What do experimental animal models of mood disorders tell clinicians about influence of probiotics on the gut-brain axis?

The interaction between the central nervous system (CNS) and the intestine and its implications for mental health is nowadays considered an interesting domain of research [1]. So far, numerous approaches have been made to develop animal models of depression/anhedonia, which allowed imitating at least some clinical symptoms of depression, in laboratory conditions. Thus, the data concerning, and proving, the linkage of signaling paths between the gastrointestinal (GI) tract and CNS activity are still missing. In general, behavioral models in psychopharmacology are used for different purposes. The main goal of psychopharmacologists is to develop new drugs and improve existing drugs' efficiency in the treatment of mental disorders, as well as to investigate their mechanisms of action. By considering anxiety, depression, schizophrenia, feeding disorders, neurodegeneration, and drug dependence, psychopharmacologists are able to model psychiatric disorders. The use of animal behavior to study psychoactive drugs is based on the critical need for sensitive, selective, and reliable predictors of drug activity in humans. Behavioral models are particularly appropriate because they represent the integrated activity of the intact living organism [2]. Animal behavior models and tests can help to provide an empirical basis for evaluating the potential clinical application of drugs and, as a consequence, should also be helpful in clarifying the neurobiological basis of various disorders [3].

The aim of the paper was to review and discuss various mechanisms linking specific probiotic bacteria with behaviors related to anxiety and depression (i.e., anhedonia) and cognition, and the exact mechanisms of their action, especially based on data provided from animal models and tests. We would like to point out the potential clinical impact resulting from future studies investigating the gut-brain axis (GBA) activity, with respect to the efficacy of probiotic treatment of mental disorders, that is, depression and anxiety.

The signaling path between the GI tract and the CNS is called GBA or even microbiota gut-brain axis (microbiota GBA), in reference to the fact that recently it has been demonstrated that intestinal microbiota are significantly involved in the modification of GBA functioning [4, 5, 6]. The gut and the brain remain in permanent contact with each other via various direct and indirect routes, including not only the nervous system but also humoral transmission (cytokines, hormones, neurotransmitters, bacterial metabolites released into the systemic circulation) and immune system.

Microbiota affect the CNS mainly through the enteral nervous system (ENS), which innervates the gastrointestinal wall, covering the entire length from esophagus to rectum. Intestinal microbiota regulate electrophysiological thresholds in ENS neurons [7] and are capable of modulating gut motility and pain perception [8, 9]. Intriguingly, it was found that numerous neurons run from the intestines to the prefrontal cortex and limbic system, that is, the hippocampus and the cingulate gyrus, thus to the structures responsible for emotional processing, morality, self-awareness, motivation, and memory.

One of the more important neuronal pathways in intestinal-brain communication is based on the vagus nerve, which has a role in control of GI motility and immunology and also affects the behavior of humans and animals. The research [7, 10, 11] demonstrated that the manipulation of intestinal microbiota changed the behavior of animals in tests used to assess anxiety, depression, and memory behavior, and that vagotomy caused the influence of microbiota on animal behavior to disappear in some experiments [10]. However, other research indicates that the activity of the vagus nerve is not necessary for some of these effects [12].

The cells of the immune system and the cytokines produced by them are also significant agents of communication between the intestines and the brain [13, 14]. The balance of intestinal microbes is able to regulate the inflammatory responses of the host. The gut microbiota population, with its metabolites and microbial-associated molecular patterns (MAMPs), such as lipopolysaccharide (LPS), bacterial lipoprotein (BLP), flagellin, and CpG sequences in bacterial DNA, affects circulating levels of proand anti-inflammatory cytokines [15].

Cytokines are able to act indirectly on the brain by activating receptors on nerves, the vagus nerve among others. Another way in which cytokines, chemokines, and immune factors influence the CNS are the circumventricular organs, drainage of the lymphatic system into the brain and the blood-brain barrier (BBB) via both diffusion and cytokine transporters [15, 16]. In the brain, there are resident immune cells such as macrophages, lymphocytes, and microglia, which are sensitive to immunological stimuli. It has been proven that gut microbiota can regulate the activity of these resident immune cells, for example through the TL-4 receptor widely distributed in lymphoid tissue [16].

