Review Reports - Probiotics as Modulators of Adult Neurogenesis and Synaptic Plasticity: New Perspectives in the Pathophysiology and Treatment of Affective Disorders

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper by Rosas-Sánchez et al. on the use of probiotics as modulators of neurogenesis and synaptic plasticity is well written and comprehensive. It provides an in-depth summary of the state of the art and current knowledge on preclinical and clinical findings in this field.

Author Response

Comments 1: [This paper by Rosas-Sánchez et al. on the use of probiotics as modulators of neurogenesis and synaptic plasticity is well written and comprehensive. It provides an in-depth summary of the state of the art and current knowledge on preclinical and clinical findings in this field.]

Response 1: [Dear reviewer, we appreciate your feedback on our manuscript.]

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript provides a timely and comprehensive narrative review on the emerging role of probiotics as modulators of adult neurogenesis and synaptic plasticity via the microbiota-gut-brain axis, with particular relevance to the pathophysiology and treatment of affective disorders. The topic is highly diverse, given the limitations of current therapies for depression and anxiety and the growing interest in psychobiotics. Despite its strengths and mechanistic detail, the manuscript would benefit from greater methodological transparency, more balanced presentation of conflicting evidence, and stronger emphasis on safety and translational limitations. With targeted revisions to address these issues, the work has strong potential to make a meaningful contribution to the psychobiotic and neuroplasticity literature.

Major Comments

1. The methods section (Section 2) describes this as a narrative review without predetermined research questions or a specific search strategy, relying instead on keyword searches across databases like PubMed and Scopus. However, narrative reviews can still introduce bias if not transparent. The inclusion/exclusion criteria are hardly described (e.g., focusing on "chronic treatment" without defining duration thresholds), and there is no flow diagram or assessment of study quality. This undermines the review's objectivity and reproducibility.

2. The review cites studies up to around 2023-2024 but misses key recent advancements on probiotics in depression. Sections on clinical evidence (Section 8) and translational considerations (Section 10) feel dated, with over-reliance on older preclinical models. The author should broaden and update the search and discuss emerging topics, such as fecal microbiota transplantation (FMT) as a comparator to probiotics.

3. Multiple sections (Sections 4-7) heavily emphasize rodent and in vitro models (germ-free mice, C57BL/6 models) but fail to critically address translational gaps, such as species differences in microbiota composition or the blood-brain barrier. Claims like "probiotics exert a comprehensive influence across the entire neurogenic cascade" (Section 4.3) are not sufficiently strengthened by human evidence limitations.

4. The molecular mechanisms (Section 7) are well-described (SCFAs as HDAC inhibitors), but the review lacks specificity on strain-dependent effects. For instance, it groups Lactobacillus and Bifidobacterium broadly, without distinguishing strains (e.g., L. rhamnosus JB-1 vs. others). This could mislead readers on therapeutic applicability. The author should provide a table comparing strain-specific effects on neurogenesis/synaptic plasticity, and discuss potential off-target effects.

5. The review predominantly highlights beneficial effects of probiotics (Sections 4, 5, and 8), with minimal attention to null or negative results (studies showing no BDNF changes or increased inflammation). Section 6.2 mentions one negative study but downplays it. The author should balance the narrative by including a subsection on conflicting evidence and, if meta-analytic elements are added, by including a subsection on meta-analysis.

6. Figures 1 and 2 are referenced but not adequately integrated into the text. The review would benefit from tables summarizing the mechanisms or biomarkers of key studies. Currently, the text is dense and hard to follow without visual aids. The author should revise to include tables and ensure figures are high-resolution with detailed legends.

7. Section 8.4 briefly mentions variability but does not explore underlying factors deeply, such as sex differences, age, or microbiome enterotypes. The author should expand this section and include these in the text. Additionally, the author should discuss implications for precision medicine, including omics-based predictors.

8. The introduction and Section 3 establish neurobiological foundations, but the connection to affective disorders is superficial in later sections. The author should discuss how synaptic plasticity deficits directly map to depression symptoms.

9. Probiotics are presented as largely beneficial, but potential risks are ignored. Section 10 should include a safety profile and discuss contraindications for patients with affective disorders.

10. Section 11 conclusions are generic. The author should expand on future directions, including specific research recommendations, and propose a roadmap for clinical implementation.

Minor Comments

1. The author has used terms like "psychobiotics" and "MGB axis" interchangeably without clear definitions early on (not defined in the Introduction).

2. Several typos (e.g., "germen free" in Section 4.1 should be "germ-free"). Use consistent italics for genus/species (e.g., Bifidobacterium longum).

3. Sections 3.1 and 4.1 overlap on hippocampal neurogenesis; condense to avoid repetition.

4. In Section 6.1, "the decision to include only studies with chronic treatment was made because..." is awkwardly phrased; clarify the rationale.

5. Figure legends mention BioRender but lack alt-text descriptions for accessibility; add these.

Recommendation

I recommend a major revision. The manuscript has strong potential as a timely synthesis of an emerging field, but the major issues, particularly methodological transparency, balanced evidence presentation, and translational depth, require substantial rework to meet publication standards.

Comments on the Quality of English Language

The English could be improved, and a couple of typo mistakes were detected.

Author Response

Major Comments

Comments 1: [The methods section (Section 2) describes this as a narrative review without predetermined research questions or a specific search strategy, relying instead on keyword searches across databases like PubMed and Scopus. However, narrative reviews can still introduce bias if not transparent. The inclusion/exclusion criteria are hardly described (e.g., focusing on "chronic treatment" without defining duration thresholds), and there is no flow diagram or assessment of study quality. This undermines the review's objectivity and reproducibility.]

Response 1: [We thank the reviewer for this thoughtful and constructive comment. We acknowledge that, although narrative reviews do not follow the same systematic protocols as systematic reviews or meta-analyses, transparency in the selection process remains essential to minimize bias and improve reproducibility. Based on this feedback, we have revised Section 2 as follows:

Regarding inclusion/exclusion criteria: We agree that the term "chronic treatment" required a more precise operational definition. We have now added a duration threshold to clarify this criterion. Specifically, we define chronic treatment as interventions lasting four weeks or longer, consistent with the minimum period reported in the literature as necessary for probiotics to exert measurable effects on gut microbiota composition and host physiology. This definition has been added explicitly to Section 2.2.

Regarding the search strategy: We have expanded Section 2.3 to provide greater detail on the search process, including the Boolean operators and combinations of terms used across each database (PubMed, ScienceDirect, Web of Science, and Scopus) and the language restriction (English only). This additional transparency allows readers to better evaluate and, if desired, replicate the search strategy.

Regarding a flow diagram: We recognize that a PRISMA-style flow diagram enhances reproducibility even in narrative reviews. We are aware of the potential limitations of the methods report in a narrative review and the importance of your suggestion to include additional elements from PRISMA. However, we have assumed that this review is a narrative review in which some items requested by PRISMA have not been included from the outset. We are concerned about the inclusion of PRISMA elements as we may omit some information and confuse potential readers as this narrative review may contain a mixture of methods. We respectfully request to the reviewer to consider this possibility and permit us to omit this suggestion from our report. To improve our methods report, we have added some clarifications that have been included in the methods.

Regarding study quality assessment: We acknowledge this as a genuine limitation of narrative reviews. As we note in the original manuscript, narrative reviews by design do not include formal quality appraisal tools (e.g., GRADE, Cochrane RoB). However, we have added a paragraph to the limitations section explicitly acknowledging that the absence of a formal quality assessment represents a constraint on the conclusions drawn, and we encourage future systematic reviews on this topic to address this gap.

We believe these revisions substantially improve the transparency, objectivity, and reproducibility of the methods section while remaining consistent with the narrative review design.]

The review cites studies up to around 2023-2024 but misses key recent advancements on probiotics in depression. Sections on clinical evidence (Section 8) and translational considerations (Section 10) feel dated, with over-reliance on older preclinical models. The author should broaden and update the search and discuss emerging topics, such as fecal microbiota transplantation (FMT) as a comparator to probiotics.

Response 2: [We sincerely thank the reviewer for this valuable observation. We agree that ensuring the currency and comprehensiveness of the cited literature is essential to the quality and relevance of the review. In response, we have taken the following actions:

Regarding the updating of the literature: We have conducted a supplementary search of PubMed, ScienceDirect, Web of Science, and Scopus, filtering for publications from 2024–2025, using the same keyword combinations described in Section 2.3. As a result, several recent studies have been incorporated into Sections 8 and 10, including updated meta-analyses and randomized controlled trials examining the effects of probiotic supplementation on depressive and anxiety symptoms, as well as more recent findings on peripheral biomarkers of brain plasticity such as BDNF. Specifically, we have added references that strengthen the clinical evidence base and reflect current discussion in the field.

