1. IntroductionStressful events provoke a chain of biochemical and neurochemical responses in the body and brain [1,2,3,4,5,6,7]. Some of these changes can be protective or adaptive in nature, while other may play a significant role in the development of dysfunctions followed by the progression to diseases [1,3,5,8,9,10,11,12]. The stressed-derived aberrations in the dopamine transmission have received particular attention due to the involvement of this neurotransmitter in pathological conditions, such as drug and alcohol addictions [8,13,14], anxiety, depression [15,16], and post-traumatic stress disorder (PTSD) [17,18,19,20]. In fact, these psychopathologies may be significantly escalated by chronic as well as by single exposure to stress [3,21,22]. Insights into the neurochemical responses to stress can be gained from valid animal models. Such a translational model, which has the etiological relevance for examining stress-related pathological conditions, is a social defeat [3,12,23,24,25,26,27,28]. The applicability of this paradigm is exceptionally simple given the almost ubiquitous presence of territorial or hierarchical aggression in males [6]. The interaction of the tested male with an aggressive subject results in increased heart rate, blood pressure, an elevation of body temperature and a powerful endocrine response [7,29,30]. These physiological changes in combination with evoked defensive and submissive behaviors that are clearly observed in a defeated animal indicate an acute stress condition. During exposure of an intruder rat to the social defeat procedure, the burst firing of the VTA dopamine neurons is increased [31], and as a consequence, dopamine release is enhanced in terminal fields including the nucleus accumbens [31,32].To reproduce the phenotype of stress-related psychopathologies, repeated social defeat was predominantly employing [3,6,26,27,28,33,34]. Such a paradigm is more equivalent to the current theories on the development of depression in human rather than a single acute stress [35]. On the other hand, a strong acute trauma might trigger conditions such as a PTSD. The mechanisms of the development of this pathology are still unclear and social defeat model can be useful in this regard. In fact, robust and relatively long lasting behavioral and physiological changes can be elicited even by a single exposure to social defeat [3,6,36]. According to the study performed by Meerlo and colleagues, a profound increase in body temperature during the circadian resting phase along with changes in spontaneous home cage activity of rats can continue for up to 10 days after social defeat [29]. The same work revealed that a food intake and growth in such animals were suppressed for a number of days, whereas the decrease in locomotion under a novel environment was evident 2 days after stress [29]. Currently, it is well known that the exposure to a single acute stressor induces neuroadaptations in VTA dopamine cells [3]. Thus, the induction of long-term potentiation (LTP) at GABAA synapses on VTA dopamine neurons was blocked 24 h following acute exposure to stress [37]. Since dopaminergic transmission is under a potent control of VTA GABA neurons [38,39], stress-triggered loss of LTP is expected to increase dopamine release in the terminal field [37]. In agreement with this speculation, electrophysiological recordings revealed that burst firing of VTA dopamine neurons increases with acute restrain stress and persists at least for 24 h [40]. It was also speculated that the loss of LTP at inhibitory synapses in combination with the induction of LTP at excitatory synapses can result in enhanced responsivity of VTA dopamine neurons to future stimulations [3]. However, direct evidence that increased dopamine release is a consequence of single stress is currently lacking.Therefore, the present study was designed to explore the consequences of a single social defeat on accumbal dopamine release evoked by an electrical stimulation of the VTA measured by a fast-scan cyclic voltammetry (FSCV). Based on the results of previous studies [37,40], neurochemical measurements as well as behavioral assessments performed on separate group of rats, were made 24 h following the stress procedure. 4. Discussion

The present study was mainly focused on the short-term consequences of a single social defeat on accumbal dopamine dynamics, while behavioral assessments were also achieved. Expectedly, robust stress-associated behaviors were evident in intruder rats during their confrontation with an aggressive resident. However, the majority of performed tests did not reveal significant alterations in behaviors of defeated rats 24 h following the interaction. Thus, a sucrose intake, locomotor activity, rearing and grooming were identical to those in control animals. Nevertheless, stressed animals demonstrated a substantial increase in the immobility time during the forced swim test. This social defeat consequence, which indicates a deficiency in natural escape behavior, was paralleled with changes in dopamine release evoked by an electrical stimulation of the VTA. Remarkably, the increased dopamine efflux was observed following the VTA activation at high frequencies (40, 50 and 60 Hz), while lowest frequencies (5, 10, 20 and 30 Hz) did not result in the differences in accumbal dopamine response between defeated and control rats. Furthermore, the administration of dopamine D2 receptor antagonist, raclopride (2 mg/kg, i.p.) caused a significantly weaker effect on electrically evoked dopamine release in stressed subjects compared to control. The use of the dopamine depletion protocol revealed no alteration of the reduction or recovery of the amplitude of dopamine release following stress triggered by a social defeat.