It is worth highlighting, especially in the context of CNS diseases, that increased levels of proinflammatory cytokines in the general circulation may lead to increased permeability of BBB and further neuroinflammation in the nervous system, mainly by stimulation of microglial cells, release of of inflammatory cytokines, as well as recruitment of peripheral immune cells into the brain. In turn, chronic inflammation may lead to abnormal changes in mood and behavior [17]. Moreover, cytokines change the concentration of some neurotransmitters in the brain, such as dopamine, serotonin (5-HT), and glutamate [18]. All these factors together may seriously affect neuronal function. Administration of proinflammatory cytokines directly into rodents' brains induces sickness behaviors, which are commonly observed in systemic infection, such as changes in motivational state, sleep disorders, reduced appetite, decreased social/sexual interactions, and attenuation of cognitive behavior. Interestingly, in recent years scientists have found that sickness behaviors are very similar to those observed in anxiety and stress disorders, major depressive disorder, schizophrenia, autism, and learning disabilities [19].

Interestingly, gut microbiota are able to release a number of neuroactive compounds, precursors to hormones and neurotransmitters as well as neurotransmitters themselves, including gamma-aminobutyric acid (GABA), 5-HT, dopamine, noradrenaline, acetylcholine and histamine. The levels of many neurotransmitters found in the intestine are equivalent to or greater than those in the brain [15], which could indicate that bacterial production of neurotransmitters is a significant form of bacteria-neuron communication [20].

Metabolites of bacteria can transmit signals to the CNS via receptors in the enteric and autonomic nervous system or through enteroendocrine cells (EECs) [16, 21]. EECs influenced by contact with microbial by-products produce several neuropeptides, such as neuropeptide Y (NPY), cholecystokinin, glucagon-like peptide-1 and -2, and substance P, diffusing throughout the lamina propria, which is occupied by various neural and immune cells [22].

Gut microbiota also indirectly affect the production of neurotransmitters in host EEC and neuroendocrine cells by regulating the available precursors of neuroactive compounds. It has been proven that bacteria-derived short chain fatty acids (SCFAs), among others, can activate sympathetic neurons and receptors on EECs, resulting in increasing colonic and blood concentrations of 5-HT [23, 24]. Although 5-HT is not known to cross the BBB, its precursor tryptophan produced by the gut microbiota and present in peripheral blood is capable of crossing the BBB, and then can be used in 5-HT synthesis [15].

SCFAs, one of the more important metabolites produced in the gut due to the bacterial fermentation process, can also reach the systemic circulation, where they are capable of inducing T regulatory cell (Treg) differentiation and of regulating the secretion of interleukins. It is noteworthy that SCFAs are capable of passing through the BBB and influencing neuroinflammation in the brain. They can affect microglial maturation and can provide the cells with energy by alerting the levels of neurotransmitters and neurotrophic factors, increasing neurogenesis, brain-derived neurotrophic factor (BDNF) expression, neural proliferation, and promotion of memory consolidation [17]. Finally, SCFAs influence the integrity and function of intestinal and blood-brain barriers, which is speculated to play one of the most important roles in the etiology of inflammation [25].

Other metabolites that possess immunomodulatory effect and impact on brain physiology are bacterial neurotoxins and formyl peptides blocking neurotransmission [20]. In this and many other cases, it is beneficial to limit their contact with the interior of the human body, which is conditioned by a well-functioning intestinal barrier.

The proper integrity of the intestinal barrier is especially important for the processes associated with the functioning of the immune system in gut-associated lymphoid tissue (GALT). The intestinal barrier is a structure built of one layer of epithelial cells connected with tight junctions (TJs) [26].

In cases where the epithelial barrier is permeated by inflammation or other mechanisms, bacteria and bacterial products may also pass paracellularly between the cells, whose TJs are damaged [15]. In such cases, increased exposure of bacterial antigens and undigested nutrients in the lymphatic tissue and blood results in the production of cytokines, which mediate the inflammatory reaction, including impact on the BBB [17, 27].

The BBB is similar in its structure and functioning to the intestinal barrier [28]. It is noteworthy that germ-free (GF) mice have greatly increased permeability of the BBB because of lowered synthesis of proteins building TJs: occludin and claudin-5. Further colonization of the gut with either Clostridium tyrobutyricum or Bacteroides thetaiotaomicron reinstated BBB integrity, probably due to SCFAs produced by these species [25]. One of SCFAs–butyrate–might also be effective in enhancing epithelial barrier function because of its ability to increase production of secreted mucus [29].

There is evidence of the essential role of intestinal barrier integrity dysfunction, and associated with it, elevated levels of proinflammatory cytokines in the pathogenesis of CNS diseases such as depression, anxiety, or neurodegenerative diseases. Serum concentrations of IgM and IgA against LPS (lipopolysaccharide–bacterial endotoxin, activating the mechanism of immunological activation) is much higher in the major depressive disorder (MDD) patients than in healthy volunteers [30].