Regarding over-reliance on older preclinical models: We acknowledge that some of the foundational preclinical evidence cited in these sections relied on earlier animal model studies. We have made an effort to complement these with more recent preclinical findings where available, while also more explicitly contextualizing the limitations of translating rodent model outcomes to human clinical settings. We have added language in Section 8.3 that more critically addresses this translational gap.

Regarding fecal microbiota transplantation (FMT) as a comparator: We agree that FMT represents a highly relevant and emerging intervention that warrants discussion in this review, particularly given that it shares mechanistic overlap with probiotic interventions through microbiota modulation. We have added a dedicated paragraph in Section 11 that discusses FMT in comparative perspective with probiotics. Specifically, we note that while FMT involves the transfer of a complete donor microbiota and has shown promising results in treatment-resistant depression in early clinical trials, probiotics offer a more controllable, reproducible, and scalable alternative, with a well-characterized safety profile. We also highlight the complementarity of these approaches, noting that emerging research is beginning to explore their sequential or combinatorial use. This discussion is supported by recent references, including the clinical trial registry data available at clinicaltrials.gov referenced in Section 9.1, as well as recent reviews of FMT in neuropsychiatric conditions.

We believe these revisions substantially modernize the clinical and translational sections of the manuscript and position it more competitively within the current state of the field.]

Comments 3: [Multiple sections (Sections 4-7) heavily emphasize rodent and in vitro models (germ-free mice, C57BL/6 models) but fail to critically address translational gaps, such as species differences in microbiota composition or the blood-brain barrier. Claims like "probiotics exert a comprehensive influence across the entire neurogenic cascade" (Section 4.3) are not sufficiently strengthened by human evidence limitations.].

Response 3: [We thank the reviewer for this important observation. We agree that Sections 5–8, which constitute the mechanistic core of the review, rely heavily on preclinical evidence from rodent models (germ-free mice, C57BL/6 models) and in vitro systems, and that this reliance was not always accompanied by an explicit critical discussion of its translational limitations. In response, we have made the following targeted revisions to each affected section, drawing on contextualizing language and caveats that were already present elsewhere in the manuscript and consolidating them into the sections where the preclinical claims are made.

Regarding Section 5.3 specifically ("probiotics exert a comprehensive influence across the entire neurogenic cascade"): We acknowledge that this statement, as originally written, overstates the strength of the evidence by not distinguishing between what has been demonstrated in rodent systems and what remains to be validated in humans. We have revised this sentence to read: "In preclinical models, probiotics appear to exert influence across multiple stages of the neurogenic cascade, from initial cell proliferation to functional integration, although direct human evidence for these specific neurogenic effects remains limited and largely inferred from peripheral biomarkers such as BDNF" — a reformulation that preserves the mechanistic content while being appropriately epistemically qualified.

Regarding translational gaps in Sections 5–8 more broadly: Rather than adding new references — which would be outside the scope of this revision round — we have inserted brief contextualizing statements at the close of each mechanistic subsection, drawing on limitations and caveats that were already explicitly discussed in Section 5.4 (translational studies and peripheral biomarkers), Section 9.3 (methodological limitations), and Section 9.4 (interindividual variability), where the manuscript already acknowledged that: (a) directly assessing neurogenesis in the living human brain is challenging due to ethical and technical limitations; (b) significant heterogeneity exists in microbiota composition between laboratory rodents and humans, influenced by standardized diets, controlled housing, and inbred genetic backgrounds that have no human equivalent; (c) the blood-brain barrier permeability and SCFA transport mechanisms characterized in rodent systems may not translate directly to human physiology; and (d) GF mouse models, while mechanistically invaluable, represent an extreme microbiological condition with no clinical human counterpart.

Specifically, we have added the following types of qualifying statements within Sections 5-8, grounded entirely in content already present in the manuscript:

In Section 5 (Probiotics and adult neurogenesis): Following descriptions of GF mouse findings, we now include an explicit note that GF animals represent a non-physiological extreme absent in human clinical reality, and that the neurogenic effects observed in these models should be interpreted as mechanistic proofs-of-concept rather than direct clinical predictions — consistent with the caveats already stated in Section 5.4.

In Section 6 (Synaptic plasticity): Following mechanistic claims derived from rodent LTP/LTD studies, we now clarify that synaptic plasticity measurements in rodent hippocampal slice preparations and in vivo electrophysiology do not have direct non-invasive equivalents in human research, and that neuroimaging correlates (fMRI, DTI) discussed in Section 9.2 represent only indirect functional proxies — a point already developed in Section 3.4 of the manuscript.

In Section 6 (Synaptic receptor expression): Following descriptions of receptor modulation studies conducted exclusively in male rodent models, we now note the sex composition limitation and the documented species differences in GABA-A receptor subunit distribution between rodents and humans — both of which are consistent with the heterogeneity limitations discussed in Section 9.3.

In Section 8 (Molecular mechanisms): We have added a closing paragraph to Section 8 that consolidates these cross-cutting translational caveats, noting explicitly that the epigenetic mechanisms, SCFA signaling pathways, and neuroimmune interactions described in this section have been characterized predominantly in rodent and in vitro systems, and that while these mechanistic findings provide a compelling biological framework, their translation to human clinical contexts is conditioned by important species-level differences in microbiota composition, BBB architecture, and metabolite bioavailability — all of which are acknowledged in Section 8 of the current manuscript.

We believe these revisions, which draw exclusively on content and caveats already present in the manuscript, meaningfully address the reviewer's concern without altering the mechanistic content of Sections 5-8 or introducing new claims that would require additional references. The overall effect is to transform what were previously uncaveated mechanistic summaries into appropriately qualified preclinical findings that are honestly situated within their translational context.]

Comments 4: [The molecular mechanisms (Section 7) are well-described (SCFAs as HDAC inhibitors), but the review lacks specificity on strain-dependent effects. For instance, it groups Lactobacillus and Bifidobacterium broadly, without distinguishing strains (e.g., L. rhamnosus JB-1 vs. others). This could mislead readers on therapeutic applicability. The author should provide a table comparing strain-specific effects on neurogenesis/synaptic plasticity, and discuss potential off-target effects.]

Response 4: [We thank the reviewer for this important observation regarding the lack of strain-level specificity in the mechanistic sections. We fully agree that grouping Lactobacillus and Bifidobacterium at the genus level, without distinguishing between individual strains, risks misleading readers about the therapeutic applicability of the findings reviewed. In response, we have compiled Table 1, which systematically organizes the strain-specific effects of all probiotic interventions already discussed across Sections 5-8 of the manuscript, including 15 distinct strains and consortia (e.g., L. rhamnosus JB-1, L. fermentum ATCC 9338, L. rhamnosus GR-1, B. infantis, L. helveticus, E. faecalis EC-12, among others), detailing for each: the experimental model and duration, neurogenesis-related effects, synaptic plasticity outcomes, and the specific receptors or intracellular pathways modulated, along with the corresponding references already cited in the original manuscript. Additionally, we have added a paragraph at the end of Section 7.4 that explicitly addresses potential off-target and null effects documented in the reviewed studies, drawing on three observations already present in the manuscript: the absence of dopaminergic or serotonergic receptor changes with E. faecalis EC-12 despite behavioral improvement; the paradoxical upregulation of inhibitory GABAA subunits following antibiotic-induced dysbiosis; and the dendritic spine loss induced by propionic acid via MAPK/ERK signaling. This paragraph concludes by reinforcing that strain-level mechanistic specificity — rather than genus-level generalization — must guide future therapeutic applications of psychobiotics. No new references were required for any of these additions, as all data were already present in the manuscript.]

Comments 5: [The review predominantly highlights beneficial effects of probiotics (Sections 4, 5, and 8), with minimal attention to null or negative results (studies showing no BDNF changes or increased inflammation). Section 6.2 mentions one negative study but downplays it. The author should balance the narrative by including a subsection on conflicting evidence and, if meta-analytic elements are added, by including a subsection on meta-analysis.]

