Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein’s function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties.

Neurons inevitably rely on a proper repertoire and distribution of membrane-bound ion‑conducting channels. Among these proteins, the family of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels possesses unique properties giving rise to the corresponding Ih-current that contributes to various aspects of neural signaling. In mammals, four genes (hcn1-4) encode subunits of HCN channels. These subunits can assemble as hetero- or homotetrameric ion-conducting channels. In order to elaborate on the specific role of the HCN2 subunit in shaping electrical properties of neurons, we applied an Adeno-associated virus (AAV)-mediated, RNAi-based knock-down strategy of hcn2 gene expression both in vitro and in vivo. Electrophysiological measurements showed that HCN2 subunit knock-down resulted in specific yet anticipated changes in Ih-current properties in primary hippocampal neurons and, in addition, corroborated that the HCN2 subunit participates in postsynaptic signal integration. To further address the role of the HCN2 subunit in vivo, we injected recombinant (r)AAVs into the dorsal hippocampus of young adult male mice. Behavioral and biochemical analyses were conducted to assess the contribution of HCN2-containing channels in shaping hippocampal network properties. Surprisingly, knock-down of hcn2 expression resulted in a severe degeneration of the CA1 pyramidal cell layer, which did not occur in mice injected with control rAAV constructs. This finding might pinpoint to a vital and yet unknown contribution of HCN2 channels in establishing or maintaining the proper function of CA1 pyramidal neurons of the dorsal hippocampus.