A very important factor disturbing the integrity of the intestinal barrier is the increased hypothalamic-pituitary-adrenal (HPA) axis tension and increased level of cortisol due to stress, especially when the initiated stress reactions are maintained for hours or even days.

Cortisol, by acting on immunological cells, modulates the secretion of cytokines and influences the composition and functions of microbiota. The interaction of cortisol with mast cells contributes to the release of their granularity and the digestion of the protein elements of the TJ, which in turn leads to the development of intestinal barrier integrity disorders. The increased level of gamma IFN under stress has also an adverse effect on intestinal barrier integrity. The penetration of bacterial antigens into the lamina propria and the bloodstream is then increased [21, 31].

On the other hand, higher amounts of secreted noradrenaline increases the pathogenicity of bacteria and viruses and improves their adhesion to the intestinal epithelium. As a result, there is an increased uptake of pathogenic bacteria by dendritic cells located in the intestinal wall, which leads to the presentation of antigens to lymphocytes and the enhancement of the inflammation. For example, chronic psychological stress has been shown to increase Escherichia coli uptake by GALT 30 times [32]. The secreted cytokines (interferon gamma (IFN-γ), IL1 beta and tumor necrosis factor alpha (TNF-α)), influence afferent neurons, transmitting inflammation-related signals to the brain. They also stimulate the activity of HPA axis, which maintains inflammation with loss of intestinal barrier function. This results in adverse physiological, psychological, and behavioral changes [22].

It is also noteworthy that stress itself also affects the composition and functions of microbiota, as numerous studies have shown a quantitative reduction of Lactobacillus and Bifidobacteria populations under emotional and physical stress [31]. CNS alerting the mucosal habitat and regulating release of signaling molecules, cytokines, and antimicrobial peptides into the gut lumen significantly affects microbial environment and the relative proportions of the main microbiotic phyla [21].

It is generally accepted that depression and anxiety disorders are neuropsychiatric disorders with well-known etiological connections to traumatic life incidents, particularly when experienced in childhood and especially during periods of chronic stress. The relationship between gut microbiota and chronic stress disorders can be of great importance here. Beside the well-known association between stress and neuropsychiatric disorders, the difficulty in understanding these complex processes by which stress increases vulnerability to disease still takes place [33]. Although stress is a natural occurrence, chronic, uncontrollable physical and mental stress can generate abnormal changes in behavior, brain structure, and CNS function [3, 34, 35]. Chronic stress has been commonly observed in several GI disorders and it could play a key role in GBA dysregulation of the stress-related CNS disturbances [36, 37, 38]. Animal studies have confirmed that emotional stressors, such as maternal separation, or some unpredictable mild stressors, like immobilization, crowding, changes in temperature, and sound signal can have a negative impact on the components of microbiota in the gut [39]. Among stressors used in mice, repeated social defeat has often been studied to show the effects of psychosocial stressors on behavioral changes. Some experiments [40] performed in a group of mice showing strong avoidance behaviors, called susceptibility to social defeat, significantly differed in the composition of the gut microbiota compared to another group of resilient mice. Additionally, the differences in the microbiome were directly linked to IL-1β and IL-6 levels as well as avoidance behavior. These results suggested that different classes of bacteria were linked to vulnerability to social stress because of chronic stressor exposure [40]. One can see that inflammation processes are common in models of social stress in mice and multiple studies now confirm that the microbiota are involved in these behavioral and biochemical changes induced by stress. In addition to increasing proinflammatory cytokine levels, social stressors can also provoke increased inflammation in the gut. Moreover, changes in the microbiota were linked with changes in anxiety-related behavior, especially in the aged animals [1]. These animals also demonstrated increased gut permeability, which was directly related to blood inflammatory cytokine levels (particularly IL-6 and IL-1β, as already stated) [41]. Thus, when considered together, these data can suggest that the changes in microbiome and increased gut permeability, also associated with aging, may cause impairments in behavior, including anhedonia-related disorder and cognitive disturbances, either seen with aging.

