); thalamus (d); hypothalamus (e); cerebellar cortex (f); cerebellar medulla (g).

Western blot analysis

Western blot analysis was performed to validate the cross-reactivity of the rabbit anti-5-HT1A receptor antibody (ab85612; Abcam, Cambridge, UK) to the 5-HT1A receptor present in horse brains. This analysis was performed as previously described with minor modifications (Choi et al., 2020). The protein from each brain tissue sample was extracted and diluted with radioimmunoprecipitation assay buffer (WE324435; ThermoFisher Scientific, Massachusetts, USA) to 2 mg/mL. Samples were loaded onto 10% SDS-polyacrylamide gels and separated using a Mini-Protean system (Bio-Rad, Hercules, CA, USA). The membrane was incubated with 5% blotto (2.5 g nonfat milk, 50 mL PBS, and 0.1% Tween 20) for blocking, and then incubated with 1 μg/mL of anti-5-HT1A receptor antibody and 0.1 μg/mL β-actin (positive control) diluted with BLOTTO milk for 12 h at 4℃.

Immunofluorescence

Immunofluorescence was performed to evaluate the expression of 5-HT1A receptors in horse brains using a previously described protocol, with minor modifications (Choi et al., 2020). Brain tissue slides were dehydrated using phased ethanol concentrations of 100%, 95%, 80%, 70%, 50%, and 25% and then treated in citrate antigen retrieval buffer for 30 min at 97.5℃. After antigen retrieval, the slides were incubated with blocking buffer (5% donkey serum; Sigma, St. Louis, MO, USA). The anti-5-HT1A receptor antibody (5 μg/mL) was used as the primary antibody diluted in the blocking buffer. In addition, 1 μg/mL donkey anti-rabbit IgG (Alexa FluorTM 488; ThermoFisher Scientific, USA) was used as a secondary antibody.

Imaging

The slides were immunolabeled using a Leica DM 2500 fluorescent microscope (Leica, Wetzlar, Germany). The microscope was equipped with an external light source (EL 6000; Leica). Green fluorescence was observed using a dual-emission fluorescein isothiocynate/tetramethylrhodamine isothiocyanatete filter. Images were captured using a Leica DFC 450 C digital camera.

Cross-reactivity of the rabbit anti-5-HT1A receptor antibody in horse brain tissues

The cross-reactivity of the anti-5-HT1A receptor antibody in the brain tissues was evaluated using western blot analysis. The 5-HT1A receptor protein bands of the frontal, central, and posterior cerebral cortices; cerebellar cortex; thalamus; and hypothalamus, but not the cerebellar medulla, appeared at approximately 50 kDa (Fig. 2). The β-actin band was detected at an approximate molecular weight of 45 kDa in all horse brain tissue samples.

Figure 2. Cross-reactivity of serotonin 1A (5-HT1A) receptor in horse brains. The 5-HT1A receptor protein band appeared at a molecular size of approximately 50 kDa. The β-actin band was detected at approximately 45 kDa in horse brains.

Expression of the 5-HT1A receptor in horse brain tissues

Immunohistochemistry was performed to investigate 5-HT1A receptor expression in the horse brain tissues. Positive expression of 5-HT1A receptors was found in the frontal, central, and posterior cerebral cortices; cerebellar cortex; thalamus; and hypothalamus (Fig. 3A to 3D, 3F and 3G). Immunolabeling of 5-HT1A receptors was found in the cytoplasm of neuronal cells. In the cerebellar cortex, 5-HT1A receptors were also expressed in the cytoplasm of Purkinje cells (Fig. 3D). Expression of 5-HT1A receptors was not detected in the cerebellar medulla (Fig. 3E).

Figure 3. Localization of serotonin 1A (5-HT1A) receptor in horse brains. Immunolabeling of the 5-HT1A receptor was detected in the cytoplasm of neurons in the cerebral cortex (A-C), thalamus (F), hypothalamus (G), and cytoplasm of Purkinje cells in the cerebellar cortex (D). The expression of the 5-HT1A receptor was not detected in the cerebellar medulla (E). DAPI, 4’6-diamidino-2-phenylindole; N, neuron; PC, Purkinje cell.

