Giacomo Maddaloni

Changes in the amount of daylight (photoperiod) alter physiology and behaviour^1,2. Adaptive responses to seasonal photoperiods are vital to all organisms—dysregulation associates with disease, including affective disorders³ and metabolic syndromes⁴. The circadian rhythm circuitry is implicated in such responses^5,6, yet little is known about the precise cellular substrates that underlie phase synchronization to photoperiod change. Here we identify a brain circuit and system of axon branch-specific and reversible neurotransmitter deployment that are critical for behavioural and sleep adaptation to photoperiod. A type of neuron called mrEn1-Pet1⁷ in the mouse brainstem median raphe nucleus segregates serotonin from VGLUT3 (also known as SLC17A8, a proxy for glutamate) to different axonal branches that innervate specific brain regions involved in circadian rhythm and sleep–wake timing^8,9. This branch-specific neurotransmitter deployment did not distinguish between daylight and dark phase; however, it reorganized with change in photoperiod. Axonal boutons, but not cell soma, changed neurochemical phenotype upon a shift away from equinox light/dark conditions, and these changes were reversed upon return to equinox conditions. When we genetically disabled Vglut3 in mrEn1-Pet1 neurons, sleep–wake periods, voluntary activity and clock gene expression did not synchronize to the new photoperiod or were delayed. Combining intersectional rabies virus tracing and projection-specific neuronal silencing, we delineated a preoptic area-to-mrEn1Pet1 connection that was responsible for decoding the photoperiodic inputs, driving the neurotransmitter reorganization and promoting behavioural synchronization. Our results reveal a brain circuit and periodic, branch-specific neurotransmitter deployment that regulates organismal adaptation to photoperiod change.

Physiology and behavior are structured temporally to anticipate daily cycles of light and dark, ensuring fitness and survival. Neuromodulatory systems in the brain—including those involving serotonin and dopamine—exhibit daily oscillations in neural activity and help shape circadian rhythms. Disrupted neuromodulation can cause circadian abnormalities that are thought to underlie several neuropsychiatric disorders, including bipolar mania and schizophrenia, for which a mechanistic understanding is still lacking. Here, we show that genetically depleting serotonin in Tph2 knockout mice promotes manic-like behaviors and disrupts daily oscillations of the dopamine biosynthetic enzyme tyrosine hydroxylase (TH) in midbrain dopaminergic nuclei. Specifically, while TH mRNA and protein levels in the Substantia Nigra (SN) and Ventral Tegmental Area (VTA) of wild-type mice doubled between the light and dark phase, TH levels were high throughout the day in Tph2 knockout mice, suggesting a hyperdopaminergic state. Analysis of TH expression in striatal terminal fields also showed blunted rhythms. Additionally, we found low abundance and blunted rhythmicity of the neuropeptide cholecystokinin (Cck) in the VTA of knockout mice, a neuropeptide whose downregulation has been implicated in manic-like states in both rodents and humans. Altogether, our results point to a previously unappreciated serotonergic control of circadian dopamine signaling and propose serotonergic dysfunction as an upstream mechanism underlying dopaminergic deregulation and ultimately maladaptive behaviors.