During social defeat in rodents, acute physiological adaptations, which include an activation of cardiovascular and endocrine systems [7,52] along with neurochemical responses, such as an increased dopamine and norepinephrine release [6,31,53], as well as striking changes in patterns of behavioral activity [6,31] are well recognized. Most of these changes are reduced or eliminated within 24 h after stress exposure. However, some behavioral and neurobiological effects can be determined even after a single episode of social defeat [6]. For example, single social defeat resulted in a significant decrease in locomotion of Tryon Maze Dull S3 rats in the open field test, which lasted for several days after a single exposure [54]. In contrast to these findings, no changes in locomotor activity, as well as in many other behaviors (e.g., sucrose intake, grooming, rearing and others) were observed in the present study. The strain difference (Sprague Dawley versus Tryon Maze Dull S3) and, more likely, the divergence in the social defeat procedure could be accounted for the discrepancy in results. Specifically, in the previous study rats were placed in the cage of an aggressive male for 1 h, while a total time of stress exposure in our experimental approach was 20 min. The longer confrontation between a resident and intruder also resulted in a reduction in the amplitude of the daily temperature rhythm, the profound increase in body temperature during the circadian resting phase, the decrease in spontaneous home cage activity and the decrease in locomotion in a novel environment [29,55]. Perhaps, a lengthier single defeat could trigger more behavioral and physiological consequences.Nevertheless, the immobility time in the Porsolt forced swim test (FST), which is considered an indicator of a depression-like state [35,46], was affected in the current study. Though previous studies on a single social defeat did not focus on floating behavior [29,54,55], it is well documented that repeated social defeat prolongs an immobilization time in FST, indicating the development of helplessness and despair in animals. Remarkably, recent study in mice revealed a possible link between striatal dopamine turnover and floating behavior [4].In the current study this behavioral consequence of social defeat was paralleled with changes in the accumbal dopamine release evoked by an electrical stimulation of the VTA. Noticeably, dopamine release was enhanced under the condition, when the stimulations were performed at high frequencies, which trigger phasic pattern of dopamine neurotransmission [56,57]. The increased phasic dopamine signaling in rat nucleus accumbens was previously revealed during a social confrontation and following a defeated subject was returned into its home cage [31]. In agreement with these neurochemical changes, substantial increases in burst frequency were detected in the VTA dopamine firing patterns during an aggressive confrontation as well as in home cage [31]. Furthermore, it was found that neurons with higher burst levels under normal conditions (before social defeat procedure) did not switch from non-bursting to bursting types, whereas cells with higher burst activity displayed augmented increases in bursting [31]. This is consistent with the results on electrophysiological consequences of single restraint, which is also a highly stressful condition for conscious animals [40]. Moreover, the evidence was obtained that the shift to the increased burst firing persists 24 h after initial exposure to restraint stress. Because dopamine bursts result in enhanced efflux at synaptic terminals [31,58], increased phasic dopamine signaling should be predictable under this circumstance. However, no neurochemical exploration of changes in fast dopamine transmission was performed in this earlier study [40]. Together, these findings suggest that the increased phasic dopamine response that develops during a single stress exposure can be preserved at least for 24 h after the termination of stress. Perhaps, this consequence may disrupt normal dopamine dynamics in the nucleus accumbens and therefore endorsing some depressive-like behaviors.A single stress promotes several neuroadaptations, which can be responsible for observed dopamine changes. Thus, acute stress-induced blockade of the LTP induction at GABAA synapses on VTA dopamine neurons [37,59] may result in enhanced responsiveness of dopamine cell bodies. The same outcome can be reached due to the induction of LTP at excitatory synapses in the VTA. In fact, the increases in AMPA/NMDA ratio were triggered within 2 h of acute stress and have been continued at least 24 h [9,60]. These fast neuroplasticity alterations in the VTA, which independently predict an enhanced dopamine release in the nucleus