Response 5: [We thank the reviewer for this substantive and well-founded observation. We acknowledge that, as originally written, Sections 5, 6, and 9 predominantly emphasized the beneficial effects of probiotic interventions, with null and negative findings either absent or underemphasized relative to their actual prevalence in the reviewed literature. We agree that this imbalance in narrative framing could give readers an inflated impression of the consistency and robustness of the evidence base. In response, we have carefully reviewed the content already present throughout the manuscript and identified a number of null, negative, and conflicting findings that were either mentioned only in passing or dispersed across different sections without being consolidated into a coherent critical discussion. Rather than introducing new references, we have added a dedicated subsection — Section 9.5: Conflicting evidence, null results, and cautionary findings — that brings together these observations from across the manuscript into a single, balanced critical synthesis. Specifically, this subsection draws on: (1) the already-cited finding in Section 8.1 that meta-analyses have generally found only small pooled effects and that positive results in major depressive disorder remain preliminary [186]; (2) the observation in Section 7.3 that Enterococcus faecalis EC-12 produced no significant changes in dopaminergic (Drd5) or serotonergic (Htr2b) receptors despite reducing anxiety-like behavior [164], illustrating that behavioral improvement does not necessarily reflect broad neuroplasticity; (3) the negative finding in Section 7.2 — which the reviewer correctly notes was downplayed — that antibiotic-induced dysbiosis paradoxically increased inhibitory GABAA subunit expression, worsening depressive-like behavior and spatial memory [147]; (4) the finding in Section 6.3 that propionic acid, a SCFA produced by gut bacteria, induces dendritic spine loss via MAPK/ERK signaling [141], demonstrating that not all microbial metabolites are uniformly pro-neuroplastic; and (5) the statement already present in Section 11.3 that a pilot study in treatment-resistant depression reported symptom recurrence after probiotic discontinuation [187], raising unresolved questions about the persistence of any neuroplastic effects. Regarding the reviewer's suggestion to include meta-analytic elements: while a formal meta-analysis falls outside the methodological scope of this narrative review as declared in Section 2.1, we have added a sentence in Section 8.5 explicitly acknowledging this limitation and directing readers to the most recent meta-analyses already cited in the manuscript [129, 183, 186] for quantitative synthesis of pooled effect sizes. No new references were required for any of these additions.]

Comments 6: [Figures 1 and 2 are referenced but not adequately integrated into the text. The review would benefit from tables summarizing the mechanisms or biomarkers of key studies. Currently, the text is dense and hard to follow without visual aids. The author should revise to include tables and ensure figures are high-resolution with detailed legends.]

Response 6: [We thank the reviewer for this observation regarding the integration of visual elements and the density of the text. We would like to address each point raised separately.

Regarding Figures 1 and 2, we respectfully note that both figures were designed as comprehensive visual syntheses of the mechanistic content developed across the sections in which they are cited, and that their placement and function are explicitly described in the corresponding figure legends. Figure 1 (cited in Section 6) provides a four-panel visual summary of the mechanisms by which probiotics modulate synaptic plasticity — covering structural synaptic proteins (Panel A), neurotrophic factor regulation by probiotic-derived metabolites (Panel B), effects on dendritic and spinal cord morphology (Panel C), and modulation of LTP and LTD (Panel D) — directly complementing the subsections 6.1 through 6.4 in which each panel is individually referenced and described. Figure 2 (cited in Section 8) similarly organizes the underlying molecular mechanisms into four panels: epigenetic mechanisms of probiotics on neurogenesis (Panel A), modulation of inflammatory pathways and plasticity (Panel B), SCFA-mediated signaling (Panel C), and neuroactive peptides derived from the microbiota (Panel D), corresponding directly to subsections 8.1 through 8.4. In this sense, rather than being peripheral to the text, both figures serve as panel-by-panel visual counterparts to the prose developed in their respective sections, and the text explicitly directs the reader to the corresponding panel at each relevant point. We have revised the figure legends to make this structural correspondence more explicit and have ensured that both figures are submitted at a resolution of at least 300 dpi in accordance with the journal's technical requirements.

Regarding the inclusion of tables, we fully agree with the reviewer that tabular summaries enhance navigability and reduce the interpretive burden imposed by dense mechanistic prose. In response to a previous comment you made on strain-specific effects (addressed separately in our response), we have already incorporated Table 2, which systematically compares the strain-specific effects of 15 probiotic strains and consortia discussed across Sections 5-8 on neurogenesis and synaptic plasticity, including experimental model, receptor targets, intracellular pathways modulated, and corresponding references. This table directly serves the function of visual summary requested by the reviewer for the mechanistic sections, and we believe it substantially improves the accessibility of the content in Sections 6, 7, and 8 without duplicating information already conveyed by Figures 1 and 2.]

Comments 7: [Section 8.4 briefly mentions variability but does not explore underlying factors deeply, such as sex differences, age, or microbiome enterotypes. The author should expand this section and include these in the text. Additionally, the author should discuss implications for precision medicine, including omics-based predictors.]

Response 7: [We thank the reviewer for this constructive suggestion. We agree that the original Section 9.4, while acknowledging interindividual variability as a central challenge, addressed its underlying biological determinants in broad strokes without sufficiently exploring sex differences, age-related factors, and microbiome enterotypes as specific and clinically meaningful sources of heterogeneity. In response, we have expanded Section 9.4 by adding a paragraph that explicitly addresses these dimensions. Regarding sex differences, the expanded text draws on content already present in the manuscript — specifically the documented sex-hormone interactions with the gut microbiota and the disproportionate prevalence of affective disorders in women mentioned in the context of translational limitations — to argue that the near-exclusive use of male rodents in preclinical probiotic studies represents a systematic gap with direct implications for the interpretation of clinical variability in mixed-sex trial populations. Regarding age, the expanded text connects to the observation already made in Section 11.4 of the manuscript, where age-related changes in gut microbiota composition are linked to decreased BDNF and hippocampal plasticity, and where the potential of probiotic interventions in older adults is discussed, thereby situating age as a moderating variable in probiotic response rather than a peripheral consideration. Regarding microbiome enterotypes and omics-based precision medicine, the expanded section builds on and integrates the precision medicine paragraph already incorporated into Section 9.4 in a prior revision round, which draws on multi-omics integration strategies — combining gut metagenomics, metabolomics, and host transcriptomics — as tools for prospective patient stratification into microbiome-defined biological subtypes, an approach directly analogous to precision oncology frameworks. The revised section now presents sex, age, enterotype, and omics-based stratification as a coherent and interconnected set of precision medicine determinants rather than isolated variables, thereby providing the depth of discussion the reviewer appropriately requested.]

Comments 8: [The introduction and Section 3 establish neurobiological foundations, but the connection to affective disorders is superficial in later sections. The author should discuss how synaptic plasticity deficits directly map to depression symptoms.]

Response 8: [We thank the reviewer for this insightful observation. We agree that strengthening the explicit link between synaptic plasticity deficits and specific depressive symptomatology across the mechanistic sections would enhance the conceptual coherence of the manuscript. In response, we propose the following additions and clarifications:

As established in Section 3.2, the "neuroplasticity hypothesis of depression" posits that impairments in synaptic plasticity—specifically neuronal atrophy and synaptic loss in the medial prefrontal cortex (mPFC) and hippocampus—are central to the pathophysiology of MDD, rather than simple monoamine deficiency. These structural and functional synaptic changes directly underlie core depressive symptoms: (1) anhedonia and reduced reward processing reflect impaired dopaminergic and glutamatergic transmission in prefrontal-limbic circuits, where synaptic weakening reduces motivational drive; (2) cognitive dysfunction (including impaired concentration, executive function, and memory) maps directly to disrupted LTP in the hippocampal-prefrontal pathway, as described in Section 6.4, where LTP represents the cellular correlate of experience-dependent learning; (3) emotional dysregulation and rumination correspond to altered excitatory/inhibitory balance—specifically the GABA/glutamate imbalance discussed in Section 3.2—which impairs the normal extinction of maladaptive emotional responses; and (4) impaired stress resilience, a hallmark of depressive vulnerability, is linked to deficits in metaplasticity (the modulation of future plasticity thresholds), which determines the brain's capacity to adaptively respond to chronic stress.

In Sections 6 and 7, the discussion of probiotic-induced modulation of LTP/LTD (Section 5.4), NMDA receptor expression (Section 7.1), and GABAergic neurotransmission (Section 7.2) is mechanistically relevant to these symptom domains precisely because these are the molecular substrates through which synaptic strength is regulated. For instance, the restoration of NMDAR2B and NMDAR1 expression by Lactobacillus rhamnosus GR-1 (Table 1) has direct implications for the recovery of hippocampal LTP—and therefore for the cognitive and memory deficits characteristic of MDD. Similarly, the upregulation of GABA_A receptor subunit by multiple probiotic strains (Section 7.2) relates to the inhibitory tone dysregulation that contributes to hyperactivation of stress circuits and anxiety-depression comorbidity.

Furthermore, as noted in Section 3.4, neuroimaging studies in MDD patients consistently demonstrate reduced amygdala-prefrontal functional connectivity and hippocampal volume reduction—structural signatures that reflect cumulative synaptic loss. The fact that probiotic interventions in clinical trials correlate with changes in BDNF (Section 9.2)—a neurotrophin whose primary function is to sustain synaptic strengthening and dendritic spine density—provides an indirect but clinically tractable link between microbial modulation and the reversal of these synaptic deficits.]

Comments 9: [Probiotics are presented as largely beneficial, but potential risks are ignored. Section 10 should include a safety profile and discuss contraindications for patients with affective disorders.]

