**3. Neuroendocrine regulation of equine stress reaction**

In contrast to rodents or humans, horses are not as well studied with regards to the neuroendocrine regulation of stress response, especially at the acellular and molecular level in brain structures, therefore rodent and human results will be partially extrapolated to horses in this review. For a long time, researchers suggested that the sympathetic-adrenal medullary (SAM) and hypothalamic–pituitary–adrenal (HPA) axis play the main role in stress responses [25–27]. This theory, up to the present, seems to limit or oversimplify the weight aspect of the animal's stress response.

There is almost no tissue or cell in animals (including microbiota) that, directly or indirectly, does not play some role in the maintenance of homeostasis during the acute or chronic stress response [28]. In a simplified version, the stress systems in horses have two essential components—controllers and effectors. The controllers or receptors of *sensory* systems monitor the value of the regulated homeostatic parameter which they are customized, compare it to the reference value and generate a neural (or hormonal) signal that is proportional to the absolute value of the difference. If the sensory signal is interpreted in the CNS as a threat, this will be acted upon through various pathways affecting different effectors, which creates physiological reactions into or out of the system in order to bring the controlled variable closer to the reference value [29]. Equine homeostasis is usually maintained through complex, coordinated mechanisms of self-regulation, among which feedback plays an important, but not determinative role.

The first step in the stress response is the perception of a stressor through the sensory system, which is composed of sensory receptor cells, neural and blood pathways, and parts of the brain involved in sensory perception. The brain interprets them as either a real or a potential threat, which triggers nonspecific and specific stress responses that are commensurate with the nature of the stimulus (**Figure 1**) [30]. Thus, the physical stressors which are well studied, for example, pain and blood loss, require an immediate "systemic" reflex reaction. On the other hand, the equine brain also responds to non-physical or "psychogenic" stressors (for example transport or weaning) based on prior experience [20, 31].

Sensory systems code for four aspects of different stress stimuli—type, intensity, location, and duration [32]. There are different receptors monitoring equine homeostasis, and among them, the nociceptors have the most important role in the induction of the acute stress reaction. As a reaction to physical trauma or another *noxious stimulu*s*,* nociceptors send sensory *stimulation* primarily through to the preganglionic sympathetic neurons in the intermediolateral cell column of the thoracolumbar spinal cord*,* and from there further through the neospinothalamic, paleospinothalamic and archispinothalamic tract to different thalamic "relay" neurons [33]. Then, the thalamus sends the noxious signal to other brain structures, which initiate, spread, *memorize,* and cessation in an equine stress reaction [34]. As a reaction to homeostatic imbalance or inflammation, the brainstem is also able to generate rapid stress responses via direct projections of neurons in the paraventricular nucleus of the hypothalamus (PVN) or to preganglionic autonomic neurons [35]. It is considered that information on blood volume or oxygenation is communicated via baro- and chemoreceptors to the nucleus of the tractus solitarius (*NTS*), which then send direct noradrenergic projections to the PVN, ensuring a rapid HPA axis response [31]. The forebrain limbic regions (which mediate psychogenic stressors) have no direct connections with the HPA axis or the SAM, and thus require intervening synapses (primarily to the locus coreleus, amygdala, and bed nucleus of the stria terminalis), prior to initiating a stress response [35, 36].

Nociceptors, interneurons, and "relay" neurons of the thalamus release a variety of excitation (pain) neurotransmitters, primarily, the substance P (SP), neurokinin A, Glutamat, calcitonin-gene-related peptide (CGRP), and cholecystokinin [37]. Recently, in the equine thalamic reticular neurons (TRN neurons), unique dopaminergic projections to the thalamic relay neurons were found, whereas in primates this input arises from a variety of dopaminergic neurons within the classically defined catecholaminergic system [38]. This possibly novel, potentially dopaminergic, projection upon thalamic relay neurons within the equids may play a modulatory role in the output of

*Equine Stress: Neuroendocrine Physiology and Pathophysiology DOI: http://dx.doi.org/10.5772/intechopen.105045*