The present study was performed to investigate the regional expression of 5-HT1A receptors in horse brains. The western blot results demonstrated that the 5-HT1A receptor protein was present in the brain tissues of horses, and the rabbit anti 5-HT1A receptor antibody had cross-reactivity to 5-HT1A receptors in horse brains. The immunofluorescence results showed that 5-HT1A receptors were expressed in the cytoplasmic area of neuronal cells in the brain tissues. These data are comparable to those of 5-HT1A receptor expression in the brains of mice (Rasul et al., 2013), primates (Azmitia et al., 1996), and humans (Hadjighassem et al., 2009). A study on the distribution of mRNA coding for 5-HT1A receptors showed that the overall pattern of 5-HT1A receptor distribution is comparable across species (Lanfumey and Hamon, 2000). Thus, the results of the present study suggest that 5-HT1A expression patterns in the neuronal cells of the brain are conserved among species.

The 5-HT1A receptor is one of the most abundant subtypes expressed in the mammalian brain. Immunofluorescence revealed that antigen-specific binding sites were widely distributed in the cerebral cortex, cerebellar cortex, thalamus, and hypothalamus. This result is in accordance with those of previous studies which investigated the distribution of 5-HT1A receptors in the CNS (de Almeida and Mengod, 2008). In the present study, the cerebrum of horses was divided into three different regions (frontal, central, and posterior parts of the cerebrum), and anti-5-HT1A receptor antibody expression was detected in all three regions. According to previous studies on rodents and humans, 5-HT1A receptors generally exist in the cortical and limbic brain areas associated with memory functions (Hall et al., 1997). The 5-HT1A receptors influence the activity of glutamatergic, cholinergic, and GABAergic neurons in the cerebral area, affecting declarative and nondeclarative memory functions (Ogren et al., 2008). In horses, 5-HT1A receptor gene polymorphisms affect tractability, which is the ability of horses to be trained or easily controlled (Hori et al., 2016). Horses perform a range of cognitive tasks, including discriminative training, memorization, and concept formation (Brubaker and Udell, 2016). Our present study suggests that 5-HT1A receptors in the cerebral cortex are highly related to the memory and cognition of horses and affect their tractability.

The cerebellum plays an important role in the cognitive function and motor control of animals (Wolf et al., 2009). Immunofluorescence analysis demonstrated 5-HT1A receptor expression in the cytoplasm of neurons in the cerebellar cortex, suggesting their association with cognition and motor learning in horses. The presence of immunoreactivity of 5-HT1A receptors in the cerebellum is in accordance with previous data showing high densities of 5-HT1A binding sites (Parsey et al., 2005). In addition, innervation of the cerebellar cortex by serotonergic fibers has been well documented using fluorescence histochemistry, autoradiography in serotonergic nerve terminals, and immunocytochemistry (Bishop and Ho, 1985). In contrast, anti-5-HT1A receptor antibody-positive cells were not observed in the cerebellar medulla. These data agree with the western blot results showing that the protein band of the 5-HT1A receptor was not detected in the cerebellar medulla sample. These results may be due to a lack of cells in the cerebellar medulla. The inner medulla of white matter contains only nerve fiber except cell bodies (Voogd and Glickstein, 1998); thus, 5-HT1A receptor expression was not found.

A 5-HT1A-immunoreactive response was also observed in Purkinje cells in this study. Purkinje cells are distinctive neurons in the brain that comprise the Purkinje layer of the cerebellar cortex (Voogd and Glickstein, 1998). These cells have been reported to express 5-HT1A receptors (Rasul et al., 2013), which is consistent with our data. Purkinje cells are a class of GABAergic inhibitory neurons. As previously described, 5-HT1A receptors regulate the activity of GABAergic neurons; thus, 5-HT1A receptors may be related to the GABA system in horse brains.