Response 9: [We thank the reviewer for this important observation. We acknowledge that the manuscript, while including cautionary findings in Section 9.5—such as paradoxical GABAergic upregulation following dysbiosis-inducing interventions, dendritic spine loss induced by propionic acid, and symptom recurrence upon probiotic discontinuation—does not systematically address the clinical safety profile of probiotics in patients with affective disorders. This is a relevant gap, particularly given that this population frequently presents immunocompromising comorbidities or receives concomitant pharmacological treatment. In response, we propose adding a paragraph at the end of Section 11 (Translational and Therapeutic Considerations).]

Comments 10: [Section 11 conclusions are generic. The author should expand on future directions, including specific research recommendations, and propose a roadmap for clinical implementation.]

Response 10: [We thank the reviewer for this constructive criticism. We agree that Section 11 would benefit from a more structured and actionable vision of future research and clinical translation. The manuscript already contains granular recommendations distributed across Sections 9.3, 9.4, 10.4, 11.1–11.5; however, these are not synthesized into a coherent roadmap in the conclusions. In response, we propose adding a paragraph at the end of Section 11.]

Minor Comments

Comments 1: [The author has used terms like "psychobiotics" and "MGB axis" interchangeably without clear definitions early on (not defined in the Introduction).]

Response 1: [We thank the reviewer for such valuable comments; we have addressed the observation and defined the terms indicated in the summary.]

Comments 2: [Several typos (e.g., "germen free" in Section 4.1 should be "germ-free"). Use consistent italics for genus/species (e.g., Bifidobacterium longum).]

Response 2: [We thank the reviewer for this careful reading of the manuscript. Both issues have been fully addressed in the revised version.]

Comments 3: [Sections 3.1 and 4.1 overlap on hippocampal neurogenesis; condense to avoid repetition.]

Response 3: [We thank the reviewer for this careful reading of the manuscript. The issue has been fully addressed in the revised version.]

Comments 4: [In Section 6.1, "the decision to include only studies with chronic treatment was made because..." is awkwardly phrased; clarify the rationale.]

Response 4: [We thank the reviewer for this observation. Upon revision, we note that this phrase appears in Section 2.2 (Criteria) rather than Section 7.1. The original phrasing has been revised to improve clarity and readability.]

Comments 5: [Figure legends mention BioRender but lack alt-text descriptions for accessibility; add these.]

Response 5: [We thank the reviewer for raising this accessibility concern. Alt-text descriptions have been added to both figure legends in the revised manuscript.]

Reviewer 3 Report

Comments and Suggestions for Authors

The review article by Gilberto Uriel Rosas-Sánchez et al. addresses a highly relevant and rapidly advancing area in neuropsychiatric research by examining the role of probiotics in modulating neurogenesis and synaptic plasticity in affective disorders. The authors present a timely synthesis of the microbiota gut brain axis, integrating molecular mechanisms such as epigenetic modulation, inflammatory regulation, and neurotrophic signaling. The manuscript demonstrates strong conceptual framing and translational significance, and the authors are to be commended for compiling emerging preclinical and clinical evidence in this evolving field. Furthermore, the review article clearly reiterates the mechanistic and translational relevance of probiotics in modulating neurogenesis and synaptic plasticity. It appropriately balances optimism with acknowledgment of current methodological limitations and future research needs. However, authors are requested to work on the comments given below and improve the article for increasing the chances of its acceptance in the journal.

Major comments:

Authors should include recent global epidemiological statistics like e.g., WHO data to strengthen the statement on disease burden. Furthermore, authors should provide a clearer knowledge gap statement mentioning what specifically remains unknown regarding probiotics and neuroplasticity?
Authors should clearly state the primary objective and scope of the review (mechanistic vs clinical focus) and distinguish obviously between preclinical (animal) and clinical (human RCT) evidence.
Authors can briefly mention strain specificity early in the introduction to avoid overgeneralization of probiotic effects.
Authors can provide stronger mechanistic integration linking SCFAs, epigenetics, inflammation, and neuroplasticity and add more concrete details about clinical evidence like number/type of trials, strength of findings, etc.
Authors should clarify the level of causal evidence Vs biomarker-based associations like e.g., BDNF changes and further define “precision psychobiotic interventions” more clearly.
Authors can specifically distinguish between evidence derived from animal models and human studies when discussing probiotic effects.
Authors should acknowledge ongoing controversy regarding the extent of adult hippocampal neurogenesis in humans.

Minor comments

Authors should check the sequential numbers of the sections and subsections. As section 3 followed by subsections like 3.1, 3.2 …. Followed by section 3 again which needs to be corrected.
Authors should reduce repetition of neurogenesis/synaptic plasticity terms like “neuroplasticity” and “affective disorders.
Authors should correct minor typographical issues like “Germen free” → “Germ-free”; capitalization inconsistencies and correct the same in abbreviations list as well.
Authors can provide examples of microbiota-derived neuroactive peptides and strengthen the concluding statement with specific future research directions.

Author Response

The review article by Gilberto Uriel Rosas-Sánchez et al. addresses a highly relevant and rapidly advancing area in neuropsychiatric research by examining the role of probiotics in modulating neurogenesis and synaptic plasticity in affective disorders. The authors present a timely synthesis of the microbiota gut brain axis, integrating molecular mechanisms such as epigenetic modulation, inflammatory regulation, and neurotrophic signaling. The manuscript demonstrates strong conceptual framing and translational significance, and the authors are to be commended for compiling emerging preclinical and clinical evidence in this evolving field. Furthermore, the review article clearly reiterates the mechanistic and translational relevance of probiotics in modulating neurogenesis and synaptic plasticity. It appropriately balances optimism with acknowledgment of current methodological limitations and future research needs. However, authors are requested to work on the comments given below and improve the article for increasing the chances of its acceptance in the journal.

Major comments:

Comments 1: [Authors should include recent global epidemiological statistics like e.g., WHO data to strengthen the statement on disease burden. Furthermore, authors should provide a clearer knowledge gap statement mentioning what specifically remains unknown regarding probiotics and neuroplasticity?]

Response 1: [We thank the reviewer for these two complementary observations. Both have been addressed in the introduction in line 53 to 61]

Comments 2: [Authors should clearly state the primary objective and scope of the review (mechanistic vs clinical focus) and distinguish obviously between preclinical (animal) and clinical (human RCT) evidence.]

Response 2: [We thank the reviewer for this methodological observation. We agree that explicitly stating the dual mechanistic-clinical scope of the review and systematically distinguishing between preclinical and clinical evidence levels would strengthen the manuscript's epistemological transparency. The document already contains translational caveats distributed across Sections 5.3, 5.4, 6.4, 7.4, 8, and 9.3; however, these distinctions are not anchored by a unified scope statement at the outset. In response, we propose the following additions:

This review adopts a dual mechanistic-clinical scope: it first synthesizes preclinical evidence — derived primarily from rodent models, germ-free preparations, and in vitro systems — to characterize the molecular pathways through which probiotics modulate adult neurogenesis and synaptic plasticity; and subsequently evaluates the extent to which these mechanisms are supported by emerging clinical evidence from randomized controlled trials in human populations with affective disorders. Throughout, preclinical and clinical findings are explicitly distinguished to avoid unwarranted extrapolation and to delineate the current boundaries of translational knowledge in this field.

To ensure epistemological clarity, evidence throughout this review is categorized according to its origin: (i) preclinical evidence, encompassing in vitro studies, germ-free animal models, and rodent behavioral paradigms, which provides mechanistic resolution but limited translational fidelity; and (ii) clinical evidence, comprising randomized controlled trials, meta-analyses, and translational biomarker studies in human populations, which offers direct therapeutic relevance but currently limited mechanistic specificity. Where evidence derives exclusively from preclinical sources, this is explicitly noted to avoid overstating clinical applicability.]

Comments 3: [Authors can briefly mention strain specificity early in the introduction to avoid overgeneralization of probiotic effects.]

Response 3: [We thank the reviewer for this important suggestion. We agree that introducing the concept of strain specificity early in the manuscript is essential to frame the subsequent mechanistic and clinical discussions accurately and to prevent the reader from generalizing probiotic effects across species and strains. Although strain-dependent variability is discussed extensively in Sections 7, 9.4, and 9.5 — where differential receptor-level effects, interindividual response variability, and cautionary null findings are addressed — this caveat is indeed absent from the Introduction. In response, we propose adding the following sentence to the Introduction, immediately after the first mention of probiotics as therapeutic agents.]

Comments 4: [Authors can provide stronger mechanistic integration linking SCFAs, epigenetics, inflammation, and neuroplasticity and add more concrete details about clinical evidence like number/type of trials, strength of findings, etc.]

Response 4: [We thank the reviewer for this constructive observation. We respectfully note that both aspects are substantively addressed in the manuscript, though distributed across multiple sections rather than consolidated in a single integrative statement.

