Shank2/3 double knockout-based screening of cortical subregions links the retrosplenial area to the loss of social memory in autism spectrum disorders.

Members of the Shank protein family are master scaffolds of the postsynaptic architecture and mutations within the SHANK genes are causally associated with autism spectrum disorders (ASDs). We generated a Shank2-Shank3 double knockout mouse that is showing severe autism related core symptoms, as well as a broad spectrum of comorbidities. We exploited this animal model to identify cortical brain areas linked to specific autistic traits by locally deleting Shank2 and Shank3 simultaneously. Our screening of 10 cortical subregions revealed that a Shank2/3 deletion within the retrosplenial area severely impairs social memory, a core symptom of ASD. Notably, DREADD-mediated neuronal activation could rescue the social impairment triggered by Shank2/3 depletion. Data indicate that the retrosplenial area has to be added to the list of defined brain regions that contribute to the spectrum of behavioural alterations seen in ASDs.

STEM tomography of high-pressure frozen and freeze-substituted cells: a comparison of image stacks obtained at 200 kV or 300 kV.

Scanning transmission electron microscopic (STEM) tomography of high-pressure frozen, freeze-substituted semi-thin sections is one of multiple approaches for three-dimensional recording and visualization of electron microscopic samples. Compared to regular TEM tomography thicker sample sections can be investigated since chromatic aberration due to inelastic scattering is not a limit. The method is ideal to investigate subcellular compartments or organelles such as synapses, mitochondria, or microtubule arrangements. STEM tomography fills the gap between single-particle electron cryo-tomography, and methods that allow investigations of large volumes, such as serial block-face SEM and FIB-SEM. In this article, we discuss technical challenges of the approach and show some applications in cell biology. It is ideal to use a 300-kV electron microscope with a very small convergence angle of the primary beam ("parallel" beam). These instruments are expensive and tomography is rather time consuming, and therefore, access to such a high-end microscope might be difficult. In this article, we demonstrate examples of successful STEM tomography in biology using a more standard 200-kV microscope equipped with a field emission tip.

Nutrient limitation affects presynaptic structures through dissociable Bassoon autophagic degradation and impaired vesicle release.

Acute mismatch between metabolic requirements of neurons and nutrients/growth factors availability characterizes several neurological conditions such as traumatic brain injury, stroke and hypoglycemia. Although the effects of this mismatch have been investigated at cell biological level, the effects on synaptic structure and function are less clear. Since synaptic activity is the most energy-demanding neuronal function and it is directly linked to neuronal networks functionality, we have explored whether nutrient limitation (NL) affects the ultrastructure, function and composition of pre and postsynaptic terminals. We show that upon NL, presynaptic terminals show disorganized vesicle pools and reduced levels of the active zone protein Bassoon (but not of Piccolo). Moreover, NL triggers an impaired vesicle release, which is reversed by re-administration of glucose but not by the blockade of autophagic or proteasomal protein degradation. This reveals a dissociable correlation between presynaptic architecture and vesicle release, since restoring vesicle fusion does not necessarily depend from the rescue of Bassoon levels. Thus, our data show that the presynaptic compartment is highly sensitive to NL and the rescue of presynaptic function requires re-establishment of the metabolic supply rather than preventing local protein degradation.