In the present study, immunoreactive expression of 5-HT1A receptors was also observed in the thalamus and hypothalamus. The existence of 5-HT1A receptors in the thalamus and hypothalamus has been well documented in various animals such as rats (Chalmers and Watson, 1991), ferrets (Monckton and McCormick, 2002), and primates (Gundlah et al., 1999). The present immunofluorescence results showed the similarity of the distribution of 5-HT1A receptors in the diencephalon of horses compared to that in other species. The thalamus is located in the central brain area and consists of a large number of neuronal clusters with diverse functions and connections (Jones, 2012). As nerve fibers project from the thalamus to the cerebral cortex in all directions, the thalamus is often thought to act as a gate that regulates the exchange of information to the central cortex. This regulation is mediated by the input of ascending activating systems that release neurotransmitters, including 5-HT, from the brainstem and hypothalamus (Steriade et al., 2013). The hypothalamus is the main link between the endocrine and nervous systems (Saper and Lowell, 2014). The results of the present study revealed the presence of 5-HT1A receptors in both the thalamus and hypothalamus of horses. These data are supported by a previous study that investigated the influence of training and simulated race activities on 5-HT levels in horses (Medica et al., 2020). The 5-HT level increased after simulated race and training activities. Exercise triggers physical stress that affects homeostasis, which is regulated by the hypothalamic-pituitary-adrenal system (Zschucke et al., 2015). Thus, each part of the diencephalon of horses seems to be connected to serotonergic fibers and regulate neuroendocrine function.

In summary, the present study demonstrated the localization of 5-HT1A receptors in the CNS of horses for the first time. The data showed that 5-HT1A receptors are generally distributed in the brains of horses, including in the cerebrum, cerebellum, and diencephalon. Furthermore, 5-HT has been proposed to modulate cognitive and behavioral functions in animals, and the results of this study indicate that 5-HT1A receptors are highly related to these functions. These data provide insights into the possible physiological functions of this receptor subtype.

In conclusion, the present study found that 5-HT1A receptor is present in various regions of the horse brain, including cerebral cortices, cerebellar cortex, thalamus, and hypothalamus. The study suggests that the 5-HT and 5-HT1A receptor systems may play significant roles in the central nervous system of horses, based on the widespread distribution of 5-HT1A receptors. Understanding the 5-HT1A receptors in horses may provide insights into horse behavior and its associated mechanism.

The authors thank Heejun Jung, Junyoung Kim, Sungmin Kim, Youngwook Jung, and Yubin Song for their assistance with sample collection.

Conceptualization, Y.C. and M.Y.; methodology, Y.C.; Investigation, Y.C.; data curation, Y.C.; writing—original draft preparation, Y.C.; writing—review and editing, M.Y; supervision, M.Y.; project administration, Y.C.; funding acquisition, M.Y.

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Article

Original Article

Journal of Animal Reproduction and Biotechnology 2023; 38(2): 77-83

Published online June 30, 2023 https://doi.org/10.12750/JARB.38.2.77

Copyright © The Korean Society of Animal Reproduction and Biotechnology.

Expression of the serotonin 1A receptor in the horse brain

Yeonju Choi1 and Minjung Yoon2,*

1Department of Animal Science and Biotechnology, Kyungpook National University, Sangju 37224, Korea
2Department of Horse, Companion and Wild Animal Science, Kyungpook National University, Sangju 37224, Korea

Correspondence to:Minjung Yoon
E-mail: mjyoonemail@gmail.com

Received: April 25, 2023; Revised: June 14, 2023; Accepted: June 14, 2023

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background: Serotonin receptors can be divided into seven different families with various subtypes. The serotonin 1A (5-HT1A) receptor is one of the most abundant subtypes in animal brains. The expression of 5-HT1A receptors in the brain has been reported in various animals but has not been studied in horses. The 5-HT1A receptor functions related to emotions and behaviors, thus it is important to understand the functional effects and distribution of 5-HT1A receptors in horses to better understand horse behavior and its associated mechanism.
Methods: Brain samples from seven different regions, which were the frontal, central, and posterior cerebral cortices, cerebellar cortex and medulla, thalamus, and hypothalamus, were collected from six horses. Western blot analysis was performed to validate the cross-reactivity of rabbit anti-5-HT1A receptor antibody in horse samples. Immunofluorescence was performed to evaluate the localization of 5-HT1A receptors in the brains.
Results: The protein bands of 5-HT1A receptor appeared at approximately 50 kDa in the frontal, central, and posterior cerebral cortices, cerebellar cortex, thalamus, and hypothalamus. In contrast, no band was observed in the cerebellar medulla. Immunofluorescence analysis showed that the cytoplasm of neurons in the cerebral cortices, thalamus, and hypothalamus were immunostained for 5-HT1A receptors. In the cerebellar cortex, 5-HT1A was localized in the cytoplasm of Purkinje cells.
Conclusions: In conclusion, the study suggests that 5-HT and 5-HT1A receptor systems may play important roles in the central nervous system of horses, based on the widespread distribution of the receptors in the horse brain.