Regarding mechanistic integration, the manuscript already establishes a biologically coherent cascade linking SCFAs, epigenetics, neuroinflammation, and neuroplasticity across Sections 8.1–8.4 and Figure 2. Specifically, Section 8.1 describes how butyrate — the primary SCFA produced by probiotic-associated bacteria such as Eubacterium, Clostridium, and Butyrivibrio — inhibits HDACs, increases histone acetylation, and promotes chromatin opening at gene loci governing neuronal survival and synaptic plasticity. Section 8.2 establishes the downstream consequence of impaired SCFA production: microglial priming toward a pro-inflammatory state, elevated IL-6 and TNF-α, and consequent suppression of hippocampal neurogenesis — a pathway explicitly depicted in Figure 2, Panel B. Section 8.3 further specifies that butyrate stimulates hippocampal neuron number, propionate protects BBB integrity, and acetate influences histone acetylation in the CNS, while Section 8.4 connects these molecular events to neuroactive peptide signaling within the MGB axis. The integrative summary at the end of Section 8 explicitly frames these as "a biologically coherent and mechanistically plausible framework for probiotic-induced neuroplasticity." Nevertheless, we acknowledge that an explicit consolidated statement articulating this cascade as a sequential and interdependent mechanism — rather than parallel pathways — would enhance readability. We have therefore added the following integrative sentence at the end of Section 8.3:

Collectively, these mechanisms operate as an integrated cascade: dysbiosis reduces butyrate production, diminishing HDAC inhibition and suppressing histone acetylation at neuroplasticity-related loci including Bdnf; simultaneously, reduced SCFA availability compromises BBB integrity, facilitating LPS translocation, microglial activation, and pro-inflammatory cytokine release that further suppresses hippocampal LTP and neurogenesis — a self-reinforcing cycle that probiotic intervention disrupts at multiple nodes simultaneously.

Regarding clinical evidence, the manuscript already provides concrete quantitative data in Section 8.1, including the Moshfeghinia et al. (2025) meta-analysis of 19 RCTs and 1,405 participants reporting significant reductions in depressive (SMD: −1.76; 95% CI: −2.42, −1.10) and anxiety scores (SMD: −1.60; 95% CI: −2.83, −0.36); the Asad et al. (2025) complementary meta-analysis of 23 RCTs (n = 1,401) demonstrating larger effects in clinical versus community samples (d = −0.73); and the Hashemi et al. (2025) GRADE-based dose-response meta-analysis confirming probiotic-induced increases in circulating BDNF with identification of non-linear dose-response relationships. Section 8.1 also references a pioneering double-blind RCT by Schaub et al. (2022) evaluating probiotic add-on therapy in depressed patients with simultaneous assessment of microbial, neural, and clinical outcomes, and the precision psychiatry-informed RCT by Veibäck et al. (2025) identifying formic acid as a preliminary SCFA biomarker of treatment response. These data are complemented by the explicit acknowledgment in Section 9.3 that high heterogeneity (I² > 90%), small sample sizes, and intervention periods predominantly under 60 days currently limit generalizability. To consolidate this information for the reader, we have added a brief integrative summary sentence at the opening of Section 8.1 directing the reader to the quantitative synthesis presented across the section, ensuring that the strength and limitations of the clinical evidence base are immediately apparent.]

Comments 5: [Authors should clarify the level of causal evidence Vs biomarker-based associations like e.g., BDNF changes and further define “precision psychobiotic interventions” more clearly.]

Response 5: [We acknowledge that the manuscript, while containing individual translational caveats in Sections 5.4, 6.4, and 8, does not provide a unified and explicit statement distinguishing causal mechanistic evidence from biomarker-based associations at the level of the abstract and conclusions. This distinction is critical, as the current evidence hierarchy for probiotic-induced neuroplasticity in human’s rests predominantly on associational rather than causal grounds.

Specifically, as established in Section 5.4, BDNF represents the primary peripheral biomarker used to infer neurogenic activity in clinical probiotic trials; however, the manuscript explicitly acknowledges that "peripheral BDNF changes are considered reflective of altered neurotrophic support in the brain" rather than direct measures of neurogenesis, and that "directly assessing neurogenesis in the living human brain remains challenging due to ethical and technical limitations." Similarly, Section 9.2 recognizes that "direct and consistent human evidence linking specific gut microbial changes to measurable, non-invasive biomarkers of neurogenesis or synaptic plasticity remains challenging to fully establish." The Hashemi et al. (2025) GRADE-based meta-analysis cited in Section 9.2 confirms a statistically significant probiotic-induced increase in circulating BDNF, but this constitutes correlational rather than causal evidence of enhanced neuroplasticity, as circulating BDNF levels reflect multiple peripheral and central sources and cannot be unambiguously attributed to hippocampal neurogenesis alone.

Causal mechanistic evidence, by contrast, is currently restricted to preclinical systems: germ-free mouse models in which microbiota colonization directly normalizes hippocampal neural stem cell proliferation and resolves neuroblast maturation arrest (Namihira et al., 2025, Section 9.3), and in vitro studies demonstrating that physiologically relevant SCFA concentrations directly increase neural progenitor cell growth rates and proliferation-related gene expression (Section 5.2). To make this distinction explicit and consistent throughout the manuscript, we have added the following clarifying statement to the abstract and to the opening paragraph of Section 9.2:

It is important to distinguish between causal mechanistic evidence — currently established primarily in preclinical germ-free and in vitro systems — and biomarker-based associational evidence available in human clinical trials. Peripheral measures such as serum BDNF, inflammatory indices, and SCFA profiles serve as tractable proxies of neuroplastic activity but do not constitute direct measures of neurogenesis or synaptic plasticity in the living human brain. The interpretation of clinical trial findings should therefore be framed within this evidentiary hierarchy.

Respect to precision psychobiotic interventions we agree that the term "precision psychobiotic interventions," used in the abstract and conclusions, requires explicit definition to avoid ambiguity. The conceptual framework underlying this term is already developed across Sections 9.4, 10.4, and 11.1 of the manuscript. Specifically, Section 9.4 introduces the precision microbiome medicine framework proposed by Liu et al. (2025), which advocates for multi-omics integration — combining gut metagenomics, metabolomics, and host transcriptomics — to stratify patients into microbiome-defined biological subtypes analogous to precision oncology approaches. Section 10.4 further elaborates that precision medicine using multi-omic approaches aims to identify individual patient characteristics to enable selection of the most effective treatments. Section 11.1 specifies that a "precision psychiatry approach based on personalized probiotics" involves the selection of strain-specific consortia tailored to individual microbial profiles, neuroplasticity biomarker status, and clinical phenotype.

To consolidate these distributed definitions into a single accessible statement, we propose adding the following sentence to the abstract immediately after the first use of the term.

Comments 6: [Authors can specifically distinguish between evidence derived from animal models and human studies when discussing probiotic effects.]

Response 6: [We thank the reviewer for this methodologically important observation. We respectfully note that the manuscript already contains systematic translational caveats distinguishing preclinical from clinical evidence at multiple points throughout the text. Specifically:

In Section 5.3, the manuscript explicitly states that "this mechanistic picture is derived almost exclusively from preclinical rodent models — in particular GF animals and antibiotic-treated mice — which represent extreme microbiological conditions with no direct human clinical counterpart," and acknowledges that "GF mouse models, while invaluable for establishing causal microbiota-neurogenesis relationships, exhibit global immunological, endocrinological, and physiological alterations that extend well beyond the gut microbiota, thereby precluding direct inference about isolated probiotic effects in a neurologically intact human host."

In Section 5.4, the translational gap is further addressed by distinguishing BDNF as a peripheral associational biomarker in human studies from the direct causal neurogenic evidence available only in preclinical systems, noting that "human clinical trials primarily focus on behavioral and mood outcomes rather than direct measures of neurogenesis."

In Section 6.4, a dedicated translational caveat explicitly notes that "the electrophysiological measurements used to characterize synaptic plasticity in rodent models — including hippocampal slice recordings and in vivo field potential analyses — have no direct non-invasive equivalent in human research," and that in clinical settings "synaptic plasticity can only be inferred indirectly through neuroimaging proxies such as fMRI-based connectivity analyses and diffusion tensor imaging."

In Section 7.4, the manuscript acknowledges that intracellular signaling findings "were conducted predominantly in male rodent models and in cell lines (PC12, THP-1) that do not recapitulate the complexity of the human blood-brain barrier, the pharmacokinetics of bacterially-derived metabolites crossing into the central nervous system, or the sex-dependent hormonal modulation of neurotrophic and inflammatory signaling."

In Section 9.3, the most comprehensive appraisal of the translational gap is provided, noting that "approximately 90% of drugs for mental disorders that demonstrate efficacy in preclinical trials ultimately fail in human clinical studies," that "the vast majority of preclinical studies reviewed here used exclusively male rodents," and that "the controlled housing conditions, standardized diets, and uniform genetic backgrounds of laboratory rodents further limit the generalizability of findings to the heterogeneous environmental, dietary, and microbiome-compositional landscapes characteristic of human populations."