#### **Figure 1.**

*Schematic representation of the central and peripheral components with regulatory pathways involved in the equine stress response caused by colic disease. LC/NE, locus coeruleus/norepinephrine system; SNS, sympathetic nervous system; PVN, paraventricular nucleus; BST, bed nucleus of the stria terminalis; POMC, proopiomelanocortin; CRH, corticotropin-releasing hormone; VP, vasopressin; GABA, γ-aminobutyric acid; ACTH, adrenocorticotropic hormone; NPY, neuropeptide Y; SP, substance P; Ach, acetylcholine; PACAP, pituitary adenylate cyclase-activating polypeptide; LPS, lipopolysaccharide; NA, norepinephrine; E, epinephrine; DA, dopamine. Activation is represented by green lines, inhibition by red lines and auto regulatory feedback loop by red dashed lines.*

thalamic relay neurons to other structures of the brain [38]. It is considered, that these neurons have a strong influence on the processing of neural information, potentially providing the equid cerebral cortex with neural information that has a lower signalto-noise ratio, making the extraction of salient neural information more precise than observed in other mammals [39]. The presence of catecholamines in the TRN neurons modifies stress behavior and may play a role in the various aspects of sleep observed in equids. It is well known that sleep in equids appears to be unusual, as they are short sleepers (around 2.9–3.3 hours per day), and have brief sleep cycles of around 15 min, with a non-rapid eye movement (REM) phase followed by a brief REM phase (less than 30 s), mostly while standing [40]. Undoubtedly, this evolutionary trait in horses on a daily basis supports a quick and effective alarm response to various acute stress factors.

After receiving a threat sensory information, most researchers argued that two areas of the brain have distinctive important roles in the stress reaction—the catecholaminergic neurons in the locus coeruleus (LC-NA system), which are mainly responsible for activation of the SAM axis, and the hypothalamus, which is responsible for activation of HPA axis (**Figure 1**) [41–43]. Furthermore, other brain circuits modulate and fine-tune the adaptive or protective stress responses, including the amygdala-hippocampus complex, the mesocortical and mesolimbic components of the dopaminergic system, the noradrenergic cell group A2/C2 in the solitary nucleus, the A1/C1 cell groups in the ventrolateral medulla, the cuneiform nucleus and dorsal raphe nucleus, the parabrachial nucleus, and the bed nucleus of the stria terminalis [44, 45]. These structures are responsible for releasing various excitatory and inhibitory neurotransmitters, via overlapping brain circuits, in accordance with stressor modality or intensity. In addition, an alteration of the parasympathetic nervous system (PNS) with attenuation of the "vagal tone" of the heart and lungs occurs to help control the duration of activation of the SAM axis. Reconciliation of the PNS response to stress is mediated via the nucleus ambiguus and dorsal motor nucleus of the vagus nerve, possibly via input from the nucleus of the solitary tract [45].