Changes in daylight amount (photoperiod) drive pronounced alterations in physiology and behaviour ^1,2 . Adaptive responses to seasonal photoperiods are vital to all organisms – dysregulation is associated with disease, from affective disorders ³ to metabolic syndromes ⁴ . Circadian rhythm circuitry has been implicated ^5,6 yet little is known about the precise neural and cellular substrates that underlie phase synchronization to photoperiod change. Here we present a previously unknown brain circuit and novel system of axon branch-specific and reversible neurotransmitter deployment that together prove critical for behavioural and sleep adaptation to photoperiod change. We found that the recently defined neuron type called mr En1-Pet1 ⁷ located in the mouse brainstem Median Raphe Nucleus (MRN) segregates serotonin versus VGLUT3 (here proxy for the neurotransmitter glutamate) to different axonal branches innervating specific brain regions involved in circadian rhythm and sleep/wake timing ^8,9 . We found that whether measured during the light or dark phase of the day this branch-specific neurotransmitter deployment in mr En1-Pet1 neurons was indistinguishable; however, it strikingly reorganizes on photoperiod change. Specifically, axonal boutons but not cell soma show a shift in neurochemical phenotype upon change away from equinox light/dark conditions that reverses upon return to equinox. When we genetically disabled the deployment of VGLUT3 in mr En1-Pet1 neurons, we found that sleep/wake periods and voluntary activity failed to synchronize to the new photoperiod or was significantly delayed. Combining intersectional rabies virus tracing and projection-specific neuronal silencing in vivo , we delineated a Preoptic Area-to-mr En1Pet1 connection responsible for decoding the photoperiodic inputs, driving the neurochemical shift and promoting behavioural synchronization. Our results reveal a previously unrecognized brain circuit along with a novel form of periodic, branch-specific neurotransmitter deployment that together regulate organismal adaptation to photoperiod changes.

Serotonin (5-HT)-releasing fibers show substantial structural plasticity in response to genetically-induced changes in 5-HT content. However, whether 5-HT fibers appear malleable also following clinically-relevant variations of 5-HT levels that may occur throughout an individual's life has not been investigated. Here, using confocal imaging and 3D modeling analysis in Tph2GFP knock-in mice, we show that chronic administration of the antidepressant fluoxetine dramatically affects the morphology of 5-HT fibers innervating the hippocampus resulting in a reduced density of fibers. Importantly, GFP fluorescence levels appeared unaffected in the somata of both dorsal and median raphe 5-HT neurons, arguing against potential fluoxetine-mediated down-regulation of the Tph2 promoter driving GFP expression in the Tph2GFP mouse model. In keeping this notion, mice bearing the pan-serotonergic driver Pet1-Cre partnered with a Cre-responsive tdTomato allele also showed similar morphological alterations in hippocampal 5-HT circuitry following chronic fluoxetine treatment. Moreover 5-HT fibers innervating the cortex showed proper density and no overt morphological disorganization, indicating that the reported fluoxetine-induced rearrangements were hippocampus specific. On the whole, these data suggest that 5-HT fibers are shaped in response to subtle changes of 5-HT homeostasis, and may provide a structural basis by which antidepressants exert their therapeutic effect.

Abnormal hippocampal neural plasticity has been implicated in behavioural abnormalities and complex neuropsychiatric conditions, including bipolar disorder (BD). However, the determinants of this neural alteration remain unknown. This work tests the hypothesis that the neurotransmitter serotonin (5-HT) is a key determinant of hippocampal neuroplasticity, and its absence leads to maladaptive behaviour relevant for BD. Depletion of brain 5-HT in Tph2 mutant mice resulted in reduced behavioural despair, reduced anxiety, marked aggression and lower habituation in novel environments, reminiscent of bipolar-associated manic behaviour. Treatment with valproate produced a substantial improvement of the mania-like behavioural phenotypes displayed by Tph2 mutants. Brain-wide fMRI mapping in mutants revealed functional hippocampal hyperactivity in which we also observed dramatically increased neuroplasticity. Importantly, remarkable correspondence between the transcriptomic profile of the Tph2 mutant hippocampus and neurons from bipolar disorder patients was observed. Chronic stress reversed the emotional phenotype and the hippocampal transcriptional landscape of Tph2 mutants. These changes were associated with inappropriate activation of transcriptional adaptive response to stress as assessed by gene set enrichment analyses in the hippocampus of Tph2 mutant mice. These findings delineate 5-HT as a critical determinant in BD associated maladaptive emotional responses and aberrant hippocampal neuroplasticity, and support the use of Tph2−/− mice as a new research tool for mechanistic and therapeutic research in bipolar disorder.