Depression is a severe disorder, often manifested by many psychological, behavioral, and physiological disturbances, including anhedonia, defined as “the decreased ability to experience pleasure from positive stimuli or a degradation in the recollection of pleasure previously experienced” [42]. So far, numerous attempts have been made to develop animal models of depression/anhedonia, which allowed imitating at least some clinical symptoms of depression, in laboratory conditions. These models enable testing many factors involved in the pathophysiology, etiology, symptomatology, and pharmacological treatment of depression; thus, the ideal animal model of depression should have as many criteria specific to depression in humans as possible and should be sensitive to antidepressants.

Currently, many various animal models of depression are used to mimic the depressive state in humans. Among them, chronic unpredictable mild stress (CUMS) seems to be considered one of the best models of depression [43]. This model intends to use chronic stress, which leads to depressive state and anxiety-like behaviors. It imitates stressful situations in people's everyday lives, and its aim is to cause the state of anhedonia, which is the main sign of depression in humans. Stimuli that initiate the stress response in laboratory animals, so-called stressors, act usually from two to four weeks, and are potentially deleterious to the organism, causing many physiological reactions to stress. The exposure to stressors causes an increased level of corticosteroids in plasma, and behavioral anhedonia-related changes in tested animals evaluated as reduction in sucrose preference, locomotor activity impairment, decreased food or water consumption or responsiveness to rewarding stimuli which can be diminished by administration of antidepressants [3, 34]. CUMS is now a valuable tool to investigate the neurobiological, behavioral, and hormonal changes underlying the psychopathology associated with stress and efficacy of antidepressant therapy.

Beside the animal models described above, we can dispose of some predictive tests of antidepressant activity. Thus, tests are used to prove the validity of the model and the potential effect of a treatment. Behavioral despair is primarily induced in rodents by exposure to unpredictable stressors, since anhedonia, following its definition, is a core sign of depression that can be assessed in rodents, as stated.

Among them, one of the most widely used screening tests for antidepressants is the forced swimming test (FST) or the tail suspension test (TST) [3]. In both tests exposure to the CUMS leads animals to exhibit some behavioral alteration, such as increased immobility (despair) time. However, in both mentioned paradigms, a depression state is not induced, in contrast to the CUMS model in which exposure to various chronic stressors induces numerous changes leading consequently to a depressed state of the animals.

Additionally, two other animal tests could be mentioned here in the context of anhedonia-related behavior. The first one is the sucrose preference test for rodents. Its assumptions are based on the animal's natural preference for a sweet solution, which is in proportion to the pleasure that the animal experiences while drinking [44]. In the case of examining the effect of chronic stress (e.g., CUMS) on preference for sweetened solutions, post-treatment preference can be compared to a baseline one.

In order to measure anxiety-like behavior, including that related to anhedonia as well as the influence of the chronic stress [3], the elevated plus maze (EPM) or the light-dark box test (LDB) is used. In the first, an increased time spent in the open arms or increased number of entries to the open arms can reflect anxiolytic activity, whereas the opposite is true for the anxiogenic effects. The second is as an approved animal test to measure unconditioned anxiety responses in rodents. Mice and rats prefer darker compartments to lighter areas; however, when presented in a novel surrounding, they have a tendency to explore. These two conflicting emotions lead to anxiety-related symptoms.

In order to measure cognitive effects, including those related to anhedonia as well as the influence of chronic stress on memory formation, many different animal tests can be used, for instance, open mazes but also the passive avoidance (PA) test, which is a fear-motivated test classically used to assess short-term or long-term memory in rodents [2, 3, 12, 45, 46]. This paradigm requires the animal to behave contrary to its natural tendency to prefer dark areas and to avoid lighted ones. Cognitive performance is positively correlated with the latency to move from the white compartment: the better the remembrance, the greater the latency.

For many years, probiotics, defined as “living microorganisms that, when ingested in adequate quantities, confer a health benefit on the host” [9, 47], have been used to address physiological abnormalities in developmental programming of epithelial barrier function, gut homeostasis, and immunological responses [37, 48]. Recently, data have demonstrated that probiotics are also capable of altering the CNS function of the host via the GBA. As such, much evidence suggests that the microbiome-GBA axis can regulate behavior and neuropsychiatric symptoms [37, 49, 50], as stress and anxiety seem to be strongly linked to a dysfunction of this axis, and commensal bacteria consumption can have a positive impact on stress-related neuropsychiatric disorders. Thus, modulating the enteric microbiota is increasingly considered a new therapeutic approach for these disorders [51]. However, the specifics of all probiotic bacteria influencing anxiety- and depression-related behaviors (i.e., anhedonia) and the exact mechanisms of their action are still being investigated.

