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Understanding the association between autonomic nervous system [ANS] function and brain morphology across the lifespan provides important insights into neurovisceral mechanisms underlying health and disease. Resting-state ANS activity, indexed by measures of heart rate [HR] and its variability [HRV] has been associated with brain morphology, particularly cortical thickness [CT]. While findings have been mixed regarding the anatomical distribution and direction of the associations, these inconsistencies may be due to sex and age differences in HR/HRV and CT. Previous studies have been limited by small sample sizes, which impede the assessment of sex differences and aging effects on the association between ANS function and CT. To overcome these limitations, 20 groups worldwide contributed data collected under similar protocols of CT assessment and HR/HRV recording to be pooled in a mega-analysis (N = 1,218 (50.5% female), mean age 36.7 years (range: 12-87)). Findings suggest a decline in HRV as well as CT with increasing age. CT, particularly in the orbitofrontal cortex, explained additional variance in HRV, beyond the effects of aging. This pattern of results may suggest that the decline in HRV with increasing age is related to a decline in orbitofrontal CT. These effects were independent of sex and specific to HRV; with no significant association between CT and HR. Greater CT across the adult lifespan may be vital for the maintenance of healthy cardiac regulation via the ANS-or greater cardiac vagal activity as indirectly reflected in HRV may slow brain atrophy. Findings reveal an important association between CT and cardiac parasympathetic activity with implications for healthy aging and longevity that should be studied further in longitudinal research.

Alpha7 neuronal nicotinic acetylcholine receptors (alpha7-nAChR) form Ca(2+)-permeable homopentameric channels modulating cortical network activity and cognitive processing. They are located pre- and postsynaptically and are highly abundant in hippocampal GABAergic interneurons. It is unclear how alpha7-nAChRs are positioned in specific membrane microdomains, particularly in cultured neurons which are devoid of cholinergic synapses. To address this issue, we monitored by single particle tracking the lateral mobility of individual alpha7-nAChRs labeled with alpha-bungarotoxin linked to quantum dots in live rat cultured hippocampal interneurons. Quantitative analysis revealed different modes of lateral diffusion of alpha7-nAChR dependent on their subcellular localization. Confined receptors were found in the immediate vicinity of glutamatergic and GABAergic postsynaptic densities, as well as in extrasynaptic clusters of alpha-bungarotoxin labeling on dendrites. alpha7-nAChRs avoided entering postsynaptic densities, but exhibited reduced mobility and long dwell times at perisynaptic locations, indicative of regulated confinement. Their diffusion coefficient was lower, on average, at glutamatergic than at GABAergic perisynaptic sites, suggesting differential, synapse-specific tethering mechanisms. Disruption of the cytoskeleton affected alpha7-nAChR mobility and cell surface expression, but not their ability to form clusters. Finally, using tetrodotoxin to silence network activity, as well as exposure to a selective alpha7-nAChR agonist or antagonist, we observed that alpha7-nAChRs cell surface dynamics is modulated by chronic changes in neuronal activity. Altogether, given their high Ca(2+)-permeability, our results suggest a possible role of alpha7-nAChR on interneurons for activating Ca(2+)-dependent signaling in the vicinity of GABAergic and glutamatergic synapses.

Efficient and long-lasting transfection of primary neurons is an essential tool to address many questions in current neuroscience using functional gene analysis. Neurons are sensitive to cytotoxicity and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA interference (shRNA) vectors, using magnetofection, into rat hippocampal neurons (E18/19) cultured for several hours to 21 days in vitro. This protocol even allows for double-transfection of DNA into a small subpopulation of hippocampal neurons (GABAergic interneurons), as well as achieving long-lasting expression of DNA and shRNA constructs without interfering with neuronal differentiation. The protocol, which uses inexpensive equipment and reagents, takes 1 h, utilizes mixed hippocampal cultures, a transfection reagent, CombiMag and a magnetic plate, shows low toxicity and is suited for single cell analysis. Modifications as done by our three laboratories are detailed.