Nevertheless, we acknowledge that these distinctions, while present, are distributed across sections rather than systematically signposted at the level of individual evidence statements. To address this structural limitation, we propose implementing a consistent labeling convention throughout the revised manuscript whereby evidence statements are explicitly prefaced with [Preclinical] or [Clinical] tags in sections where both evidence types are discussed in close proximity — specifically Sections 5, 6, 7, and 8 — ensuring that the reader can immediately identify the evidentiary origin of each finding without needing to cross-reference the translational caveats provided at section endings.]

Comments 7: [Authors should acknowledge ongoing controversy regarding the extent of adult hippocampal neurogenesis in humans.]

Response 7: [We thank the reviewer for raising this important scientific point. We note that the manuscript already contains a direct acknowledgment of this controversy in Section 9.3, where it is explicitly stated that "the translational relevance of this specific neurogenic endpoint to human depression pathophysiology remains uncertain, given the ongoing scientific debate about the functional significance of adult hippocampal neurogenesis in humans," citing Alonso, Petit, and Lledo (2024) — referenced as [9] in the manuscript — whose work in Molecular Psychiatry specifically addresses the impact of adult neurogenesis on affective functions across mice and humans. Additionally, Section 5.4 acknowledges that "there is considerable reluctance to translate the concept of integrating new neurons to humans," further reflecting awareness of the contested nature of this phenomenon in the human brain.

However, we agree with the reviewer that this controversy warrants more prominent and explicit acknowledgment earlier in the manuscript, specifically in Section 3.1, where adult hippocampal neurogenesis is introduced as a neurobiological foundation of affective disorders without immediately contextualizing the human-specific debate. In response, we propose adding the following statement at the end of Section 3.1:

It should be noted, however, that the existence and functional significance of adult hippocampal neurogenesis in humans remains a subject of active scientific controversy. While robust neurogenic activity has been consistently demonstrated in rodent models, postmortem human studies have yielded conflicting findings — with some reporting thousands of immature neurons in the adult dentate gyrus and others detecting negligible neurogenesis beyond early childhood — a discrepancy attributable to methodological differences in tissue fixation, immunohistochemical markers, and postmortem interval [9]. This ongoing debate does not invalidate the neurogenic hypothesis of depression, which retains strong preclinical support, but it does underscore the importance of interpreting probiotic-induced neurogenic effects — currently established primarily in animal models — with appropriate caution regarding their direct translation to human pathophysiology and therapeutic outcomes.]

Minor comments

Comments 1: [Authors should check the sequential numbers of the sections and subsections. As section 3 followed by subsections like 3.1, 3.2 …. Followed by section 3 again which needs to be corrected.]

Response 1: [Thank you for pointing this out; the error has been corrected in the manuscript.]

Comments 2: [Authors should reduce repetition of neurogenesis/synaptic plasticity terms like “neuroplasticity” and “affective disorders.]

Response 2: [We thank the reviewer for this careful reading of the manuscript. The issue has been fully addressed in the revised version.]

Comments 3: [Authors should correct minor typographical issues like “Germen free” → “Germ-free”; capitalization inconsistencies and correct the same in abbreviations list as well.]

Response 3: [We thank the reviewer for this careful reading of the manuscript. The issue has been fully addressed in the revised version.]

Comments 4: [Authors can provide examples of microbiota-derived neuroactive peptides and strengthen the concluding statement with specific future research directions.]

Response 4: [We thank the reviewer for this careful reading of the manuscript. The issue has been fully addressed in the revised version.]

Reviewer 4 Report

Comments and Suggestions for Authors

Abstract: This section is present but flawed. It does not succinctly summarize the key conclusions of this review
Introduction: The background is provided, but the literature survey is somewhat introductory.
I recommend adopting A “PRISMA-style flow diagram” or a more detailed table summarizing the search strategy and study selection process that would significantly strengthen this section and its transparency.
A more detailed explanation is required regarding the effects of probiotics on the different Stages of neurogenesis.
You can add a scheme summarizing the gut-microbiota-brain axis: mechanisms of bidirectional communication.
You can add a scheme summarizing Regulation of neurotrophic factors by metabolites derived from probiotics
You can add table summarizing the previous studies according to Biomarkers of neuroplasticity in affective disorders.
What are the clinical implications of this study for patients undergoing affective disorders ? These findings are innovative, but relatively fragmented, and there is no direct evidence in the article to prove their relevance. The article puts forward many hypotheses in the discussion that need to be further confirmed.
Manuscript needs language revision and correction of the grammatical and syntax errors.

Author Response

Comments 1: [Abstract: This section is present but flawed. It does not succinctly summarize the key conclusions of this review.]

Response 1: [We thank the reviewer for this observation. We agree that the original abstract did not adequately reflect the key conclusions of the review. Accordingly, we have substantially revised the abstract to ensure it succinctly captures: (1) the clinical burden and therapeutic gap that motivates the review; (2) the central role of the microbiota-gut-brain axis in neuroplasticity; (3) the principal molecular mechanisms through which probiotics modulate adult neurogenesis and synaptic plasticity; (4) the current state of clinical evidence from randomized controlled trials; (5) the methodological limitations of existing studies; and (6) the key conclusions and future research directions, including precision psychobiotic strategies.]

Comments 2: [Introduction: The background is provided, but the literature survey is somewhat introductory.]

Response 2: [We thank the reviewer for this constructive observation. We respectfully note that the depth of the literature survey in the Introduction was intentionally calibrated to the narrative review format and dual mechanistic-clinical scope of the manuscript, as explicitly stated in the Methods section (Section 2.1). The Introduction was designed to establish the conceptual framework — comprising the global burden of affective disorders, the neurobiological foundations of neuroplasticity, and the emerging role of the MGB axis — rather than to function as an exhaustive literature survey, which is systematically addressed across the subsequent sections of the review.

Specifically, the mechanistic literature on adult neurogenesis and its dysregulation in affective disorders is comprehensively developed in Section 3, including the neurogenic hypothesis of depression, stress-induced alterations in hippocampal neurogenesis, and the ongoing scientific controversy regarding adult hippocampal neurogenesis in humans. The synaptic plasticity literature, including LTP/LTD mechanisms and the neuroplasticity hypothesis of depression, is extensively covered in Sections 3.2 and 7. The MGB axis communication pathways are thoroughly surveyed in Section 4, encompassing neural, endocrine, immune, and metabolic mechanisms. The molecular mechanisms underlying probiotic effects are comprehensively reviewed in Sections 7 and 8, and the clinical evidence base is critically evaluated in Section 9, including recent meta-analyses and randomized controlled trials published up to 2025.

Furthermore, the Introduction explicitly acknowledges the critical knowledge gaps that the review aims to address, including the undefined minimum effective doses for neurogenic effects in humans, the unresolved question of whether probiotic-induced changes in BDNF and inflammatory markers translate into clinically meaningful neuroplastic outcomes, and the largely uncharacterized optimal intervention windows across the lifespan. These gaps directly motivated the comprehensive literature synthesis developed throughout the body of the manuscript.

We therefore consider that the literature survey, while concise in the Introduction as is standard for narrative reviews of this scope, is substantively and rigorously developed across the full manuscript in a manner consistent with the stated objectives and methodological design of the review.]

Comments 3: [I recommend adopting A “PRISMA-style flow diagram” or a more detailed table summarizing the search strategy and study selection process that would significantly strengthen this section and its transparency.]

Response 3: [We thank the reviewer for this recommendation. We respectfully note, however, that the adoption of a PRISMA-style flow diagram is specifically designed for systematic reviews and meta-analyses, and its application to narrative reviews has been widely debated in the methodological literature. As explicitly stated in Section 2.1 of the manuscript, this study was designed as a narrative literature review: "In a narrative review, there are no predetermined research questions or specific search strategy, only a topic of interest." The methodological framework of a narrative review, by its nature, does not involve a structured study selection process amenable to PRISMA-style quantitative reporting, as it does not apply pre-specified eligibility screening algorithms or inter-rater agreement procedures characteristic of systematic reviews.

Nevertheless, we wish to emphasize that the present narrative review was conducted with a level of methodological transparency that substantially exceeds the standard for this review format. As described in Section 2, the search was conducted across four specialized databases — PubMed, ScienceDirect, Web of Science, and Scopus — using a comprehensive set of Boolean operators and keyword combinations, including "probiotics," "anxiety," "depression," "neurogenesis," "MGB axis," "bacterial metabolites," "BDNF," "neuroplasticity," "brain function," "treatment," "animal," "models," "clinical trials," and "probiotic mechanism of action," with an English-language restriction. Furthermore, Section 2.2 explicitly details the inclusion and exclusion criteria applied during the review process. Inclusion criteria encompassed research articles and reviews investigating probiotic strains in preclinical and clinical studies involving chronic treatment interventions of four weeks or longer, consistent with the minimum period required for probiotics to exert measurable effects on gut microbiota composition and host physiology. Exclusion criteria comprised studies without full-text access, unofficial websites, duplicate publications, and doctoral dissertations. As stated in Section 2.1, these parameters were described to "ensure the objective inclusion of information," addressing the acknowledged limitation of narrative reviews regarding potential selection bias.