Keywords: brain, horse, receptor, serotonin

INTRODUCTION

Serotonin is a biogenic monoamine present in a variety of organisms. It functions as a neurotransmitter in animals and found in a variety of sites in the central and peripheral nervous system (Berger et al., 2009). It has been implicated in various physiological and behavioral processes.

Serotonergic signaling appears to play a key role in the generation and modification of various cognitive and behavioral functions such as sleep, mood, pain, addiction, locomotion, sexual activity, depression, anxiety, alcohol abuse, aggression, and learning. Novel roles for serotonin in heart disease, asthma, and phagocytosis have been recently reported.

The serotonin pathway plays a crucial role in brain development processes such as neurogenesis and axonal branching during various stages of development. Disruptions in serotonergic system have been implicated in the etiology of mental disorders such as schizophrenia, migraine, depression, suicidal behavior, infantile autism, eating disorders, and obsessive-compulsive disorder.

Several documents have reported that 5-HT influences animal behaviors such as social interaction, aggressiveness, and anxiety (Bacqué-Cazenave et al., 2020). Thus, investigation of the serotonergic system is important for understanding animal behavior and psychology.

5-HT receptors are groups of G protein-coupled receptors and are divided into seven families with at least 14 subtypes. 5-HT receptors are widely distributed and mediate the action of 5-HT on brain functions (Hannon and Hoyer, 2008; Mosienko et al., 2015). These receptor subtypes vary in their roles in controlling the serotonergic system and in their regional expression. Among these subtypes, the 5-HT1A receptor has been studied the most and is one of the most abundant and widely distributed 5-HT receptors in the brain (Albert and Vahid-Ansari, 2019).

The 5-HT1A receptor functions related to emotions and behaviors, such as decreasing aggression (de Boer and Koolhaas, 2005) and increasing sociality (Thompson et al., 2007), have been described well in several studies. Our previous study showed that 5-HT was negatively correlated with dominance in horses and positively correlated with their trainability (Kim et al., 2021). This suggests that 5-HT1A receptor activation may be directly related to the temperament of horses. Thus, to better understand horse behavior and its associated mechanisms, it is important to understand the functional effects and distribution of 5-HT1A receptors in the brain.

The existence of 5-HT1A receptors has been well described in various animals, including humans (Ito et al., 1999), monkeys (de Almeida and Mengod, 2008), and rodents (Bonnin et al., 2006). However, the precise regional patterns of 5-HT1A receptors have not been reported in horses. Therefore, the present study was designed to immunohistochemically localize 5-HT1A receptors in the horse brain.

MATERIALS AND METHODS

Sample preparation

Brain tissues were collected from six Thoroughbred horses, including three mares and three geldings (1-2 years old). The horses were sacrificed at a local slaughterhouse in Jeju, South Korea. The brains were removed from the skulls and transported immediately to the laboratory in a 4℃ ice box. Samples were obtained from seven different regions of the brain, categorized as frontal, central, and posterior cerebral cortices; cerebellar cortex; cerebellar medulla; thalamus; and hypothalamus (Fig. 1). For the immunofluorescence assay, each tissue sample was cut into pieces of approximately 1 cm3 and immersed in 4% paraformaldehyde for 24 h at room temperature for tissue fixation. Following dehydration using a series of ethanol concentrations (25%, 50%, 70%, 80%, 90%, and 100%), tissues were embedded in paraffin. In addition, tissues (1 mg of each piece) were snap-frozen in liquid nitrogen and stored at -80℃ for western blot analysis.

Figure 1.The right lateral side of the horse brain. Frontal cerebral cortex (a); central cerebral cortex (b); posterior cerebral lobe (c); thalamus (d); hypothalamus (e); cerebellar cortex (f); cerebellar medulla (g).