Much valuable data underlying these phenomena have been provided by using animal models and tests. Changes in the gut microbiome have been revealed in rodents to be connected with disturbances in emotional behavior and changes in brain activity or neurotransmitters, as stated [10, 12, 37, 51, 52]. Many different approaches including the use of dietary changes, germ-free rodent strains, exposure to stressful stimuli, animals with infections of pathogenic bacterias, or those exposed to GBA–modulating agents such as pro-, pre- and antibiotics have been used to confirm the influence of microbiota on brain and behavior. These data, including those cited below, have yielded promising results reflecting the pivotal role of the microbiome in both health and illness.

For instance, probiotics have been suggested to restore stress-induced alterations to normal, including clearing the post-infectious stress. Some data revealed that probiotics can improve anxiety and stress-related response in a strain of anxious BALB/c mice, connected with changes in the expression of GABA receptors [10, 12, 50, 52]. Other data also showed that Bifidobacteria and Lactobacilli could cause positive effects on anxiety in rodents and humans, and can have a potential role in neurodegenerative and neuropsychiatric disorders [10, 12, 53]. The data from studies of an antidepressant, escitalopram, proved that the Bifidobacteria used in the study had the potential to induce better effects on anxiety and depression-related behavior in BALB/c mice than other antidepressants. These authors have demonstrated that two Bifidobacterium strains, B. longum 1714 and B. breve 1205, can improve changes in behaviors related to stress in this innately anxious mouse strain, suggesting that Bifidobacteria can reduce anxiety in mice without previous stress or any physiological manipulation. Furthermore, B. longum 1714 can ameliorate stress-induced hyperthermia and despair in the TST, an animal test for depression-related behavior, suggesting a role in sensitivity to an acute stressor and depression. B. breve 1205 provoked an anxiolytic effect in the EPM and reduced body weight gain, which can suggest a role in general anxiety and metabolism regulation [50]. Lactobacillus rhamnosus JB-1, Bifidobacterium longum NCC3001, Lactobacillus helveticus R0052, and B. longum R0175 also showed anxiolytic effects in mice and rats [10, 12, 54, 55, 56]. Such findings may have some clinical implications for all patients with psychiatric illnesses resistant to therapy using antidepressants. These results strengthen the role of gut microbiota modulation as an important strategy for stress-related GBA disorders, opening new approach in neurogastroenterology.

In thinking about possible neurochemical mechanisms underlying this phenomenon, it has been commonly accepted that the behavioral anhedonia-related changes were associated with alterations in neurotransmitter pathways in the CNS. Accordingly, it has been suggested that, at a molecular level, Bifidobacteria can possibly act on the serotoninergic system. Earlier studies revealed that tryptophan level was increased by B. infantis, providing evidence that enteric microbiota appear to be important for tryptophan availability and metabolism, causing an indirect influence on 5-HT level in the rats' brains [57, 58]. Other animal studies concerning the consequences of chronic probiotic treatment, both in unperturbed and stressed animals, have also served as valuable tools in confirming the specific influence of gut bacteria on anhedonia-related disorders. Probiotic treatment during the period of postnatal stress in maternally separated rat offspring has been shown to restore normal basal corticosterone levels [59]. For instance, when administered to naive animals, L. rhamnosus reduced stress-induced corticosterone concentration, which was connected to alterations in GABA receptor gene expressions in the specific CNS regions, that is, the hippocampus and cortex [10]. All these findings cited in this part of the review give further confidence to the concept of “psychobiotics” recently introduced as a novel strategy for treating neuropsychiatric disorders [60, 61, 62]. Some probiotics could be useful in treating a wide range of psychiatric diseases in a strain-dependent manner, just as conventional psychotropic treatment does. These data show that bacteria exert a pivotal role on the functioning and relationship of GBA, stress, and behavior [37, 48], and can open up new directions for treating neuropsychiatric diseases, in a way that may be as effective as or even better than current pharmacological treatments.