### **3.1 LC/NE-sympathetic systems in the equine stress reaction**

The locus coeruleus (LC) with noradrenergic neurons have been expressly implicated in the initiation and speed of acute physiological and behavioral stress changes in rodents and humans (likely in horses) [41, 42, 46, 47]. Extrapolation of results from other species to a horse should be performed with caution, to obtain a remarkable difference between equid and other animals because catecholamine metabolites are mostly glucuroconjugated and not sulfoconjugated [48]. LC receives inputs, not only from the spinal cord and thalamus, but also from the hypothalamus, medial prefrontal cortex, nucleus prepositus hypoglossi, and nucleus paragigantocellularis [49]. LPS (endotoxin) associated release proinflammatory cytokines (IL-1β, IL-6, and TNF-α) also facilitate norepinephrine (NA) release in LC [50]. Likely, this endotoxic activation of LC is especially important in horses with strangulation intestinal obstruction. After input activation, the noradrenergic neurons in the LC send then excitatory signals to different areas of the brain and to the spinal cord that are accompanied primarily by the alarm phase of the acute stress response [49]. Amplification of LC activity leads to increased signs of alertness in electroencephalographic (EEG) analysis [51]. The LC-NA system, through projections to the sympathetic preganglionic neurons in the spinal cord with activation of α1-adrenoceptors, increases sympathetic activity and reduces parasympathetic activity, via the activation of α2-adrenoceptors on preganglionic parasympathetic neurons [49, 52, 53]. Therefore, activation of the LC-NA system within seconds leads to the activation of the equine adrenal medulla (SAM), with a distant release of norepinephrine (NA) and other catecholamines—epinephrine (E) and dopamine (**Figure 1**)*.* Consequently, this chain process in the alarm stress phase is more correctly denoted as activation of the LC-NA-sympathetic system, instead of activation of the SAM. In addition to NA release, the sympathetic nerve fibers also secrete adenosine triphosphate (ATP) and the neuropeptide Y (NPY), which enhance the systemic action of the catecholamines [54]. Accompanying the LC-NA-sympathetic system, the synthesis of E in the adrenal medulla partially stimulates the ACTH, cortisol, and pituitary adenylate cyclase-activating polypeptide (PACAP) [55].

### *3.1.1 Concentrations of equine epinephrine*

The concentration of equine E in the blood depends on the tone of the sympathetic system, as it is associated with active escape, attack, and fear. In nonstress horses,

### *Equine Stress: Neuroendocrine Physiology and Pathophysiology DOI: http://dx.doi.org/10.5772/intechopen.105045*

blood concentrations of E show a circadian rhythm. The mean plasma E concentrations were highest in the morning (~ 30 pg./ml at 8:00 hr), with a significant nadir in the sleeping phase at 04:00 hr. (~18 ng/ml) [56]. The concentrations of E increase up to 21 times higher during severe acute stress (fear, trauma) and intense physical activity [57]. The circadian rhythm of NA also exists, at 08:00 in the nonstressed horse, it was found to be 70–80 pg./ml, with the nadir observed at night (~50 pg./ml) [56]. In equine physical exercise stress, the NA concentration increases approximately 13-fold [58]. Thus, the response plasma levels of E and NA during exercise or other forms of acute stress in the horse is considerably greater than in people. The difference between horses and humans in SAM activity may help explain the superiority of the athletic performance of equine athletes compared to that of human athletes [59]. In horses during exercise, the increase in plasma NA is almost linearly proportional to exercise intensity, being higher after brief maximal exercise than after an endurance ride [58]. On the contrary, a marked increase in plasma E only occurs during strenuous exercise, especially if it is accompanied by psychogenic stress.

### *3.1.2 Roles of equine catecholamines*

Basically, the equine catecholamines regulate many biochemical processes involved in energy metabolism, as well as the physical homeostasis adaptation associated with acute and rapid stress responses. Equine catecholamines have a strong impact on the bone marrow and spleen to enable the mobilization of additional blood. It has long been known that during the resting phase of horses, approximately 50–60% of blood (i.e., more than 20 L) is kept within reservoirs, in comparison to dogs (20–25%) and humans (12%) [60]. Consequently, during the alarm phase of the equine stress response, the splenic contraction under adrenergic control, ejects reservoir blood into the circulation, following significant increases in the hematocrit levels and concentrations of the erythrocytes, leucocytes, thrombocytes, hemoglobin, and plasma protein. The normal human spleen, unlike the horse spleen, does not contain many smooth muscle adrenergic receptors; therefore, it cannot strongly contract. Thus, the equine splenic contractile response to the various stress factors is more sensitive than that of any other species [60].