Western blot analysis

Western blot analysis was performed to validate the cross-reactivity of the rabbit anti-5-HT1A receptor antibody (ab85612; Abcam, Cambridge, UK) to the 5-HT1A receptor present in horse brains. This analysis was performed as previously described with minor modifications (Choi et al., 2020). The protein from each brain tissue sample was extracted and diluted with radioimmunoprecipitation assay buffer (WE324435; ThermoFisher Scientific, Massachusetts, USA) to 2 mg/mL. Samples were loaded onto 10% SDS-polyacrylamide gels and separated using a Mini-Protean system (Bio-Rad, Hercules, CA, USA). The membrane was incubated with 5% blotto (2.5 g nonfat milk, 50 mL PBS, and 0.1% Tween 20) for blocking, and then incubated with 1 μg/mL of anti-5-HT1A receptor antibody and 0.1 μg/mL β-actin (positive control) diluted with BLOTTO milk for 12 h at 4℃.

Immunofluorescence

Immunofluorescence was performed to evaluate the expression of 5-HT1A receptors in horse brains using a previously described protocol, with minor modifications (Choi et al., 2020). Brain tissue slides were dehydrated using phased ethanol concentrations of 100%, 95%, 80%, 70%, 50%, and 25% and then treated in citrate antigen retrieval buffer for 30 min at 97.5℃. After antigen retrieval, the slides were incubated with blocking buffer (5% donkey serum; Sigma, St. Louis, MO, USA). The anti-5-HT1A receptor antibody (5 μg/mL) was used as the primary antibody diluted in the blocking buffer. In addition, 1 μg/mL donkey anti-rabbit IgG (Alexa FluorTM 488; ThermoFisher Scientific, USA) was used as a secondary antibody.

Imaging

The slides were immunolabeled using a Leica DM 2500 fluorescent microscope (Leica, Wetzlar, Germany). The microscope was equipped with an external light source (EL 6000; Leica). Green fluorescence was observed using a dual-emission fluorescein isothiocynate/tetramethylrhodamine isothiocyanatete filter. Images were captured using a Leica DFC 450 C digital camera.

RESULTS

Cross-reactivity of the rabbit anti-5-HT1A receptor antibody in horse brain tissues

The cross-reactivity of the anti-5-HT1A receptor antibody in the brain tissues was evaluated using western blot analysis. The 5-HT1A receptor protein bands of the frontal, central, and posterior cerebral cortices; cerebellar cortex; thalamus; and hypothalamus, but not the cerebellar medulla, appeared at approximately 50 kDa (Fig. 2). The β-actin band was detected at an approximate molecular weight of 45 kDa in all horse brain tissue samples.

Figure 2.Cross-reactivity of serotonin 1A (5-HT1A) receptor in horse brains. The 5-HT1A receptor protein band appeared at a molecular size of approximately 50 kDa. The β-actin band was detected at approximately 45 kDa in horse brains.

Expression of the 5-HT1A receptor in horse brain tissues

Immunohistochemistry was performed to investigate 5-HT1A receptor expression in the horse brain tissues. Positive expression of 5-HT1A receptors was found in the frontal, central, and posterior cerebral cortices; cerebellar cortex; thalamus; and hypothalamus (Fig. 3A to 3D, 3F and 3G). Immunolabeling of 5-HT1A receptors was found in the cytoplasm of neuronal cells. In the cerebellar cortex, 5-HT1A receptors were also expressed in the cytoplasm of Purkinje cells (Fig. 3D). Expression of 5-HT1A receptors was not detected in the cerebellar medulla (Fig. 3E).

Figure 3.Localization of serotonin 1A (5-HT1A) receptor in horse brains. Immunolabeling of the 5-HT1A receptor was detected in the cytoplasm of neurons in the cerebral cortex (A-C), thalamus (F), hypothalamus (G), and cytoplasm of Purkinje cells in the cerebellar cortex (D). The expression of the 5-HT1A receptor was not detected in the cerebellar medulla (E). DAPI, 4’6-diamidino-2-phenylindole; N, neuron; PC, Purkinje cell.