Serotonin-synthetizing neurons, which are confined in the raphe nuclei of the rhombencephalon, provide a pervasive innervation of the central nervous system and are involved in the modulation of a plethora of functions in both developing and adult brain. Classical studies have described the post-natal development of serotonergic axons as a linear process of terminal field innervation. However, technical limitations have hampered a fine morphological characterization. With the advent of genetic mouse models, the possibility to label specific neuronal populations allowed the rigorous measurement of their axonal morphological features as well as their developmental dynamics. Here, we used the Tph2GFP knock-in mouse line, in which GFP expression allows punctual identification of serotonergic neurons and axons, for confocal microscope imaging and we performed 3-dimensional reconstruction in order to morphologically characterize the development of serotonergic fibers in specified brain targets from birth to adulthood. Our analysis highlighted region-specific developmental patterns of serotonergic fiber density ranging from a linear and progressive colonization of the target (Caudate Putamen, Basolateral Amygdala, Geniculate Nucleus and Substantia Nigra) to a transient increase in fiber density (medial Prefrontal Cortex, Globus Pallidus, Somatosensory Cortex and Hippocampus) occurring with a region-specific timing. Despite a common pattern of early post-natal morphological maturation in which a progressive rearrangement from a dot-shaped to a regular and smooth fiber morphology was observed, starting from post-natal day 28 serotonergic fibers acquire the region specific morphological features present in the adult. In conclusion, we provided novel, target-specific insights on the morphology and temporal dynamics of the developing serotonergic fibers.

Modeling biological systems in vitro has contributed to clarification of complex mechanisms in simplified and controlled experimental conditions. Mouse embryonic stem (mES) cells can be successfully differentiated toward specific neuronal cell fates, thus representing an attractive tool to dissect, in vitro, mechanisms that underlie complex neuronal features. In this study, we generated and characterized a reporter mES cell line, called Tph2^GFP, in which the vital reporter GFP replaces the tryptophan hydroxylase 2 (Tph2) gene. Tph2^GFP mES cells selectively express GFP upon in vitro differentiation toward the serotonergic fate, they synthesize serotonin, possess excitable membranes, and show the typical morphological, morphometrical, and molecular features of in vivo serotonergic neurons. Thanks to the vital reporter GFP, we highlighted by time-lapse video microscopy several dynamic processes such as cell migration and axonal outgrowth in living cultures. Finally, we demonstrated that predifferentiated Tph2^GFP cells are able to terminally differentiate, integrate, and innervate the host brain when grafted in vivo. On the whole, the present study introduces the Tph2^GFP mES cell line as a useful tool allowing accurate developmental and dynamic studies and representing a reliable platform for the study of serotonergic neurons in health and disease.

The endogenous NMDA receptor (NMDAR) agonistd-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebrald-aspartate levels is due to the concomitant onset ofd-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylicd-amino acids. In the present work, we show thatd-aspartate content in the mouse brain drastically decreases after birth, whereasDdomRNA levels concomitantly increase. Interestingly, postnatalDdogene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control onDdoexpression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistentDdogene hypermethylation in the brain, we usedDdoknock-out mice (Ddo^−/−), which show constitutively suppressedDdoexpression. In these mice, we found for the first time substantially increased extracellular content ofd-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation ofd-aspartate levels inDdo^−/−brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules inDdo^−/−brains, highlights an unexpected importance ofDdodemethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of freed-aspartate.SIGNIFICANCE STATEMENTThe enzymed-aspartate oxidase (DDO) catalyzes the degradation of the NMDA receptor agonist,d-aspartate. In the brain, DDO is expressed only during postnatal life, thus reducing the embryonic storage ofd-aspartate and keeping thisd-amino acid at low levels during adulthood. Although the presence of DDO in mammals is long established, its biological role in the brain and the mechanism regulating its expression are still unclear. Here, we found thatDdopromoter demethylation enables the postnatal expression ofDdo. Moreover, persistent suppression ofDdoexpression leads to persistent spillover of extracellulard-aspartate and produces precocious cell death in the mouse brain, thus suggesting a key role for DDO in preventing early neurodegeneration triggered by excessive NMDA receptor stimulation.