Elsevier

Neuroscience

Volume 162, Issue 1, 4 August 2009, Pages 208-221
Neuroscience

Systems Neuroscience
Research Paper
Altered synchrony and connectivity in neuronal networks expressing an autism-related mutation of neuroligin 3

https://doi.org/10.1016/j.neuroscience.2009.04.062Get rights and content

Abstract

The neuroligin (NL) gene family codes for brain specific cell adhesion molecules that play an important role in synaptic connectivity. Recent studies have identified NL mutations linked to patients with autism spectrum disorders (ASD). Cognitive deficits seen in autistic patients are hypothesized to arise from altered synchronicity both within and between brain regions. Here we show how the expression of autism-associated neuroligin mutation R471C-NL3 affects synchrony in dissociated cultures of rat hippocampal neurons. Spontaneous network activity patterns of cultures expressing wild type and mutant NL3 were measured by optical techniques. Firing events were quantified and compared by cross-correlation analysis. Our results suggest that NL3 overexpression enhances synchrony of spontaneous activity patterns, however, this ability is reduced with the R471C-NL3 mutation. We investigated the structural basis of this phenomenon using fractal dimension analysis to characterize the arrangement of axon trajectories. R471C-NL3 cultures were associated with lower fractal dimensions and higher lacunarity values, indicating a decrease in the complexity of axonal architecture. Transfection of R471C-NL3 into a subpopulation of cells in a network resulted in neuronal degeneration. This degeneration likely affected the inhibitory population of neurons, as there were half as many (P<0.01, n=12) glutamate decarboxylase (GAD) 65 expressing cells in R471C-NL3 cultures compared to wild type NL3 and control cultures. Electrophysiological recordings showed a reduction of inhibitory activity in networks carrying the mutation in comparison to networks overexpressing wild-type NL3. Together, these data support the hypothesis that the autism-associated NL3 mutation affects information processing in neuronal networks by altering network architecture and synchrony.

Section snippets

Primary cell culture

Dissociated primary co-cultures of neurons and glia were grown on p-type, 12 Ω/cm, boron-doped, double-side polished, 1 cm2 pre-cut silicon wafers, 1 mm in thickness (Silicon Quest International, Reno, NV, USA) as described (Goda and Colicos, 2006). Wafers were treated with poly-d-lysine (Sigma-Aldrich, St. Louis) (50 μg/ml working dilution) overnight. Wafers were then washed in PBS 3×10 min and incubated for 3 h with mouse laminin (Sigma-Aldrich) at a final concentration of 10 μg/ml.

Recording of spontaneous network activity

Correlated spatiotemporal activity patterns of neuronal networks are suggested to be important for reliable transmission, processing and storage of information (Madhavan et al 2007, Xu and Clancy 2008). To investigate the role of NL3 in neuronal connectivity we compared the spontaneous activity patterns of networks containing cells overexpressing wild type or mutant NL3. Network activity was monitored by recording intracellular calcium (Ca2+) of neurons using the fluorescent Ca2+ sensitive dye

Discussion

Biochemical studies as well as postmortem and genetic data have provided important insights into the underlying molecular basis of ASD. However autism presents as a cognitive disorder, there are currently few functional or systems-level paradigms to assess how specific biochemical pathways affect activity patterns, and consequently information processing, in the brain. Addressing this in a model system has been difficult, for example, results collected from NL3 knockout mice suggest there is no

Acknowledgments

We thank Robyn Flynn for comments on the manuscript and Lucas Scott for technical assistance. This work was supported by CIHR and Cure Autism Now grants to M.C. M.C. is an AHFMR Scholar and an HSFC Investigator. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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