There are many other possible mechanisms involved in the effects of probiotics; any of the neurotransmitters involved in anhedonia, depression, and anxiety, such as monoamines and GABA could be involved [10, 63]. As shown in experiments using maternal separation to induce anhedonia-like disturbances in rats, administration of Bifidobacterium infantis 35624 alleviated depression-like behaviors and decreased the level of 5-HT and dopamine and their metabolites in the brain [58]. Furthermore, monoaminergic transmission was increased in the colonized germ-free mice compared with control germ-free mice [42], resulting in an altered behavioral phenotype. In another paper, authors have examined the abilities of an antidepressant, fluoxetine, and another probiotic, to attenuate responses in two known tests for depression-like behavior in animals, that is, the corticosterone response to an acute restraint stressor and the TST [54]. Authors have examined two strains of mice which differ in sensitivity to anxiety-like behavior and measures of despair (BALB/c and Swiss Webster mice, with high and normal behavioral phenotypes, respectively). While adult male BALB/c mice represented increased antidepressant-like behavior in response to both an antidepressant drug and L. rhamnosus JB-1 in both tests used, Swiss mice have not responded to either treatment as compared to control mice [55, 64]. Clinical studies examining the activity of L. rhamnosus JB-1 in patients with mood disorders are justified, together with further pre-clinical work explaining how interactions between genotype and alteration in intestinal microbiota may influence behavioral responses to stress. This study can confirm the possibility that a modification of intestinal microbiota by probiotics represents a promising potential therapeutic target in the novel treatment of neuropsychiatric diseases, as already pointed out. As such, pharmacotherapy modulating serotoninergic neurotransmission, such as antidepressants of many different pharmacological classes, including selective serotonin reuptake inhibitors (SSRIs), have shown therapeutic effects in the treatment of both neuropsychiatric and GI disorders. More studies are needed to elucidate the underlying mechanisms involved in modulation of this GBA communication pathway.

Another aspect of the physiological connection between the gut microbiota and the host reveals an important influence of probiotics on metabolic parameters such as insulin sensitivity, body weight, or parameters of chronic tissue inflammation, which are also important in stress and anhedonia [65]. Some authors aimed at investigating the interaction between habitual diet and the effect of probiotics on depression-related behavior and further examined some potential mechanisms underlying the microbe-mediated behavioral changes. Data have already been collected for rats fed a control or high-fat diet (HFD) for a few weeks and given either a multi-species probiotic formulation (“Ecologic Barrier”, mix of bacterial strains like B. bifidum W23, B. lactis W52, L. acidophilus W37, L. brevis W63, L. casei W56, L. salivarius W24, L. lactis W19, L. lactis W58) or vehicle for five weeks [66]. These promising data revealed that, independently of diet, probiotics significantly reduced depression-like behavior measured by using the FST. HFD aggravated the depressive-like behavior in rats in this test. Probiotic treatment prevented the depression-like effect of HFD, but had no effects in rats on the control diet. This antidepressant-like action of probiotics has been associated with an increased output of T cell-related cytokines by mononuclear cells in the peripheral blood at the expense of monocyte-derived cytokines such as IL-6 and TNF-a. These results further confirm an immunomodulatory action of probiotics. Similarly, authors have recently revealed that the same multi-species probiotic mixture reduced depression-like behavior in rodents independently of diet [67]. Previous studies with probiotics in rats have revealed a reduction in depression-like behavior induced by some environmental stimuli such as maternal separation and chronic restraint stress [58, 68]. It may be hypothesized, then, that a disturbance in metabolism seen in depression seems to be sensitive to a probiotic treatment. This suggestion could be of great clinical relevance, since studies in humans indicate that obesity can also be associated with weaker clinical response to commonly used antidepressants. MDD is also associated with dysmetabolic states such as obesity and diabetes mellitus type 2; thus, the gut microbiota may influence both disease characteristics. Some data point out that MDD may contain a dysmetabolic component sensitive to probiotic treatment [66, 67, 69]. The close relationship between depression-like behavior and T cell populations suggests that lymphocyte-brain interactions can be considered a promising potential target in the domain of psychoneuroimmunology.

