HCN channels are expressed differentially in retinal bipolar cells and concentrated at synaptic terminals Frank Mu ¨ller, 1 Alexander Scholten, 1 Elena Ivanova, 1 Silke Haverkamp, 2 Elisabeth Kremmer 3 and U. Benjamin Kaupp 1 1 Institut fu ¨ r Biologische Informationsverarbeitung, Forschungszentrum Ju ¨ lich, D-52425 Ju ¨ lich, Germany, 2 Max-Planck-Institut fu ¨ r Hirnforschung, Abteilung Neuroanatomie, Deutschordenstraße 46, D-60528 Frankfurt, Germany 3 Institut fu ¨ r Molekulare Immunologie, GSF-Forschungszentrum fu ¨ r Umwelt und Gesundheit, Marchioninistraße 25, D-81377 Mu ¨ nchen, Germany Keywords: bipolar cell, electrophysiology, HCN channels, I h , immunocytochemistry, retina Abstract Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels codetermine the integrative behaviour of neurons and shape their response to synaptic stimulation. We used immunohistochemistry and patch-clamp recording to study the composition and distribution of HCN channels in the rat retina. All four HCN channel isoforms (HCN1–4) are expressed differentially in the retina. In particular, different classes of bipolar cells have a different inventory of HCN channels. We found no evidence for the formation of heterooligomeric HCN channels. HCN channels are densely clustered at synaptic terminals of bipolar cells and photoreceptors. This suggests that HCN channels are involved in the control of transmitter release. Introduction In the mammalian retina, the light response of photoreceptors is processed by an elaborate neuronal network (for review see Wa ¨ssle & Boycott, 1991; Masland, 2001). At least nine types of cone bipolar cells and one type of rod bipolar cell provide the pathways for the signal flow from photoreceptors to ganglion cells (for rat see Euler & Wa ¨ssle, 1995; Hartveit, 1997). Functionally, bipolar cells fall into ON- and OFF-cells. The axons of OFF-bipolar cells, which show hyperpo- larizing light responses, stratify in the outer half of the inner plexiform layer (IPL). Axons of ON-bipolar cells, which depolarize in light, stratify in the inner half of the IPL. Within the ON- and OFF- sublamina, different bipolar cell types stratify at distinct levels. Both ON- and OFF-bipolar cells are further subdivided into cells which exhibit transient and sustained light responses (Awatramani & Slaughter, 2000; Wu et al., 2000). The output of a cone is tapped by several types of bipolar cell, each constituting a separate ‘temporal channel’ that transfers different characteristics of the light response. The light response of bipolar cells is shaped by different glutamate receptors (DeVries, 2000) and by voltage-gated channels. One class of voltage-gated cation channels present in bipolar cells (Kaneko & Tachibana, 1985, Karschin & Wa ¨ssle, 1990) is activated by hyperpolarization and gated by cyclic nucleotides (HCN channels; in previous studies; HCN currents were designated I f ,I h or I q ). HCN channels codetermine the resting potential and membrane conductance and thereby play an important role in the integrative behaviour of neurons and the sensitivity to synaptic input. HCN channels affect the cable properties of the dendrite and shape the time course and propagation of excitatory and inhibitory postsynaptic potentials (for review see Pape, 1996; Kaupp & Seifert, 2001). In rod and cone photoreceptors, HCN channels shape the light response. The channels become activated during hyperpolarization in bright light and depolarize the cell toward the dark membrane poten- tial, making the light response transient (Fain et al., 1978; Baylor et al., 1984; Hestrin, 1987). Although their function in bipolar cells is not known, HCN channels might shape the light response of OFF-bipolar cells. In mammals, four HCN channel genes (HCN1–4) have been iden- tified (Ludwig et al., 1998; Santoro et al., 1998; Ludwig et al., 1999; Seifert et al., 1999; for review see Kaupp & Seifert, 2001). When expressed heterologously, HCN subtypes form homomeric channels that differ in their kinetics and mid-point potential of activation (V 1/2 ) and in the shift of V 1/2 towards more positive potentials by cAMP. The functional diversity of HCN channels might be enhanced further either by formation of heteromultimers from different subtypes (Chen et al., 2001; Ulens & Tytgat, 2001) or coassembly of HCN subunits with minimal K þ channel-related proteins (Yu et al., 2001), previously identified in voltage-gated K þ channels (Abbott & Goldstein, 1998). In this study, we address these issues concerning the function of HCN channels in the retina: (i) which of the different bipolar cell types express HCN channels; (ii) does a given cell type express a single HCN channel isoform, or several; (iii) do different HCN channel subunits colocalize  allowing for the formation of heterooligomeric chan- nels  or are they targeted to different compartments of the cell; and (iv), do HCN channels colocalize with neurotransmitter receptors that might modulate their activity through changing cAMP levels? Given the highly polarized nature of photoreceptors and bipolar cells, the subcellular locales of HCN channels seem to be crucial for their function. For example, spatially restricted expression of K þ channels was described in bipolar cells (Klumpp et al., 1995). Our study describes for the first time the expression pattern of all four HCN channel isoforms within the entire and well-defined neuronal network of the retina. A detailed immunological and European Journal of Neuroscience, Vol. 17, pp. 2084–2096, 2003 ß Federation of European Neuroscience Societies doi:10.1046/j.1460-9568.2003.02634.x Correspondence: Dr Frank Mu ¨ller, as above. E-mail: f.mueller@fz-juelich.de Received 20 December 2002, revised 28 February 2003, accepted 3 March 2003