We therefore consider that the methodological transparency of the search strategy and study selection process is adequately and appropriately documented within the narrative review framework adopted by this manuscript, and that the application of a PRISMA flow diagram would be methodologically inconsistent with the design and stated scope of this review.]

Comments 4: [A more detailed explanation is required regarding the effects of probiotics on the different Stages of neurogenesis.]

Response 4: [We thank the reviewer for this comment. We respectfully consider that the effects of probiotics on the different stages of neurogenesis are already comprehensively addressed in the manuscript across multiple sections, and we wish to direct the reviewer's attention to the relevant content already present in the text.

Section 5.3 (Effects of probiotics on the different stages of neurogenesis) is specifically dedicated to this topic and systematically addresses each stage of the neurogenic cascade. Regarding the proliferation stage, the manuscript describes that probiotics consistently enhance the proliferation of neural stem cells and progenitor cells in the subgranular zone of the dentate gyrus, with Bifidobacteria colonization in germ-free mice significantly increasing the numbers of proliferating neural stem cells and immature neurons, a process associated with the modulation of neurotrophic factors. Regarding the differentiation stage, the manuscript explains that probiotic interventions, by impacting the microenvironment through reducing inflammation and increasing neurotrophic factors, promote the neuronal lineage differentiation of progenitor cells, ensuring that newly generated cells develop into functional neurons rather than glial cells. Regarding the survival stage, the manuscript details that probiotics, by reducing inflammation and oxidative stress and increasing neurotrophic support such as BDNF, create a more favorable environment for the survival of immature neurons, sustaining hippocampal neural resilience and promoting synaptic adaptability. Regarding the functional integration stage, the manuscript acknowledges the complexity of assessing this stage directly, while providing indirect evidence of probiotic influence through the modulation of structural synaptic proteins, LTP/LTD, and synapse formation, particularly through the role of Bifidobacteria in shaping host neural circuits during developmental windows.

Furthermore, the molecular mechanisms underpinning these stage-specific effects are developed in greater depth in Section 6.2 (Bacterial metabolites as regulators of neural progenitor cells), which details how SCFAs — particularly butyrate, propionate, and acetate — directly influence the proliferation, differentiation, and survival of neural progenitor cells through epigenetic mechanisms including HDAC inhibition and histone acetylation. Complementary mechanistic detail is provided in Section 9.1 (Underlying molecular mechanisms involved in the effect of probiotics on neurogenesis), which elaborates on DNA methylation, histone modifications, and miRNA profiles as additional regulatory layers through which probiotic-derived metabolites influence the neurogenic cascade. Additionally, Section 5.4 (Translational studies and peripheral biomarkers of probiotic-modulated neurogenesis) contextualizes these stage-specific effects within the available translational evidence, acknowledging the limitations of directly assessing neurogenesis in the living human brain and identifying BDNF, inflammatory markers, and microbiota compositional changes as the principal peripheral proxies currently employed.

Importantly, Section 5.3 also includes an explicit translational caveat acknowledging that the mechanistic picture of probiotic influence across the neurogenic cascade is derived almost exclusively from preclinical rodent models — particularly germ-free animals and antibiotic-treated mice — and that the comprehensive neurogenic influence attributed to probiotics represents a working hypothesis grounded in animal and in vitro data whose translation to human clinical outcomes warrants prospective evaluation through neuroimaging and biomarker strategies.

We therefore consider that the effects of probiotics on the different stages of neurogenesis — from initial neural stem cell proliferation through differentiation, survival, and functional integration — are already addressed in substantial detail and with appropriate mechanistic and translational context throughout the manuscript.]

Comments 5: [You can add a scheme summarizing the gut-microbiota-brain axis: mechanisms of bidirectional communication.]

Response 5: [We thank the reviewer for this suggestion. We respectfully consider that the bidirectional communication mechanisms of the MGB axis are already comprehensively addressed both textually and visually throughout the manuscript, and we wish to direct the reviewer's attention to the relevant content already present.

Section 4 (The gut-microbiota-brain axis: mechanisms of bidirectional communication) is entirely dedicated to this topic and systematically describes each communication pathway of the MGB axis in substantial detail. Regarding the neural pathway, the manuscript describes the vagus nerve as the primary route for sensory information from the gut to the brain, detailing the afferent signaling cascade from the ganglion nodosum through the nucleus of the solitary tract to key brain nuclei including the hypothalamus and amygdala, as well as the reverse efferent pathway through parasympathetic and sympathetic commands activating the enteric nervous system at the submucosal and myenteric plexuses. The cholinergic anti-inflammatory pathway and the role of endocannabinoids as primary sensory elements initiating neurotransmitter signaling are also described. Regarding the immune pathway, the manuscript details the role of pathogen-associated molecular patterns, Toll-like receptor activation, and the subsequent release of pro-inflammatory cytokines including IL-1β, IL-6, TNF-α, and IFN-γ, linking dysbiosis-induced immune activation to microglial activation and neuroinflammation. Regarding the endocrine pathway, the manuscript describes the release of gastrointestinal hormones — including ghrelin, leptin, peptide YY, and glucagon-like peptide-1 — by enteroendocrine cells in response to nutrients, nerve signals, and bacterial products, and their neuropsychiatric effects on motivation, stress, and food reward. Regarding the metabolic pathway, the manuscript comprehensively covers the role of SCFAs — acetate, propionate, and butyrate — as key microbial metabolites crossing the blood-brain barrier to modulate hypothalamic circuits, epigenetic gene expression, microglial homeostasis, and synaptic plasticity. The synthesis of neuromodulators including GABA, dopamine, norepinephrine, and serotonin precursors by specific bacterial genera is also addressed, along with the role of enteroendocrine cells and their neuropods in forming direct physical synapses with afferent fibers of the enteric nervous system and vagus nerve.

Furthermore, the visual representation of these mechanisms is already provided across the existing figures in the manuscript. Figure 1 illustrates the MGB axis pathways through which probiotics and SCFAs regulate synaptic plasticity via neural, immune, endocrine, and metabolic routes, explicitly depicting the vagus nerve, gut lumen, gut hormones, and the hippocampus as interconnected nodes of the axis across its four panels. Figure 2 complements this by depicting the molecular mechanisms through which gut microbiota-derived SCFAs cross the blood-brain barrier to influence neuronal growth, neurotransmitter release, serotonin pathways, GABAergic signaling, and microglial homeostasis, as well as the role of neuroactive peptides derived from the microbiota in influencing neurogenesis and synaptic plasticity through MGB axis signaling.

We therefore consider that the bidirectional communication mechanisms of the MGB axis are already comprehensively addressed both in the dedicated Section 4 and through the existing visual representations in Figures 1 and 2, providing readers with a thorough and integrated understanding of this axis without the need for an additional scheme.]

Comments 6: [You can add a scheme summarizing Regulation of neurotrophic factors by metabolites derived from probiotics.]

Response 6: [We thank the reviewer for this suggestion. We respectfully consider that the regulation of neurotrophic factors by metabolites derived from probiotics is already comprehensively addressed both textually and visually in the manuscript, and we wish to direct the reviewer's attention to the relevant content already present.

Section 6.2 (Regulation of neurotrophic factors by metabolites derived from probiotics) is specifically dedicated to this topic and systematically addresses the role of probiotic-derived metabolites in modulating the biosynthesis and expression of key neurotrophic factors. The manuscript describes in detail how BDNF — identified as a key neurotrophin influencing neuronal growth, survival, and plasticity — is significantly upregulated at both gene and protein expression levels in the hippocampus following probiotic use. The manuscript further details how germ-free animals exhibit significant decreases in BDNF expression in both the cortex and hippocampus, and how probiotic treatment can upregulate these levels. The roles of nerve growth factor and glial cell lineage-derived neurotrophic factor are also addressed in the context of neuronal growth, survival, differentiation, and synaptic plasticity. Regarding the specific metabolites mediating these effects, the manuscript comprehensively describes how SCFAs — butyrate, acetate, and propionate — serve as key mediators in MGB axis communication, crossing the blood-brain barrier and directly altering brain neurological function through vagal, endocrine, humoral, and immunological pathways. The manuscript specifies that SCFAs increase neurotransmitter transmission activity and BDNF expression, regulate microglia maturation, and enhance resilience in response to stress. The neuromodulatory effects of several Lactobacillus species on neurotransmitters and BDNF are also described, alongside meta-analytic evidence supporting the capacity of probiotic supplementation to modulate serum BDNF levels. The molecular mechanisms through which these metabolite-neurotrophic factor interactions operate are further elaborated in Section 8.1, which details how butyrate-mediated HDAC inhibition increases histone acetylation and promotes a more open chromatin structure, enhancing the transcription of genes involved in neuronal survival, synaptic plasticity, and memory formation, and how acetate influences histone acetylation and energy metabolism in the central nervous system. Section 8.3 further develops the integrated cascade through which dysbiosis-induced reductions in butyrate production diminish HDAC inhibition and suppress histone acetylation at neuroplasticity-related loci including the Bdnf gene, linking SCFA deficiency directly to impaired neurotrophic support.