Another mechanism underlining stress-induced disorders, described in the first part of this article, is the “leaky gut” phenomenon. An indirect role of microbiota in response to chronic stress was recently shown in an animal model of stress-induced impairment of the intestinal barrier. Prevention of gut leakiness by the modulation of intestinal microbiota with probiotics led to an attenuation of the HPA axis stimulation during stress [70]. Studies show that pretreatment with a probiotic formulation reduced the increase in intestinal cellular permeability induced by stress, which was correlated with attenuated degradation in TJ proteins' expression of colonic mucosa. Furthermore, providing that the influence of the HPA in stress-related changes is crucial, the modulation of intestinal microbiota by probiotics may also diminish stress-induced gut leakiness already mentioned, due to a probiotic-mediated impairment of HPA response in rodents exposed to an acute stress [70]. It can be hypothesized that damage to the epithelial GI barrier can be considered a consequence of either a stressful stimulus or microbiota dysbiosis, causing increased intestinal permeability and the accompanying trans-location of pathobionts from the mucosa to areas where direct interaction with immunologic system can occur [71]. This process can lead to activation of an immunologic response characterized by higher level of pro-inflammatory mediators in both circulation and the CNS. According to this hypothesis, administration of the probiotic Lactobacillus farciminis diminished the consequences of acute restraint stress on intestinal permeability and HPA axis stimulation [70]. Even though current data contain few reports discussing the role of gut microbiota in depressed patients, data from associated disorders, such as irritable bowel syndrome (IBS), which often coexists with depressive symptoms, have shown reduced Bacteroidetes and increased Firmicutes content in the fecal samples of individuals with both disorders [72, 73]. Some probiotics are capable of reducing mRNA expression of GABA receptors important for the regulation of anxiety disorders or diminishing mitogen stimulation–induced increased plasma tryptophan implicated in depression, also those accompanying IBS [74]. Although the direct link between the brain and microbiota requires further investigation, many data confirm that specific changes in microbiotic composition could ameliorate psychological distress and improve quality of life in patients suffering from IBS. According to the animal studies providing evidence of the advantage of probiotics in stress-induced GI disorders, it is very important to test specific probiotic formulations as therapeutic tools in the treatment of anxiety and depressive disorders linked to IBS symptoms.

In order to propose some more possible mechanisms in the abovementioned processes, in the context of a possible influence of the HPA and GABA-ergic pathway, in a previous study already cited [10], authors have reported that, in mice, the oral administration for 4 weeks of a particular strain of Lactobacillus rhamnosus JB-1 promoted an anxiolytic effect, attenuated the HPA/corticosterone response to stress, and caused region-specific changes in mRNA for GABA-A and -B receptor subtypes in the CNS [55]. These changes were coherent with the effects of GABA by itself, the main inhibitory neurotransmitter in the brain, and importantly are largely withdrawn by sub-diaphragmatic vagotomy. As previously mentioned, it has been hypothesized that the afferent vagal pathway to the CNS seems to be a main route by which beneficial bacteria in the gut may influence behavior [10, 75], although other data have not confirmed this hypothesis [12]. Provided results also confirm the role of the vagus nerve in supporting the neurochemical changes connected with GABA receptor level and distribution; this may be a key factor important to the anxiety-related behavioral changes observed. Once more, it seems that choosing the microbiota GBA to modulate behavior by probiotic could be important for the regulation of the stress-related disorders. Much data pointed to the pivotal role of the HPA axis, as stated [76]. Recently, data on the effect of a combination of two probiotics, that is, Bifidobacterium longum R0175 and Lactobacillus helveticus R0052, resulted in reduced anxiety level in rats and in humans. It has been revealed that beneficial properties of this probiotic formulation were correlated with decreased levels of free cortisol in the urine, suggesting impairment of HPA axis response [56].

Moreover, it can be added that probiotics precluded changes in neuronal activation and neurogenesis induced by chronic stress. In this context, induction of apoptosis in several brain regions or changes in cognition and anxiety-like behaviors were also detected in animal models of depression due to consumption of the probiotic strains or diet supplemented by a probiotic, that is, L. helveticus R0052 [53, 60]. These studies show that a treatment with the probiotic formulation Probio'Stick (L. helveticus R0052 and B. longum R0175) for 2 weeks diminished HPA axis activation and response to chronic stress as detected by a decreased plasma levels of corticosterone and also of noradrenaline and adrenaline circulated level in stressed mice. However, L. salivarius treatment had no influence on these neuroendocrine responses to chronic stress. The lack of the influence of level of stress hormone suggests that the function of HPA and response to chronic stress depends on bacterial strain. As such, the normalization of this HPA/stress hormones axis may be an important indicator in the evaluation of antidepressant activity of probiotics [70]. Other data can suggest a beneficial role of pretreatment with probiotics in modulating neuronal pathways in the hypothalamus that coordinate synaptic plasticity. It is well known that chronic stress exposure in rodents changed the function of brain areas involved in the HPA-related response to stress, especially hypothalamus and hippocampus. Chronic stress model is often used to mimic human depressive disorders in animals [3, 34], and to detect an increase in monoamines in the hippocampus associated with deficits in learning and memory tasks, also caused by stress. Monoaminergic mechanisms can also be confirmed. In rats [57], a reduced noradrenaline level in the hippocampus following chronic 14-days Bifidobacteria treatment has been shown. The same effect was observed after the administration of antidepressants to animals, suggesting that Bifidobacteria may affect the neuronal pathways under stress conditions by mechanisms similar to that observed after chronic antidepressant drugs [64]. One can suggest that these neurophysiological changes in the brain of stressed mice were consistent with a pronounced neuroplasticity of neurotransmitters in the paraventricular nucleus (PVN) of the hypothalamus that anticipate enhanced excitability of the HPA axis in response to chronic stress. Interestingly, some studies have proved the capacity of a probiotic treatment to diminish hypothalamic corticotrophin-releasing factor (CRF) production in stressed rats, or to modulate GABA-ergic pathways involved in anxiety-related behavior in a cerebral region–dependent manner [71]. Altogether, these data imply that probiotics can influence neuronal circuits (e.g., GABA-ergic and monoaminergic) involved in the HPA axis response to stress.