*An increase in catecholamine release* within the alarm phase leads to significant magnification of equine cardiac function. The cardiovascular system in horses is more stress sensitive than that of any other domestic species, as the increase in heart rate (HR), stroke *volume (SV),* cardiac output (CO), and blood pressure (BP) is enormous during an acute alarm stress reaction [61]. Independent of stress factors and its intensity, and through catecholamine binding to β1 receptors in adult thoroughbreds (TB) horses, it was noticed that increasing the HR to 240 beats per minute (at rest 30–40 beats per minute), the SV to 1200 ml (at rest 900 ml), the BP to 250/120 mmHg (at rest 155/110 mmHg), the CO reached 240–340 l/min [62]. In nonstress horses, the CO is about 40 l/min, that is, by intense acute stress in horses is possible a 7–8 fold increase in cardiac function, in contrast to human athletes at approximately 2-fold [63, 64]. Commonly, these changes in cardiovascular parameters by acute stress horses are for a short time (5–60 sec), are intensively energy-consuming and straining, and the catecholamine concentrations quickly return to their resting levels [65]. It should also be remembered that these equine cardiovascular changes in acute stress are not only related to the increasing activities of the SNS and catecholamines but are also controlled by other vasoactive hormones, for example through plasma renin activity, atrial natriuretic peptide, endothelin-1 and vasopressin in the renin–angiotensin–aldosterone system.

#### *3.1.3 Physiological changes during equine stress*

Horses have a normal resting respiratory rate (RR) of 12–20 breaths per minute. During the onset of acute severe stress, in accordance with the body's need for oxygen, the RR rises as high as 180 breaths per minute [66]. In an adult TB horse at rest, the tidal volume (TV) is about 4–7 liters, rising to a maximum of about 10 liters during intense stress exercise [67]. When breathing at rest the dead space accounts for about 70% of the TV and the alveolar volume is around 30%. With exercise stress, there is a large increase in alveolar volume and a small increase in dead space. In adult nonstressed horses, the amount of air passing in and out of the lungs per minute (MV) is approximately 100 l/ min. At maximal stress exercise, the MV reached 1500 l/min (due to a 7-fold increase in RR and a 2-fold increase in TV) [66]. Rate and depth of breathing are controlled in part by chemoreceptors in the blood vessels which respond to changes in pH, arterial oxygen, and carbon dioxide tension. When in gallop (stress flight) the RR is coupled with stride rate and so the mechanics of locomotion override the chemical control of breathing. This unique equine phenomenon is known as locomotor-respiratory coupling [68]. When exercise ceases, the RR decreases due to the cessation of the locomotor forces that drive respiration. These equine physiological and anatomic adaptations allow an extremely high maximal rate of use per minute of O2 consumption (VO2 max). By strenuous physical (stress) horse activity the VO2 max reaches up to 200 ml/kg/min [66].

In addition, via stress activation of the LC-NA-sympathetic system, it has been noticed in horses (through contraction of the m. iris dilator) mydriasis with the appearance of tunnel vision (i.e., loss of peripheral vision) and increased body temperature, following sweating and suppression of secretion of the lacrimal and saliva glands can occur. In stressed animals, it was also noticed that decreases in gastrointestinal mobility, blood flow, and secretion could happen [69]. In the alarm phase of the stress response through catecholamines (but also through glucocorticoids), the horse's blood clotting function is accelerated to prevent excessive blood loss in the event of an injury sustained during the potential "stress fight response" [70, 71]. In the alarm phase of the equine stress response, it was noted that different immune functions were enhanced, through catecholamines binding to β-2 adrenergic receptors on immune cells (primarily on the NK cells), as well as through blood mobilization and direct sympathetic innervation of lymphoid organs (**Figure 1**) [72–74].

#### *3.1.4 Catecholamines during stress responses*

Catecholamines in an alarm phase of stress response vie β-2 adrenergic receptors induce significant lipolysis with increasing concentrations of blood fatty acids, that can be used directly as energy sources primarily by the locomotor system [75]. In different animal studies, it has been found that catecholamines, through α-adrenergic receptor binding, provoke inhibition of insulin secretion and significantly increased concentrations of glucagon (mediated through binding to the β-adrenergic receptor), and glucose, as a result of increasing either glycogenolysis or by gluconeogenesis [76, 77]. Additionally, catecholamine stimulation also releases ACTH, cortisol, and renin following the retention of sodium in the bloodstream [78].

