Axonal Kainate Receptors Modulate the Strength of Efferent Connectivity by Regulating Presynaptic Differentiation.

Prasanna Sakha,Sari E Lauri,Tiina-Kaisa Kukko-Lukjanov,Ville Jokinen,Aino Vesikansa,Alexandra Shintyapina,Henri J Huttunen,Sami Franssila,Ester Orav,Joonas Heikkinen

doi:10.3389/fncel.2016.00003

Prasanna Sakha, Sari E Lauri + Show 8 more

Open Access

https://doi.org/10.3389/fncel.2016.00003

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Abstract

Kainate type of glutamate receptors (KARs) are highly expressed during early brain development and may influence refinement of the circuitry, via modulating synaptic transmission and plasticity. KARs are also localized to axons, however, their exact roles in regulating presynaptic processes remain controversial. Here, we have used a microfluidic chamber system allowing specific manipulation of KARs in presynaptic neurons to study their functions in synaptic development and function in vitro. Silencing expression of endogenous KARs resulted in lower density of synaptophysin immunopositive puncta in microfluidically isolated axons. Various recombinant KAR subunits and pharmacological compounds were used to dissect the mechanisms behind this effect. The calcium permeable (Q) variants of the low-affinity (GluK1–3) subunits robustly increased synaptophysin puncta in axons in a manner that was dependent on receptor activity and PKA and PKC dependent signaling. Further, an associated increase in the mean active zone length was observed in electron micrographs. Selective presynaptic expression of these subunits resulted in higher success rate of evoked EPSCs consistent with higher probability of glutamate release. In contrast, the calcium-impermeable (R) variant of GluK1 or the high-affinity subunits (GluK4,5) had no effect on synaptic density or transmission efficacy. These data suggest that calcium permeable axonal KARs promote efferent connectivity by increasing the density of functional presynaptic release sites.

Highlights

Ionotropic glutamate receptors are well known for their critical roles in mediating and modulating excitatory neurotransmission in the brain
To study the role of axonal Kainate type of glutamate receptors (KARs) in synaptogenesis, we used a recently developed microfluidic culture chamber where two neuronal populations are grown in isolation but connected by narrow (7.5 μm) tunnels allowing axon growth (Taylor and Jeon, 2010; Jokinen et al, 2013), (Figure 1A)
Primary hippocampal neurons strongly expressed the mRNA for KAR subunits GluK2, GluK4, and GluK5, while GluK1 and GluK3 mRNAs were detected at lower levels both at DIV7 and at DIV14 (Figure 1B)

Summary

Introduction

Ionotropic glutamate receptors are well known for their critical roles in mediating and modulating excitatory neurotransmission in the brain. Apart from the ionotropic effects, KARs modulate neuronal functions, via G-protein coupled signaling, especially during the ‘critical period’ of circuit development in the hippocampus (Lauri and Taira, 2011). At the stage when a synaptic connection is formed and is already functional, presynaptic KARs are tonically active and inhibit vesicle release; this G-protein dependent signaling regulates the short-term dynamics of transmission (Lauri et al, 2006; Clarke et al, 2014) and is suggested to have an important role in activity-dependent fine-tuning of the connectivity in the hippocampus (Lauri and Taira, 2011)

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