*3.3.4 EOS attenuation of stress*

Unaccompanied, the EOS is not able to attenuate or shut off the stress response. In addition to the EOS, there are other neuromediators and hormones that have been proposed which protect against the effects of stress. Undoubtedly, to attenuate the equine stress response, it is necessary to activate the parasympathetic nervous system (PNS) with an increased tone of the vagus nerve and release of the neurotransmitter acetylcholine. Activation of PNS is mediated, among other things, via the neurons from the double reticular nucleus and the neurons from the nucleus of the solitary pathway of the medulla oblongata [81]. But this is not enough to completely "shut off" the animal's stress reaction. Numerous animal studies have shown that the hippocampus inhibits the activation of the CRH neurons in the PVN and LA through GABAergic and endocannabinoid neurotransmitters [31]. PVN neurons in the hypothalamus produce numerous neuropeptides that may contribute to activation via local paracrine actions, either by recurrent collaterals or dendritic release [159]. As mentioned above, due to mutual reverberant neural connections a gradually decreasing intensity or autoregulatory negative feedback loops exist between the CRF and the noradrenergic neurons, as in the initial stage they stimulate each other [93, 94].

Certainly, a pivotal role in attenuation of the stress response is also played via the mechanism of negative glucocorticoid feedback. Glucocorticoids have direct and indirect pathways to negative feedback to the limbic system, hypothalamus, and pituitary gland. This attenuates or decreases the primary release of CRF and ACTH. In the PVN, the binding of glucocorticoids to its receptor causes rapid synthesis and release of endocannabinoids. The released endocannabinoids bind to CB1 receptors on presynaptic terminals, inhibiting glutamate release and thereby reducing the drive to CRH neurons [160]. The effect of negative glucocorticoid feedback on equine VP

secretion is less pronounced than that for CRF because VP secreting neurons are less sensitive to glucocorticoids. Glucocorticoids may also provide positive feedback in some brain structures, particularly under chronic stress conditions [161]. In addition, neuropeptides associated with CRF, such as urocortin, also play an important role in suppressing (or activating) the function of the HPA axis during the stress response [162]. Numerous other hormones directly or indirectly attenuate deleterious effects of prolonged activation of the LA-NA-sympathetic and HPA axis in horses, foremost in that are the hormones melatonin and insulin, and therefore they are sometimes referred to anti-stress hormones [163, 164].

Through all of the physiological processes listed above, gradual decreases in the activities of the LA-NA-sympathetic axis and the HPA-axis have commonly been noticed. Depending on the stress factors (as well as their intensity and duration) a decrease in stress hormone levels in horses does not occur immediately. Commonly, it may take several hours or days for stress hormones to drop back to their baseline levels. It has also been noticed that in horses, repeated exposure to the same stressor (novelty stress) can result in habituation of the HPA axis response, characterized by decreasing glucocorticoid responses over time [165, 166]. Habituation appears to be mediated, at least in part, by the paraventricular thalamic nucleus [167]. But, it must also be noted that not all stressors cause response habituation in horses. Responses to more 'severe' stressors (e.g., pain) are maintained, therefore here it is possibly not an adaptation but only protection that comes into play.
