JULIDE: A Software Tool for 3D Reconstruction and Statistical Analysis of Autoradiographic Mouse Brain Sections

In this article we introduce JULIDE, a software toolkit developed to perform the 3D reconstruction, intensity normalization, volume standardization by 3D image registration and voxel-wise statistical analysis of autoradiographs of mouse brain sections. This software tool has been developed in the open-source ITK software framework and is freely available under a GPL license. The article presents the complete image processing chain from raw data acquisition to 3D statistical group analysis. Results of the group comparison in the context of a study on spatial learning are shown as an illustration of the data that can be obtained with this tool

Rescue of oxytocin response and social behaviour in a mouse model of autism

A fundamental challenge in developing treatments for autism spectrum disorders is the heterogeneity of the condition. More than one hundred genetic mutations confer high risk for autism, with each individual mutation accounting for only a small fraction of cases1,2,3. Subsets of risk genes can be grouped into functionally related pathways, most prominently those involving synaptic proteins, translational regulation, and chromatin modifications. To attempt to minimize this genetic complexity, recent therapeutic strategies have focused on the neuropeptides oxytocin and vasopressin4,5,6, which regulate aspects of social behaviour in mammals7. However, it is unclear whether genetic risk factors predispose individuals to autism as a result of modifications to oxytocinergic signalling. Here we report that an autism-associated mutation in the synaptic adhesion molecule Nlgn3 results in impaired oxytocin signalling in dopaminergic neurons and in altered behavioural responses to social novelty tests in mice. Notably, loss of Nlgn3 is accompanied by a disruption of translation homeostasis in the ventral tegmental area. Treatment of Nlgn3-knockout mice with a new, highly specific, brain-penetrant inhibitor of MAP kinase-interacting kinases resets the translation of mRNA and restores oxytocin signalling and social novelty responses. Thus, this work identifies a convergence between the genetic autism risk factor Nlgn3, regulation of translation, and oxytocinergic signalling. Focusing on such common core plasticity elements might provide a pragmatic approach to overcoming the heterogeneity of autism. Ultimately, this would enable mechanism-based stratification of patient populations to increase the success of therapeutic interventions

Preprocessing procedure.

<p>(A) Sections are stored as individual 2D slices. (B) Histogram of one section. Two modes: Tissues and Background. (C) Binary images resulting from the threshold calculated using OTSU technique. (D) Results of opening operator to clean noise or separate overlapping sections. (E) Individual sections ready for 3D reconstruction.</p

Intra-subject 3D reconstruction procedure.

<p>(A) Sections are first stacks in the Z direction. (B) Consecutive sections are extracted and down-sampled by a factor of four (for the first iteration, the two central sections are extracted). (C) Consecutive sections are co-registered and replace in the stacked volume. (D) The procedure ends when all sections have been aligned to the consecutive or the previous one.</p

JULIDE framework. JULIDE is a 5 steps processing.

<p>Step 1. Pre-Processing: Cleaning of the autoradiographs from dust or overlapping sections. Step 2. Calibration: grayscale intensities are linearly calibrated using the co-exposed [14C] standard scale. Step 3. 3D reconstruction. Step 4. Spatial normalization to the chosen reference space. Step 5. Statistical analysis and results visualization.</p

Activation results during learning (Day 1).

<p>A: JULIDE results. At Day 1, the hippocampus (HP) and the parietal associative cortex (PTLp) are the most activated regions during learning, whereas after the task they are no longer activated. t-test comparison between Day 1 trained mice and Active Control (AC). Uncorrected p-value  = 0.02. Clusters circled in red show significant activation after FWE and FDR corrections (corrected p-values <0.01). B: MCID results for the parietal associative cortex (PTLp). Increase in glucose consumption, measured as 2DG uptake, in the parietal cortex (PTL) the first (Day 1) day of training. t-test comparison between Day 1 trained mice (n = 7) and Active Control (AC; n = 8). *p-value <0.01.</p

Inter-subject normalization by 3D registration.

<p>(A) Average image of a group of 8 mice brain after 3D affine registration. (B) Average image of the same group after 3D non-rigid registration.</p

Astrocytic VMAT2 in the developing prefrontal cortex is required for normal grooming behavior in mice

Astrocytes control synaptic activity by modulating peri-synaptic concentrations of ion and neurotransmitters including dopamine and, as such, can be critically involved in the modulation of some aspect of mammalian behavior. Here we report that genetic mouse model with a reduced medial prefrontal cortex (mPFC) dopamine levels, arising from astrocyte-specific conditional deletion of vesicular monoamine transporter 2 (VMAT2; aVMTA2cKO mice) shows excessive grooming and anxiety-like behaviour. The VMAT2cKO mice also develop a synaptic pathology, expressed through increased relative AMPA vs. NMDA receptor currents in synapses of the dorsal striatum receiving inputs from the mPFC. Importantly, behavioural and synaptic phenotypes are prevented by reexpression of mPFC VMAT2, showing that the deficits are driven by mPFC astrocytes. By analysing human tissue samples, we found that VMAT2 is expressed in human mPFC astrocytes, corroborating the potential translational relevance of our observations in mice. Our study shows that impairments of the astrocytic-control of dopamine in the mPFC has a profound impact on circuit function and behaviours, which resemble symptoms of anxiety disorders and obsessive compulsive disorder (OCD)

Dysfunction of homeostatic control of dopamine by astrocytes in the developing prefrontal cortex leads to cognitive impairments.

Astrocytes orchestrate neural development by powerfully coordinating synapse formation and function and, as such, may be critically involved in the pathogenesis of neurodevelopmental abnormalities and cognitive deficits commonly observed in psychiatric disorders. Here, we report the identification of a subset of cortical astrocytes that are competent for regulating dopamine (DA) homeostasis during postnatal development of the prefrontal cortex (PFC), allowing for optimal DA-mediated maturation of excitatory circuits. Such control of DA homeostasis occurs through the coordinated activity of astroglial vesicular monoamine transporter 2 (VMAT2) together with organic cation transporter 3 and monoamine oxidase type B, two key proteins for DA uptake and metabolism. Conditional deletion of VMAT2 in astrocytes postnatally produces loss of PFC DA homeostasis, leading to defective synaptic transmission and plasticity as well as impaired executive functions. Our findings show a novel role for PFC astrocytes in the DA modulation of cognitive performances with relevance to psychiatric disorders