For each animal, 20 to 30 dendrites in the apical region were reconstructed for each hippocampal field. Control mice of both genotypes were housed in standard conditions. Except for overcrowding, as well as for water and food deprivation sessions, water and food were available ad libitum. Brains were rapidly removed, hippocampal tissue dissected and quickly frozen in liquid nitrogen for Western blot and RNA-seq analyses. Biochemical studies were performed as reported in Napolitano et al. Blots were then incubated in horseradish peroxidase-conjugated secondary antibodies and target proteins visualized by ECL detection Pierce, Rockford, IL , followed by quantification by Quantity One software Biorad.
Normalized values were averaged and used for statistical analysis performed by two-way ANOVA followed by post-hoc comparison, when required. No genotype effect was observed in the sucrose preference test Supplementary Fig. Altogether, behavioural testing revealed reduced depression- and anxiety-like behaviours, increased risk-taking, unusual aggression as well as slower locomotor habituation to novelty in KO mice, which together are considered mania-like behavioural phenotypes in rodents 28 , 29 , modelling traits of the BD manic phase.
KO mice show mania-like behaviours. G Total square entries in the NHC paradigm. To examine the predictive validity of Tph2 KO mice as a novel model of bipolar mania, we tested the in vivo effect of valproate, a widely prescribed mood stabilizer used to treat BD patients.
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Moreover, valproate administration also attenuated the motor hyperactivity observed in KO mice under basal condition Fig. Overall, these experiments indicate that the manic-related phenotypes found in KO animals are sensitive to valproate treatment. Sub-chronic treatment with valproate rescued the mania-like phenotype in KO mice.
To probe the brain-wide substrates that might underlie the observed behavioural phenotype, we measured in vivo basal cerebral metabolism by means of basal cerebral blood volume bCBV weighted functional Magnetic Resonance Imaging 30 , 31 fMRI. Interestingly, fMRI scans highlighted foci of increased metabolism that were confined to dorsal and ventral hippocampal areas of KO mice with respect to WT littermates Fig.
The effect has been quantifies in hippocampal areas on slice-to-slice basis. Grey area indicates cut-offs for significance. Upper bar: number of DE genes significantly up-regulated right and significantly downregulated left in KO mice as compared to controls. Images are cropped from different blots. Full length blots are displayed in Supplementary Fig. DE genes were enriched in the Gene Ontology GO categories of ion transport, neurogenesis, neuron projection, and regulation of cell proliferation Fig.
In contrast, the expression of chloride and potassium channels Ano2 , Kcnn3 as well as of other genes involved in inhibitory pathways Gabra2 , Sstr2 , Shisa9 was decreased Fig. Among the other DE genes identified, Brain-Derived Neurotrophic Factor BDNF has been shown to play a pivotal role in both synaptic potentiation and structural hippocampal plasticity We next investigated dendritic spine density and morphology along apical dendrites of hippocampal pyramidal neurons.
Results showed in a dramatic increase of spine density in CA3 of KO animals with respect to control littermates Fig. Collectively, these data demonstrate that lack of serotonin leads to selective hippocampal hyperactivity together with increased synaptic and structural plasticity. Hippocampal-like neurons in human BD patients exhibit hyper-excitability and increased expression of neurotransmission-related genes Results showed statistically significant enrichment of the KO mouse transcriptional signature as compared to that of BD neurons. These correspondences suggest a possible mechanistic relevance of our findings for human BD states.
In BD patients abnormal mood transitions can be triggered by stress 37 , We then performed FST Fig. S6 , in keeping with the evidence that BD patients display overt aggression independently of the polarity of the mood episode Chronic stress reverses the emotional phenotype and the hippocampal transcriptional signature in Tph2 KO mice.
Increased left anterior insular and inferior prefrontal activity in post-stroke mania
To characterize the transcriptional networks underpinning the depressive-like state of KO animals, we performed RNA sequencing on the hippocampus of mice subjected to uCMS. Results showed that stress significantly affected the expression of genes in KO-S mice as compared to KO counterparts Fig.
Remarkably, 45 of them e. This change highlighted a lowering of neuroplasticity factors when KO mice switch from manic- to depressive-like behaviours Fig. As we have shown that KO mouse signature was associated with hyperactivity and increased neuroplasticity, these data suggest the establishment of a transcriptional profile in WT-S mice likely promoting neuroplasticity as a stress-adaptive strategy.
Impaired hippocampal adaptive mechanism to stress in KO mice. Here we provide evidence that congenital 5-HT deficiency results in distinct behavioural endpoints modelling bipolar-associated manic behaviour that are normalized by valproate treatment and paroxystic hippocampal functional activity. The observed abnormal transcriptomic landscape is reversed in knockout mice showing a transition from manic- to depressive-like behaviour, suggesting that hippocampal neuroplasticity is a good predictor of the mood phase experienced.
Evidence that neuroplasticity defects and behavioural abnormalities observed in depression were rescued by 5-HT-elevating pharmacological drugs such as SSRIs, led to the hypothesis that decreased 5-HT levels underlie reduced hippocampal neuroplasticity and depressive behaviour 9 , 41 , However, the advent of refined genetic mouse models allowing 5-HT synthesis abrogation challenged this view, providing evidence of opposing 5-HT manipulations via either SSRI treatment or Tph2 genetic ablation showed similar behavioural phenotype and neuromolecular response in several hippocampal neuroplasticity paradigms.
Accordingly, both boosting and depleting 5-HT content results in increased adult hippocampal neurogenesis and dendritic arborisation 21 , 22 , 45 as well as reduced behavioural despair 18 , However, while SSRI therapeutic effects have been observed in depressed patients and animal models of depression, increased hippocampal neuroplasticity and mood elevation have been described in 5-HT deficient mice housed in basal condition. Guided by the observation that BD patients and animal models of mania display heightened hippocampal neuroplasticity, we hypothesized that 5-HT deficiency may trigger the onset of manic-like phenotypes.
