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IRPHRCSTP Report Summary

Project ID: 260725
Funded under: FP7-IDEAS-ERC
Country: United Kingdom

Final Report Summary - IRPHRCSTP (Investigating the role of pre-synaptic HCN1 channels in regulating cortical synaptic transmission and plasticity.)

The hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are voltage-gated ion channels that have been implicated to have to significant roles in learning and memory as well as disorders such as epilepsy and neuropathic pain. These channels are encoded by 4 subunits, HCN1-4. In the hippocampus and cortex, HCN1 subunits are predominantly expressed. Here, they are highly localised to pyramidal neuron dendrites where they regulate synaptic potential shapes and integration and thereby neuronal firing and processes such as long-term potentiation.
Our preliminary data using immunogold labelling and electrophysiological analysis had indicated that HCN1 channels may also be present presynaptically in the medial entorhinal cortex (mEC). The aims of this project, therefore, were to establish whether these channels were located presynaptically in mEC and the function of these channels. During the course of this project, we showed that HCN1 channels are located presynaptically too within the active zone of select synaptic terminals targeting mEC layer III pyramidal neurons and not mEC layer II stellate neurons or mEC layer V pyramidal neurons. These channels were present on afferents arising from the forebrain that targeted mEC layer III and not from subcortical regions. Interestingly, electrophysiological and two-photon FM1-43 imaging data show that these channels suppressed both action potential-dependent and spontaneous (non-action potential-dependent) release of the neurotransmitter, glutamate, onto mEC layer III pyramidal neurons (Huang et al., 2011, Nature Neuroscience, 14, 478-86; Huang et al, 2017, Scientific Reports, 7, 40257). This was paradoxical given that HCN1 channels depolarize the resting membrane potential (RMP) and their presence on presynaptic terminals would, therefore, be predicted to enhance synaptic release. Unusually, we made the ground-breaking discovery that low threshold T-type Ca2+ channels are also located on the same terminals as the HCN1 channels. HCN1 channels by depolarizing the RMP was reducing the activity of T-type Ca2+ channels and thereby restricting Ca2+ entry into these synaptic terminals, resulting in a reduction of glutamate release. (Huang et al., 2011, Nature Neuroscience, 14, 478-86).
We next asked whether modulation of presynaptic and dendritic HCN1 channels was similar. HCN1 channel localisation within hippocampal pyramidal cell dendrites is determined by the expression of the tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b). Using TRIP8b null mice, their wildtype littermates, electrophysiological recordings and immunogold labelling, we found that whilst TRIP8b was required for localisation of dendritic HCN1 subunits in mEC layer III pyramidal neurons, it was not necessary for targeting of HCN1 subunits to presynaptic terminals (Huang et al., 2012, Journal of Neuroscience, 32, 14835-48). These findings, therefore, strongly suggest that presynaptic and postsynaptic HCN1 channel activity is regulated independently in vivo and thus, presynaptic and postsynaptic HCN1 channels may have distinct functions in maintaining normal neuronal excitability.
Our previous work had shown that dendritic HCN1 channels in the mEC are persistently downregulated following induction of seizures. Given that trafficking of presynaptic and postsynaptic HCN1 subunits differs substantially, we investigated if presynaptic HCN1 channel activity is also reduced following seizures. We found that seizure activity caused a sustained loss of both presynaptic and postsynaptic HCN1 channels. The enhanced neurotransmitter release occurring as a consequence of presynaptic HCN1 channel loss is likely to contribute to enhancing the process of epileptogenesis (Huang et al., 2012, Journal of Neuroscience, 32, 14835-48).
In conclusion, our work has established the existence of presynaptic HCN1 channels in excitatory synaptic terminals that arise from forebrain neurons. These terminals selectively target mEC layer III pyramidal neurons. HCN1 channels located here regulate action potential and non-action potential dependent glutamate release by restricting Ca2+ influx via T-type Ca2+ channels. The molecular mechanisms regulating trafficking and function of presynaptic and postsynaptic HCN1 channels differs. Nonetheless, both presynaptic and postsynaptic HCN1 channels are persistently downregulated following seizure activity. Thus, presynaptic HCN1 channels, like postsynaptic HCN1 channels, play an important role in maintaining mEC neural network activity.

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United Kingdom
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