[PubMed] [Google Scholar] 11. remote fear memory. GBB-18-na-s001.docx (3.7M) GUID:?A04FCE82-A9CA-48B8-8EF4-E4207A2C23C2 Abstract Hyperpolarization\activated and cyclic nucleotide\gated (HCN) channels mediate the current in the murine hippocampus. Disruption of the current by knockout of HCN1, HCN2 or tetratricopeptide repeat\made up of Rab8b\interacting protein has been shown to impact physiological processes such as synaptic integration and Apocynin (Acetovanillone) maintenance of resting membrane potentials as well as several behaviors in mice, including depressive\like and stress\like behaviors. However, the potential involvement of the HCN4 isoform in these processes is unknown. Here, we assessed the contribution of the HCN4 isoform to neuronal processing and hippocampus\based behaviors in mice. We show that HCN4 is usually expressed in various regions of the hippocampus, with unique expression patterns that partially overlapped with other HCN isoforms. For behavioral analysis, we specifically modulated HCN4 expression by injecting recombinant adeno\associated viral Rabbit Polyclonal to HLAH (rAAV) vectors mediating expression of short hairpin RNA against (shHcn4) into the dorsal hippocampus of mice. HCN4 knockdown produced no effect on contextual fear conditioning or spatial memory. However, a pronounced anxiogenic effect was obvious in mice treated with shHcn4 Apocynin (Acetovanillone) compared to control littermates. Our findings suggest that HCN4 specifically contributes to stress\like behaviors in mice. current is essential for controlling rhythmic activity in neuronal circuits (eg, in the thalamocortical system during the sleep\wake cycle) and also affects several essential constituents of neuronal processing such as resting membrane potentials, dendritic integration and synaptic transmission.11 Studies at the mRNA and protein level showed that this four HCN isoforms display distinct expression patterns in the nervous system.12, 13, 14 Especially, notable was a pronounced HCN1 and HCN2 expression gradient along apical dendrites of CA1 pyramidal neurons, increasing from proximal to distal. This gradient has been shown to substantially impact integration and temporal adjustment of synaptic inputs to CA1 Apocynin (Acetovanillone) Apocynin (Acetovanillone) pyramidal neurons.15, 16, 17, 18 Due to their diverging biophysical characteristics, HCN channels are likely candidates for directed modulation of hippocampal processing, as the individual isoforms differ strongly in for example, voltage dependence, activation kinetics and modulation by direct binding of cyclic nucleotides.1 Notably, disruption of the current by transgenic manipulation of HCN1, HCN2 or TRIP8b affects hippocampus\based behaviors including depression and anxiety.19, 20, 21, 22 However, so far only the functional contribution of HCN1 and HCN2 to hippocampal processing has been explained,22, 23, 24 while a potential role of HCN3 or HCN4 has not yet been resolved. Notably, the biophysical characteristics of HCN4 differ strongly from other isoforms, with the most hyperpolarized midpoint of activation, the slowest activation kinetics and the most substantial cAMP\induced shift of activation toward depolarized potentials.25 Transgenic mouse lines with impaired HCN4 expression have been used to show the importance of HCN4 in the context of cardiac pacemaking.26, 27, 28, 29 However, reports on HCN4 expression in the murine hippocampus are scarce and not entirely consistent,14, 30 and the effect of HCN4 disruption on hippocampal function has not been fully examined. Here, we assessed the contribution of HCN4 to neuronal processing and hippocampus\based behaviors in mice. We examined the distribution of HCN4 using immunohistochemistry (IHC) and found that expression patterns of HCN4 largely resembled that of HCN1, but unique differences were apparent at the subcellular level. For specific modulation of HCN4 expression, recombinant adeno\associated viral (rAAV) vectors mediating expression of short hairpin RNA against (shHcn4) were injected into the dorsal hippocampus of mice. Treatment with shHcn4 led to a reduction of transcript expression by about 60% compared to control\injected mice as well as a decrease of HCN4 protein expression. HCN4 knockdown produced no effect on contextual fear conditioning (FC) or spatial memory. However, a pronounced anxiogenic effect was obvious in mice treated with shHcn4 compared to control littermates. In summary, HCN4 differs clearly from other HCN isoforms in its expression patterns as well as in its contribution to hippocampus\based Apocynin (Acetovanillone) behaviors. 2.?MATERIALS AND METHODS 2.1. Antibodies Main and secondary antibodies utilized for IHC and western blot (WB) analysis are outlined in the Supplementary Material (Table S1). 2.2. Immunohistochemistry Briefly, mice were transcardially perfused with ice\chilly phosphate\buffered saline (PBS) followed by paraformaldehyde (PFA; 4% [w/v] in PBS) before dissection. Tissue was cryo\guarded in 30% (w/v) sucrose, embedded in Tissue Tek (Sakura Finetek, Zouterwoude, The Netherlands), and coronal cryo\sections (30 m) were prepared. For IHC, sections were incubated in 0.3% (v/v) H2O2 for 30 minutes at room heat before unspecific binding sites were blocked for 1 hour in PBS containing 0.75% (v/v) Triton X\100, 5% (v/v) normal goat serum and 5% (v/v) normal donkey serum. Subsequently, main antibodies were applied at 4C for 3 days, and secondary antibodies were applied at room heat for 4 hours (observe Table S1)..