Notably, 9—13 months old KO mice display reduced depression- Fig. S1A,B and anxiety-like behaviours Supplementary Fig. S1C as compared to age-matched WT littermates, arguing against potential confounding behavioural interpretation as ADHD symptoms have been reported to decrease with age in both humans 47 and animal models Moreover, the evidence that the behaviour of KO mice can be ameliorated by the mood stabilizer valproate speaks in favour of a manic-like phenotype.
Increased sucrose consumption observed in some animal models of mania has been proposed to recapitulate the hyper-hedonic behaviour displayed by BD patients We could not detect any difference between WT and KO animals in this behavioural paradigm. The condition observed in Tph2 mutant mice is likely due to serotonin depletion per se as behavioural hyperactivity has been observed in mice in which Tph2 was targeted in adulthood Therefore, a primary role of 5-HT depletion is likely to be the main causative effect of the phenotypes observed here, although a role of abnormal developmental trajectories caused by 5-HT depletion cannot be entirely ruled out.
This suggests the existence of an interplay between developmental and neuroadaptive mechanism caused by aberrant 5-HT neurotransmission in the etiology of BD. Guided by fMRI mapping, we identified the hippocampus as a key substrate for the behavioural changes observed. Strikingly, the transcriptional changes in the hippocampus of KO mice showed remarkable similarity to those observed in neurons derived from bipolar disorder patients Moreover, these transcriptional changes underlie a common hyperactive phenotype in the hippocampus of KO mice and in BD-derived hippocampal-like neurons, which is in keeping with an abnormal neuroplasticity and a reduced inhibitory drive identified in the hippocampus of preclinical models and bipolar disorder patients 14 , 15 , 56 , Owing to these similarities we propose that the hyperactivity in Tph2 mutants is phenotypically and mechanistically relevant for BD associated manic states.
Emotional instability and exaggerated fluctuations in mood are the distinctive traits in bipolar disorder.
How to Tell ADD and Bipolar Disorder Apart
Similarly to BD patients who experience abnormal mood transitions from the manic to the depressive state 38 , 58 , 59 , Tph2 mutant mice switch toward depressive-like behaviours upon exposure to stressful environment. The evidence that in Tph2 knockout mice a relatively small number of genes is reversed with stress indicates the existence of two distinct transcriptional signatures that are associated to either manic- or depressive-like behaviours, thus suggesting that hippocampal hyperactivity is mood-phase dependent. Dysregulated adaptive mechanisms to stress may be relevant for mood shifts.
Accordingly, the kindling hypothesis posits that stressful events trigger the onset of affective episode and their recurrence overburdens adaptive mechanisms to stress, thus resulting in a gradual process of sensitization 38 , According to this view, the strong transcriptional similarity between KO and WT-S mice suggests that adaptive mechanisms to stress are constitutively active in the hippocampus of KO mice housed in standard conditions.
Our evidence that recurrence of stressful episodes in KO-S reverts the transcriptional signature including the expression level of BDNF and TrkB demonstrates that the appropriate activation of neuroplasticity adaptive mechanisms to environmental insults is precluded. Such a blunted response is indicative of a possible overburden of these mechanisms.
Somewhat surprisingly, when augmented levels of serotonin were induced, higher vulnerability to adverse living environment was described in a recent study by Alboni and collaborators, who reported a stress-induced worsening of hippocampal neuroplasticity and depressive-like behaviour in fluoxetine-administered mice Taken together, these data support the hypothesis that physiological 5-HT levels are critical for an organism to adapt to the living environment and cope with stress.
Interestingly, contradictory findings regarding the assessment of depression-like behavior of Tph2 knock-out mice have been generated 62 , 63 , Indeed, both reduced and increased immobility have been observed in the FST, whereas no difference has been observed in the TST. Moreover, while Mosienko and colleagues described a decreased anxiety in these animals, other groups did not observe any change in anxiety levels. Kim, J. The stressed hippocampus, synaptic plasticity and lost memories. Nat Rev Neurosci 3 , — Pittenger, C.
Stress, depression, and neuroplasticity: a convergence of mechanisms. Neuropsychopharmacology 33 , 88— McEwen, B. Mechanisms of stress in the brain. Nat Neurosci 18 , — Gray, J. Hippocampal gene expression changes underlying stress sensitization and recovery. Mol Psychiatry 19 , — Magarinos, A. Effect of brain-derived neurotrophic factor haploinsufficiency on stress-induced remodeling of hippocampal neurons. Hippocampus 21 , — Lupien, S. Effects of stress throughout the lifespan on the brain, behaviour and cognition. Nat Rev Neurosci 10 , — Autry, A. Brain-derived neurotrophic factor and neuropsychiatric disorders.
Pharmacol Rev 64 , — Snyder, J. Adult hippocampal neurogenesis buffers stress responses and depressive behaviour. Nature , — Boldrini, M. Antidepressants increase neural progenitor cells in the human hippocampus. Neuropsychopharmacology 34 , — Hippocampal angiogenesis and progenitor cell proliferation are increased with antidepressant use in major depression.
Biol Psychiatry 72 , —71 Watanabe, Y. Stress induces atrophy of apical dendrites of hippocampal CA3 pyramidal neurons. Brain Res , — Pawlak, R. Tissue plasminogen activator and plasminogen mediate stress-induced decline of neuronal and cognitive functions in the mouse hippocampus. Mertens, J. Differential responses to lithium in hyperexcitable neurons from patients with bipolar disorder. Nature , 95—99 Walton, N. Hagihara, H. Immature dentate gyrus: an endophenotype of neuropsychiatric disorders.
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