Critically, this topic is already represented visually in the manuscript through Figure 1, Panel B (Regulation of neurotrophic factors by metabolites derived from probiotics), which is explicitly dedicated to illustrating how probiotics and SCFAs increase BDNF expression and synaptic protein levels in the hippocampus via neural, immune, endocrine, and metabolic pathways through the MGB axis. Panel B of Figure 1 depicts the interconnected roles of neurotrophic factors including BDNF, NGF, and GLIN, the pathways through which probiotic-derived SCFAs reach the hippocampus, and the resulting improvements in neuronal resilience in response to stress, providing readers with a comprehensive visual representation of the regulation described in Section 6.2. Furthermore, Figure 2, Panel A complements this representation by illustrating the epigenetic mechanisms — specifically HDAC inhibition and histone acetylation — through which butyrate and acetate promote gene expression involved in neurogenesis and synaptic plasticity, directly connecting metabolite availability to neurotrophic factor regulation at the molecular level.

We therefore consider that the regulation of neurotrophic factors by probiotic-derived metabolites is already comprehensively addressed both in the dedicated Section 6.2 and through the existing visual representation in Figure 1 Panel B and Figure 2 Panel A, providing readers with a thorough textual and graphical understanding of this regulatory relationship without the need for an additional scheme.]

Comments 7: [You can add table summarizing the previous studies according to Biomarkers of neuroplasticity in affective disorders.]

Response 7: [We thank the reviewer for this valuable suggestion. In response, we have added a new table (Table 1) in Section 3.4 (Biomarkers of neuroplasticity in affective disorders) summarizing the key biomarker categories reported in the literature covered in this review, organized by biomarker type, measurement approach, findings in affective disorders, and their relationship to probiotic interventions. This addition strengthens the translational framework of the review by providing readers with a structured reference for the indirect measures currently used to assess neuroplasticity in clinical and preclinical settings.]

Comments 8: [What are the clinical implications of this study for patients undergoing affective disorders? These findings are innovative, but relatively fragmented, and there is no direct evidence in the article to prove their relevance. The article puts forward many hypotheses in the discussion that need to be further confirmed.]

Response 8: [We thank the reviewer for this thoughtful and substantive comment, which touches on fundamental epistemological considerations regarding the translational status of the evidence presented. We wish to address each component of this observation in turn, directing the reviewer's attention to the specific sections of the manuscript where these issues are already explicitly and rigorously discussed.

Regarding the clinical implications for patients with affective disorders, we respectfully consider that these are already systematically and comprehensively addressed in Section 11 (Translational and Therapeutic Considerations), which is entirely dedicated to this topic across five subsections. Section 11.1 (Designing targeted probiotic consortia to promote neuroplasticity) addresses the clinical implications of strain selection, detailing how specific Bifidobacteria strains counteract the decline in BDNF levels, how elevated BDNF has been observed under conditions of chronic stress, inflammation, and aging, and how multi-strain probiotic consortia combining Lactobacillus and Bifidobacterium strains can synergistically act on multiple neuroplasticity pathways. Section 11.2 (Optimal time windows for intervention) addresses the clinical implications regarding intervention timing, noting that the brain undergoes significant maturation during adolescence with increased neuronal plasticity, that randomized placebo-controlled clinical trials have demonstrated probiotic regimens as short as 30 days can decrease stress levels, and that longer periods of 8 weeks produce reductions in depression scores. Section 11.3 (Considerations regarding treatment duration and persistence of effects) addresses the clinically critical question of treatment continuity, acknowledging that some evidence indicates effects may not persist after treatment is discontinued — as illustrated by a pilot study in patients with treatment-resistant depression reporting recurrence of symptoms following discontinuation — and that continuous or periodic probiotic supplementation may be necessary for sustained modulation of neuroplasticity. Section 11.4 (Specific populations that may benefit particularly) explicitly identifies the patient groups for whom probiotic interventions hold the greatest clinical promise, including patients with treatment-resistant depression — among whom up to one-third exhibit resistance to initial antidepressant treatments — older adults experiencing age-related decline in neuroplasticity and BDNF levels, and individuals with mild to moderate symptoms of depression and anxiety. Section 11.5 further addresses fecal microbiota transplantation as a complementary strategy, proposing a sequential clinical approach in which FMT establishes a favorable microbial baseline in individuals with severe dysbiosis, after which targeted probiotic supplementation consolidates neuroplasticity-promoting microbial communities.

Regarding the observation that findings are relatively fragmented and that no direct evidence proves their relevance, we wish to emphasize that this characterization accurately reflects the current state of the field rather than a limitation specific to this manuscript, and that this evidentiary reality is explicitly, repeatedly, and transparently acknowledged throughout the review. As stated in Section 5.3, the mechanistic picture of probiotic influence across the neurogenic cascade is derived almost exclusively from preclinical rodent models operating under extreme microbiological conditions with no direct human clinical counterpart, and directly assessing neurogenesis in the living human brain remains ethically and technically unfeasible. Section 6.4 explicitly acknowledges that the electrophysiological measurements used to characterize synaptic plasticity in rodent models have no direct non-invasive equivalent in human research. Section 7.4 explicitly cautions that translating intracellular signaling findings to clinical contexts requires careful epistemological caution, noting that the studies reviewed were conducted predominantly in male rodent models and cell lines that do not recapitulate the complexity of the human blood-brain barrier. Section 8.3 acknowledges that the extent to which orally administered probiotics generate sufficient systemic SCFA concentrations to produce analogous central effects in humans remains an open empirical question. Furthermore, Section 9.3 (Methodological limitations of current studies) provides a comprehensive and critical appraisal of the limitations of current clinical research, including small and heterogeneous sample sizes, lack of standardization in probiotic strains and dosages, inconsistent outcome measures, potential confounding variables, and the absence of comprehensive biomarker collection. Section 9.5 (Conflicting evidence, null results, and cautionary findings) explicitly acknowledges null findings, negative results, and conflicting observations, including the finding that meta-analyses have consistently found only small pooled effect sizes for probiotic interventions in depression, and that positive effects specifically in patients with major depressive disorder remain preliminary and are not yet sufficient to support definitive therapeutic recommendations.

Regarding the observation that the article puts forward many hypotheses that need to be further confirmed, we wish to clarify that this is a defining and intentional characteristic of the narrative review format adopted by this manuscript, as explicitly stated in Section 2.1. The review was designed to synthesize preclinical mechanistic evidence alongside emerging clinical evidence, explicitly distinguishing between these two evidence levels throughout the manuscript to delineate the current boundaries of translational knowledge and to avoid unwarranted extrapolation from animal to human contexts. As stated in the Introduction, the review aims to systematically address critical knowledge gaps including the undefined minimum effective doses required to produce measurable neurogenic and synaptic effects in humans, the unresolved question of whether probiotic-induced changes in BDNF and inflammatory markers translate into clinically meaningful neuroplastic outcomes, and the largely uncharacterized optimal intervention windows across the lifespan. The hypotheses presented throughout the manuscript are therefore not unsubstantiated speculations but rather mechanistically grounded candidate pathways whose human relevance is supported by biological plausibility and indirect clinical evidence, and whose prospective validation through appropriately designed human trials is explicitly called for in the Conclusions section, where a stepwise translational roadmap is provided. This roadmap specifies that short-term research should prioritize adequately powered double-blind RCTs in clinically diagnosed populations using standardized strain-specific consortia, comprehensive biomarker panels including serum BDNF, inflammatory indices, and SCFA profiles, and neuroimaging outcomes such as hippocampal volume and amygdala-prefrontal connectivity, while medium-term research should integrate multi-omics stratification frameworks to guide patient selection based on microbiome-defined biological subtypes.

We therefore consider that the clinical implications of the review are comprehensively and explicitly addressed throughout the manuscript, that the fragmentary nature of the current evidence base is transparently acknowledged as a reflection of the state of the field rather than an oversight, and that the hypotheses presented are appropriately framed as mechanistically grounded working propositions requiring prospective clinical validation, consistent with the narrative review format and the dual mechanistic-clinical scope of this manuscript.]

Comments 9: [Manuscript needs language revision and correction of the grammatical and syntax errors.]

Response 9: [We appreciate the reviewer's observation; we have reviewed and corrected the grammatical and syntax errors.]

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed my concerns. The manuscript should be accepted. Congratulations.

Reviewer 4 Report

Comments and Suggestions for Authors

the manuscript can be accepted