Numerous other physiological reactions of catecholamines have also been noticed, which lead to the equine body producing additional speed and strength. For example, via the binding of alpha-1 adrenergic receptors by NA, it was noted that vasoconstriction of most blood vessels occurred in the skin, digestive tract, and kidneys [78]. These receptors are inhibited and counterbalanced by beta-2 adrenergic receptors

### *Equine Stress: Neuroendocrine Physiology and Pathophysiology DOI: http://dx.doi.org/10.5772/intechopen.105045*

(stimulated by E release from the adrenal glands) in the skeletal muscles, heart, lungs, and the brain during a SAM response. At rest, in horses, only about 15% of the circulating blood is delivered to the muscles, but this increases to as much as 85% during strenuous stress exercise [66]. In other words, in the alarm phase of the stress response, through the activation of the LC-NA-sympathetic system, oxygen, and nutrient delivery is directed toward the CNS and areas of the body (primarily the cardiovascular and respiratory systems), where they are most needed to cope and escape from threat factors. In contrast, during severe acute stress responses other energyconsuming functions, such as digestion, reproduction, and growth, are temporally suppressed [59, 79, 80]. These changes are conditional and strongly depend on the type, intensity, and frequency of the stress factor. Thus, during equine stress exercise, a different picture of the hormonal background is observed in horses.

#### **3.2 HPA axis in the equine stress reaction**

In the second phase of the stress response (the stage of resistance), it is commonly observed that a strong activation of the HPA axis and the renin–angiotensin system (RAS) occurs, although a strict distinction between these stress phases is difficult to conclusively make as the reactions are dependent on the stress factor [81]. The intensity and frequency of the stressor is a major factor in determining the overall trajectory of the HPA axis response, with significantly increasing concentrations of the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), vasopressin (VP), and cortisol. Stressors of a presumptive "lesser" severity (for example the horse having 5 min exposure to a novel open field) produce lower peaks in HPA activation, in contrast to severe abdominal pain following, for example, an intestinal obstruction. The main goal of strongly activating the HPA axis during a stress reaction is the reinforcement of the homeostatic mechanisms and to provide additional energy through enhanced glycogenolysis, gluconeogenesis, and lipolysis [82, 83].

Usually, activation of the HPA axis is slightly slower than the activation of the LA-NA–sympathetic system. Commonly, after the onset of stress factor, the concentration of CRF rises immediately (as do concentrations of the NA), but the peak secretion of pituitary ACTH occurs around 5–15 s later, followed by the peak levels of cortisol 15 and 60 min later [84, 85]. There are also differences here, which are dependent on the stress factor. Commonly, various inflammatory stimuli cause prolonged HPA axis activation (2–3 hours after onset) commensurate with the need to limit immune responses [86].

### *3.2.1 Equine CRF*

Equine CRF is a 41-amino acid peptide identical in structure to human and rat CRF. CRH is produced primarily by parvocellular neuroendocrine cells within the PVN, but this neuropeptide and specific CRH receptors have been identified in numerous extra hypothalamic regions of the brain, including the pituitary and adrenal glands [81, 87]. In addition to stimulating the secretion of the ACTH, CRH coordinates various physiological and behavioral responses, for example, induced anorectic effect, stereotyped behaviors, and enhancing the activity of the SNS [88]. Thus, one of the central actions of CRF is to appropriately facilitate "fight or flight" responses [89]. Besides this, CRF during stress responses inhibits, particularly, the secretion of GnRH, LH, testosterone, and estrogen, and through the stimulation of somatostatin secretion, it also inhibits the secretion of GH, TRH, and TSH [81].

