Millimetric scale two-photon Bessel-Gauss beam light sheet microscopy with three-axis isotropic resolution using an axicon lens.

Significance: Typical light sheet microscopes suffer from artifacts related to the geometry of the light sheet. One main inconvenience is the non-uniform thickness of the light sheet obtained with a Gaussian laser beam. Aim: We developed a two-photon light sheet microscope that takes advantage of a thin and long Bessel-Gauss beam illumination to increase the sheet extent without compromising the resolution. Approach: We use an axicon lens placed directly at the output of an amplified femtosecond laser to produce a long Bessel-Gauss beam on the sample. We studied the dopaminergic system and its projections in a whole cleared mouse brain. Results: Our light sheet microscope allows an isotropic resolution of 2.4 µm in all three axes of the scanned volume while keeping a millimetric-sized field of view, and a fast acquisition rate of up to 34 mm2/s. With slight modifications to the optical setup, the sheet extent can be increased to 6 mm. Conclusion: The proposed system's sheet extent and resolution surpass currently available systems, enabling the fast imaging of large specimens.

Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons.

The LIM-homeodomain transcription factors Lmx1a and Lmx1b play critical roles during the development of midbrain dopaminergic progenitors, but their functions in the adult brain remain poorly understood. We show here that sustained expression of Lmx1a and Lmx1b is required for the survival of adult midbrain dopaminergic neurons. Strikingly, inactivation of Lmx1a and Lmx1b recreates cellular features observed in Parkinson's disease. We found that Lmx1a/b control the expression of key genes involved in mitochondrial functions, and their ablation results in impaired respiratory chain activity, increased oxidative stress, and mitochondrial DNA damage. Lmx1a/b deficiency caused axonal pathology characterized by α-synuclein(+) inclusions, followed by a progressive loss of dopaminergic neurons. These results reveal the key role of these transcription factors beyond the early developmental stages and provide mechanistic links between mitochondrial dysfunctions, α-synuclein aggregation, and the survival of dopaminergic neurons.

Mesocortical Dopamine Phenotypes in Mice Lacking the Sonic Hedgehog Receptor Cdon.

Motivated behaviors and many psychopathologies typically involve changes in dopamine release from the projections of the ventral tegmental area (VTA) and/or the substantia nigra pars compacta (SNc). The morphogen Sonic Hedgehog (Shh) specifies fates of midbrain dopamine neurons, but VTA-specific effects of Shh signaling are also being uncovered. In this study, we assessed the role of the Shh receptor Cdon in the development of VTA and SNc dopamine neurons. We find that Cdon is expressed in the proliferating progenitor zone of the embryonic ventral midbrain and that the number of proliferating cells in this region is increased in mouse Cdon(-/-) embryos. Consistent with a role of Shh in the regulation of neuronal proliferation in this region, we find that the number of tyrosine hydroxylase (TH)-positive neurons is increased in the VTA of Cdon(-/-) mice at birth and that this effect endures into adulthood. In contrast, the number of TH-positive neurons in the SNc is not altered in Cdon(-/-) mice at either age. Moreover, adult Cdon(-/-) mice have a greater number of medial prefrontal cortex (mPFC) dopamine presynaptic sites, and increased baseline concentrations of dopamine and dopamine metabolites selectively in this region. Finally, consistent with increased dopamine function in the mPFC, we find that adult Cdon(-/-) mice fail to exhibit behavioral plasticity upon repeated amphetamine treatment. Based on these data, we suggest that Cdon plays an important role encoding the diversity of dopamine neurons in the midbrain, influencing both the development of the mesocortical dopamine pathway and behavioral outputs that involve this neural circuitry.

Selective deletion of forebrain glycogen synthase kinase 3ß reveals a central role in serotonin-sensitive anxiety and social behaviour.

Serotonin (5-HT) neurotransmission is thought to underlie mental illnesses, such as bipolar disorder, depression, autism and schizophrenia. Independent studies have indicated that 5-HT or drugs acting on 5-HT neurotransmission regulate the serine/threonine kinase glycogen synthase kinase 3ß (GSK3ß). Furthermore, GSK3ß inhibition rescues behavioural abnormalities in 5-HT-deficient mice with a loss-of-function mutation equivalent to the human variant (R441H) of tryptophan hydroxylase 2. In an effort to define neuroanatomical correlates of GSK3ß activity in the regulation of behaviour, we generated CamKIIcre-floxGSK3ß mice in which the gsk3b gene is postnatally inactivated in forebrain pyramidal neurons. Behavioural characterization showed that suppression of GSK3ß in these brain areas has anxiolytic and pro-social effects. However, while a global reduction of GSK2ß expression reduced responsiveness to amphetamine and increased resilience to social defeat, these behavioural effects were not found in CamKIIcre-floxGSK3ß mice. These findings demonstrate a dissociation of behavioural effects related to GSK3 inhibition, with forebrain GSK3ß being involved in the regulation of anxiety and sociability while social preference, resilience and responsiveness to psychostimulants would involve a function of this kinase in subcortical areas such as the hippocampus and striatum.