Abstract
Communication between different sub regions of the hippocampus is fundamental to learning and memory. However accurate knowledge about information transfer between sub regions from access to the activity in individual axons is lacking. MEMS devices with microtunnels connecting two sub networks have begun to approach this problem but the commonly used 10 μm wide tunnels frequently measure signals from multiple axons. To reduce this complexity, we compared polydimethylsiloxane (PDMS) microtunnel devices each with a separate tunnel width of 2.5, 5 or 10 μm bridging two wells aligned over a multi electrode array (MEA). Primary rat neurons were grown in the chambers with neurons from the dentate gyrus on one side and hippocampal CA3 on the other. After 2–3 weeks of culture, spontaneous activity in the axons inside the tunnels was recorded. We report electrophysiological, exploratory data analysis for feature clustering and visual evidence to support the expectation that 2.5 μm wide tunnels have fewer axons per tunnel and therefore more clearly delineated signals than 10 or 5 μm wide tunnels. Several measures indicated that fewer axons per electrode enabled more accurate detection of spikes. A clustering analysis comparing the variations of spike height and width for different tunnel widths revealed tighter clusters representing unique spikes with less height and width variation when measured in narrow tunnels. Wider tunnels tended toward more diffuse clusters from a continuum of spike heights and widths. Standard deviations for multiple cluster measures, such as Average Dissimilarity, Silhouette Value (S) and Separation Factor (average dissimilarity/S value), support a conclusion that 2.5 μm wide tunnels containing fewer axons enable more precise determination of individual action potential peaks, their propagation direction, timing, and information transfer between sub networks.
Highlights
The mammalian hippocampus plays an important role in the formation of long-term episodic memories and spatial navigation, yet encoding in sub-region for different elements of memory formation remains poorly understood
To validate the clustering algorithm based on spike height and width features, we examined the waveforms for each cluster
The average dissimilarity of single clusters in 10 μm wide tunnels was 0.07 ±0.01, indicating a diffuse type, compared to 0.04 ±0.01 in 2.5 μm wide tunnels, indicating a tight cluster type (Wilcoxon signed-rank test, p = 0.05). These results suggest that 2.5 μm tunnels produce more single tight clusters than those produced by wider tunnels and the single clusters seen in 10 μm wide tunnels were more often diffuse
Summary
The mammalian hippocampus plays an important role in the formation of long-term episodic memories and spatial navigation, yet encoding in sub-region for different elements of memory formation remains poorly understood. Micro-tunnels that can approach single axon resolution and identification of connectivity between sub-regions would clarify information transmission between subregions. The tunnel-electrode construct allows simultaneous recordings with μm spatial and μs temporal resolution from a network of axons communicating between two sub regions. Key features of the information transfer are spike direction of propagation from the timing difference on the two electrodes in the same tunnel, typically spaced 200 μm apart and spike and burst dynamics to decode the information transfer. These advantages are predicated on well-isolated spikes
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