Equine CRF concentrations in pituitary venous blood are lower compared to other species. In nonstress horses, the CRF concentration ranges from 0.25 to 0.8 pmol/l, but is very changeable day-to-day [90]. The regulation of CRF and VP secretion is complex. CRH and VP neurons in the PVN have dense connections with various structures in the brain. In the rodent and humans, the LC and other NE-synthesizing cell groups belonging to the medulla and pons have reciprocal reverberatory neural connections with the CRH neurons in the PVN and stimulate the secretion of each other through CRH receptor-1 (CRH-R1) and the α1-noradrenergic receptors, respectively (**Figure 1**) [91, 92]. It was also found that auto regulatory ultrashort negative feedback loops exist in both the PVN CRH and the catecholaminergic neurons of the LC, with collateral fibers inhibiting CRH and catecholamine secretion respectively, via inhibition of the corresponding presynaptic CRH- and α2-noradrenergic receptors [93, 94].

In addition, multiple other regulatory central pathways exist, since both CRH and the catecholaminergic neurons receive stimulatory (stress-excitatory) innervation from different brain structures through various neurotransmitters, among which is the especially important pituitary adenylate cyclase-activating polypeptide (PACAP; **Figure 1**). PACAP is a key emergency neuropeptide, mediating central and peripheral components of the stress axes [95]. This neurotransmitter is primarily expressed in the CNS and also within the sympathetic nervous system including the sympathetic preganglionic neurons that innervate the adrenal gland [96]. Thus, PACAP participates in stimulating the secretion of various hormones and neurotransmitters, including ACTH, VP, epinephrine, insulin, melatonin, prolactin, MSH, brain natriuretic peptide, follistatin, and the enkephalins [95, 97].

Serotonin, cytokines, and other inflammatory factors (e.g., nitric oxide) also participate in CRF secretion in the PVN in horses, as also seen in other mammals [98, 99]. Interestingly, NPY has multiple regulatory central pathways as it stimulates CRH neurons, whereas it inhibits the LC (**Figure 1**) [100]. On the other hand, SP, as the first responder to most noxious/extreme stimuli, has reciprocal actions to those of NPY, since it inhibits CRH neurons, whereas it activates the CA-NA system (**Figure 1**) [101, 102].

CRH and AVP neurons have reciprocal reverberatory neural connections to the pro-opiomelanocortin (POMC)-containing neurons in the arcuate nucleus (AN) of the hypothalamus. POMC-containing neurons primarily secrete β-endorphin [103]. Information on horse energy balance appears to access the PVN via projections namely, from neurons in the AC, which process circulating signals relevant to metabolic status (for example, glucose and fatty acid blood concentration) [104].

The neurons of the suprachiasmatic nucleus (SCN) of the hypothalamus also have several direct projections to the CRH neurons. The SCN is known to be the main (but not the only one) coordinator of biological circadian rhythms in mammals, described as the "CLOCK system" [105]. It is well known that the SCN neurons, through photoreception, have important roles in the basal daily and seasonal variation, not only on the equine HPA axis, but also on the hypothalamic–pituitary–*gonadal axis*, melatonin, insulin, grelin and adinopectin secretion, body temperature, and other factors [105, 106]. Thus, in resting horse conditions, through SCN input the HPA axis activity has circadian and ultradian variations. In the stress-free condition, there is a pulsatile secretion of equine CRF, VP, ACTH, and glucocorticoids (one per hour), with greater amplitudes in the morning (upon waking up) than at night [107, 108]. The circadian rhythm secretion of these hormones is disrupted under equine stressful conditions, as with other animals [109].

### *3.2.2 Equine vasopressin*

Equine vasopressin (VP) is a nonapeptide, which is primarily produced in the magnocellular neurons of the PVN, its main effect is on the regulation of the blood pressure and ACTH secretion. VP is also expressed in other structures of the CNS, with functions in behavior, stress analgesia, and the regulation of circadian rhythms [110]. Normally, plasma vasopressin concentration in nonstress horses is less than 15 pg./mL [111]. An increase in equine VP concentrations is correlated with both the duration and intensity of the stress factor [112–114]. The basic physiological stimulus for a 5–10 fold increase in the secretion of VP is increased osmolality of the plasma, as well as the presence of hypotension due to hemorrhage or endotoxemia, for example in horses with colic [111]. Although VP has a short half-life (16–24 min), after a 3-day event endurance test, equine VP was elevated for 6 h [80].