DISCUSSION

The present study was performed to investigate the regional expression of 5-HT1A receptors in horse brains. The western blot results demonstrated that the 5-HT1A receptor protein was present in the brain tissues of horses, and the rabbit anti 5-HT1A receptor antibody had cross-reactivity to 5-HT1A receptors in horse brains. The immunofluorescence results showed that 5-HT1A receptors were expressed in the cytoplasmic area of neuronal cells in the brain tissues. These data are comparable to those of 5-HT1A receptor expression in the brains of mice (Rasul et al., 2013), primates (Azmitia et al., 1996), and humans (Hadjighassem et al., 2009). A study on the distribution of mRNA coding for 5-HT1A receptors showed that the overall pattern of 5-HT1A receptor distribution is comparable across species (Lanfumey and Hamon, 2000). Thus, the results of the present study suggest that 5-HT1A expression patterns in the neuronal cells of the brain are conserved among species.

The 5-HT1A receptor is one of the most abundant subtypes expressed in the mammalian brain. Immunofluorescence revealed that antigen-specific binding sites were widely distributed in the cerebral cortex, cerebellar cortex, thalamus, and hypothalamus. This result is in accordance with those of previous studies which investigated the distribution of 5-HT1A receptors in the CNS (de Almeida and Mengod, 2008). In the present study, the cerebrum of horses was divided into three different regions (frontal, central, and posterior parts of the cerebrum), and anti-5-HT1A receptor antibody expression was detected in all three regions. According to previous studies on rodents and humans, 5-HT1A receptors generally exist in the cortical and limbic brain areas associated with memory functions (Hall et al., 1997). The 5-HT1A receptors influence the activity of glutamatergic, cholinergic, and GABAergic neurons in the cerebral area, affecting declarative and nondeclarative memory functions (Ogren et al., 2008). In horses, 5-HT1A receptor gene polymorphisms affect tractability, which is the ability of horses to be trained or easily controlled (Hori et al., 2016). Horses perform a range of cognitive tasks, including discriminative training, memorization, and concept formation (Brubaker and Udell, 2016). Our present study suggests that 5-HT1A receptors in the cerebral cortex are highly related to the memory and cognition of horses and affect their tractability.

The cerebellum plays an important role in the cognitive function and motor control of animals (Wolf et al., 2009). Immunofluorescence analysis demonstrated 5-HT1A receptor expression in the cytoplasm of neurons in the cerebellar cortex, suggesting their association with cognition and motor learning in horses. The presence of immunoreactivity of 5-HT1A receptors in the cerebellum is in accordance with previous data showing high densities of 5-HT1A binding sites (Parsey et al., 2005). In addition, innervation of the cerebellar cortex by serotonergic fibers has been well documented using fluorescence histochemistry, autoradiography in serotonergic nerve terminals, and immunocytochemistry (Bishop and Ho, 1985). In contrast, anti-5-HT1A receptor antibody-positive cells were not observed in the cerebellar medulla. These data agree with the western blot results showing that the protein band of the 5-HT1A receptor was not detected in the cerebellar medulla sample. These results may be due to a lack of cells in the cerebellar medulla. The inner medulla of white matter contains only nerve fiber except cell bodies (Voogd and Glickstein, 1998); thus, 5-HT1A receptor expression was not found.

A 5-HT1A-immunoreactive response was also observed in Purkinje cells in this study. Purkinje cells are distinctive neurons in the brain that comprise the Purkinje layer of the cerebellar cortex (Voogd and Glickstein, 1998). These cells have been reported to express 5-HT1A receptors (Rasul et al., 2013), which is consistent with our data. Purkinje cells are a class of GABAergic inhibitory neurons. As previously described, 5-HT1A receptors regulate the activity of GABAergic neurons; thus, 5-HT1A receptors may be related to the GABA system in horse brains.