accumbens, can be combined in response to an acute stressor [3].Remarkably, the consequences of social defeat on dopamine dynamics can be evident in an ex vivo preparation, where the release is evoked by electrical stimulation of accumbal slices, which have no connectivity from the dopamine cell body region (VTA) [61]. In fact, the magnitude of dopamine efflux was significantly greater 1 h after the last session of the 3-day stress exposure. These data suggest the development of neuroadaptations at the level of dopamine terminals. For example, the kappa-opioid receptors and their endogenous ligand dynorphin can be involved in adaptations to stress. There is evidence for the decrease in dynorphin mRNA levels in mouse nucleus accumbens following chronic social defeat, while acute stress leads to opposite consequence [62]. It was revealed that the activation of kappa opioid receptors, which are also located on dopamine terminals, suppresses accumbal dopamine efflux [63,64]. Consequently, the reduced inhibition of dopamine release via activation of these receptors could lead to the enhanced dopamine in the nucleus accumbens due to diminished level of endogenous agonist and contrary wise. This is in agreement with the results obtained in ex vivo preparation following repeated social defeat [61]. However, the data on dopamine release changes following the current paradigm do not fit with the expectation that was based on dynorphin changes after acute stress [62].On other hand, corticotropin-releasing factor (CRF) can be responsible for the observed dopamine alterations. This neuropeptide releases in response to acute stress promoting adaptive and maladaptive behaviors [65,66] through its action on central CRF receptors [67]. Previous studies on brain slices revealed that, acting acutely within the nucleus accumbens core, CRF increases dopamine release [10,68,69]. Therefore, an emerging hypothesis can be that multiple circuit mechanisms, which are located at the level of dopamine cell bodies and terminals, may result in increased dopamine release in the nucleus accumbens. Though, the consequences of acute single trauma and chronic stress on accumbal dopamine transmission can be opposite.Altered dopamine efflux and consequent changes in its extracellular concentrations potentially may lead to presynaptic neuroadaptations, which are directed to compensate nonphysiologocal changes. This may impact the processes of synthesis, autoreceptor regulation of neurotransmitter release and reuptake. For example, the acceleration in dopamine uptake presumably due to the persistently elevated dopamine concentration in dopamine terminals was found using different models of drug addiction [13,15].To find whether the consequence of single social defeat on dopamine release is capable of affecting dopamine dynamics at the presynaptic level, we applied the dopamine depletion protocol that allows to reveal changes in synthesis, reuptake and autoreceptor regulation of dopamine [70,71,72]. This approach is based on the finding that a certain amount of time is required for the recovery of the evoked dopamine release from the effects of long electrical stimulations at high frequency. The dynamics of the decline and recovery of detected dopamine should reflect the overall regulation of presynaptic transmission. Despite the significant difference in dopamine release between control and stressed rats, the long stimulation protocol resulted in a full recovery of the depleted dopamine signal in the same levels of the depletion and following recovery of dopamine signal. Therefore, these data may suggest that the balance of intrasynaptic events in rat nucleus accumbens is not disturbed following single social defeat.Nevertheless, single social defeat resulted in a weaker effect of raclopride on electrically evoked efflux compared to the control that might suggest alteration in D2 receptor regulation of dopamine release. Interestingly, there is an evidence that acute exposure to aggression increases D2 receptor density in the nucleus accumbens of the opponent rat [73]. However, these changes were observed immediately following the exposure session. Therefore, it should still be figured out whether or not they were preserved within 24 h. Moreover, the increased D2 receptor density in the nucleus accumbens would predict rather an enhanced dopamine release following D2 receptor antagonist than its decrease. Perhaps a more simple explanation of the altered effect of raclopride can be offered. Since raclopride and dopamine compete for the D2 receptors, increased electrically evoked dopamine could modify the pharmacological effect of the drug, weakening presynaptic autoreceptor blockade in the VTA-nucleus accumbens terminals and therefore down-regulating the release.