Increasing evidence suggests that perturbation in the normal gut microbiota can be connected to GI disorders after antibiotic treatment or infection, as well as stress-related alterations in behavior, as stated [11, 74, 77]. Accordingly, it has been shown that rodents allowed to grow up in a germ-free environment had altered anxiety-related behavior, impaired HPA axis and sociability or social cognition [39, 46, 64, 78]. Chronic stress also provokes changes in the PVN of the hypothalamus, such as increase in CRF, reduction in expression of glucocorticoid receptors and other neurotransmitter receptor subunits. These neurochemical changes prove that chronic stress reinforces the excitability of HPA subjected to stress and decreases HPA negative feedback in the PVN and hippocampus. In a study [71], authors found that suppressed neurogenesis in the hippocampus after chronic stress was restored in the probiotic-treated mice. These data reveal that probiotics are able to improve survival and differentiation of neurons, suggesting that the administration of probiotics promotes neurogenesis in the dentate gyrus of the hippocampus in stressed mice, probably by improving synaptic plasticity. In the abovementioned data, it has been shown that probiotic treatment boosts neuronal activation changes in several regions in the brain, especially in the hippocampus, important for cognition, causing axonal and dendrite neuronal prolongation. It is suggested that the attenuation of HPA axis by the probiotic formulation protects neuronal plasticity in the hippocampus, sustaining CNS activity versus stress-mediated brain circuitry failure, due to the promotion of neurogenesis in the hippocampus caused by the probiotics.

The set of studies described above use depression-related models and tests in animals; these are collected in Table 1. Due to the topic of this publication, the table includes studies in which animal models were used to assess mood, that is, depression, stress, and anxiety.

It can be accepted on the basis of these studies that enteric microbiota have an important influence on the neurochemical, behavioral, and immunological parameters relevant to GBA disorders, and that “psychobiotics” can offer promising new treatments [60, 81]. The therapeutic potential of probiotics in neuropsychiatric disorders has been a matter of intense research, but more investigations are needed to fully explain their role in brain function. Despite the lack of reliable clinical data to confirm the usefulness of probiotics in treatment of affective disorders, there are quite sufficient pre-clinical data in animals to support this idea. Potential psychobiotics are capable of reducing stress-induced inflammatory response and HPA activation; to degrees similar to existing antidepressants. As probiotics differ in their effects and not all may have psychobiotic potential, further examination of their efficacy is justified. Many pre-clinical studies have now elucidated manipulations of the GI mucosa through administration of probiotics; these can affect behavior in animals, as discussed in the last part of the review [9]. Previous pre-clinical studies have also revealed that administration of probiotics may cause a reduction in depressive and anxiety-like stress-induced behavior in healthy mice independently of diet [10, 58, 68]. Indeed, from the references cited, it appears that probiotics may be useful in modulating a wide range of behaviors associated with psychiatric disorders, such as anhedonia-related behavior. What is more, researchers identified equivalent changes in many physiological and neurochemical systems that could be responsible for the observed effects: displacement in the pattern of cytokines or changes in the hippocampal function and in synaptic plasticity or in HPA axis regulation. These data therefore further suggest the microbiota as important regulators of the neuroendocrine stress axis at the central level. At present, it is well established that a large number of people suffering from depression have a dysregulated HPA axis. The gut microbiota have recently been proposed to be important regulators of the CNS function and behavior in animals, and administration of certain probiotics appear to have a potential therapeutic potential for the treatment of MDD and variety of neuropsychiatric disorders. Although many studies investigating the role of microbiota in the CNS and brain function have delivered promising results, they need further validation in clinical studies.

Fig. 1