In the present study, immunoreactive expression of 5-HT1A receptors was also observed in the thalamus and hypothalamus. The existence of 5-HT1A receptors in the thalamus and hypothalamus has been well documented in various animals such as rats (Chalmers and Watson, 1991), ferrets (Monckton and McCormick, 2002), and primates (Gundlah et al., 1999). The present immunofluorescence results showed the similarity of the distribution of 5-HT1A receptors in the diencephalon of horses compared to that in other species. The thalamus is located in the central brain area and consists of a large number of neuronal clusters with diverse functions and connections (Jones, 2012). As nerve fibers project from the thalamus to the cerebral cortex in all directions, the thalamus is often thought to act as a gate that regulates the exchange of information to the central cortex. This regulation is mediated by the input of ascending activating systems that release neurotransmitters, including 5-HT, from the brainstem and hypothalamus (Steriade et al., 2013). The hypothalamus is the main link between the endocrine and nervous systems (Saper and Lowell, 2014). The results of the present study revealed the presence of 5-HT1A receptors in both the thalamus and hypothalamus of horses. These data are supported by a previous study that investigated the influence of training and simulated race activities on 5-HT levels in horses (Medica et al., 2020). The 5-HT level increased after simulated race and training activities. Exercise triggers physical stress that affects homeostasis, which is regulated by the hypothalamic-pituitary-adrenal system (Zschucke et al., 2015). Thus, each part of the diencephalon of horses seems to be connected to serotonergic fibers and regulate neuroendocrine function.

In summary, the present study demonstrated the localization of 5-HT1A receptors in the CNS of horses for the first time. The data showed that 5-HT1A receptors are generally distributed in the brains of horses, including in the cerebrum, cerebellum, and diencephalon. Furthermore, 5-HT has been proposed to modulate cognitive and behavioral functions in animals, and the results of this study indicate that 5-HT1A receptors are highly related to these functions. These data provide insights into the possible physiological functions of this receptor subtype.

CONCLUSION

In conclusion, the present study found that 5-HT1A receptor is present in various regions of the horse brain, including cerebral cortices, cerebellar cortex, thalamus, and hypothalamus. The study suggests that the 5-HT and 5-HT1A receptor systems may play significant roles in the central nervous system of horses, based on the widespread distribution of 5-HT1A receptors. Understanding the 5-HT1A receptors in horses may provide insights into horse behavior and its associated mechanism.

Acknowledgements

The authors thank Heejun Jung, Junyoung Kim, Sungmin Kim, Youngwook Jung, and Yubin Song for their assistance with sample collection.

Author Contributions

Conceptualization, Y.C. and M.Y.; methodology, Y.C.; Investigation, Y.C.; data curation, Y.C.; writing—original draft preparation, Y.C.; writing—review and editing, M.Y; supervision, M.Y.; project administration, Y.C.; funding acquisition, M.Y.

Funding

This work was supported by National Research Foundation of Korea grant, funded by the Korean government (MSIT No. 2019R1A2C1003701).

Ethical Approval

Not applicable.

Consent to Participate

Not applicable.

Consent to Publish

Not applicable.

Availability of Data and Materials

Not applicable.

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Fig 1.

Figure 1.The right lateral side of the horse brain. Frontal cerebral cortex (a); central cerebral cortex (b); posterior cerebral lobe (c); thalamus (d); hypothalamus (e); cerebellar cortex (f); cerebellar medulla (g).
Journal of Animal Reproduction and Biotechnology 2023; 38: 77-83https://doi.org/10.12750/JARB.38.2.77

Fig 2.

Figure 2.Cross-reactivity of serotonin 1A (5-HT1A) receptor in horse brains. The 5-HT1A receptor protein band appeared at a molecular size of approximately 50 kDa. The β-actin band was detected at approximately 45 kDa in horse brains.
Journal of Animal Reproduction and Biotechnology 2023; 38: 77-83https://doi.org/10.12750/JARB.38.2.77

Fig 3.

Figure 3.Localization of serotonin 1A (5-HT1A) receptor in horse brains. Immunolabeling of the 5-HT1A receptor was detected in the cytoplasm of neurons in the cerebral cortex (A-C), thalamus (F), hypothalamus (G), and cytoplasm of Purkinje cells in the cerebellar cortex (D). The expression of the 5-HT1A receptor was not detected in the cerebellar medulla (E). DAPI, 4’6-diamidino-2-phenylindole; N, neuron; PC, Purkinje cell.
Journal of Animal Reproduction and Biotechnology 2023; 38: 77-83https://doi.org/10.12750/JARB.38.2.77

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