Expression of hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 in axon terminals of peptidergic nociceptive primary sensory neurons in the super®cial spinal dorsal horn of rats Miklo Âs Antal, 1 Ildiko  Papp, 1 Niyazi Bahaerguli, 1 Ga Âbor Veress 1 and Gyo Èrgy Vereb 2 1 Department of Anatomy, Histology and Embryology, and 2 Department of Biophysics and Cell Biology, University of Debrecen, Nagyerdei krt. 98, Debrecen, Hungary, H-4012 Keywords: CGRP, HCN2, immunocytochemistry, isolectin-B4, nociceptive primary afferents Abstract Hyperpolarization-activated cyclic nucleotide-gated cation channel proteins (HCN1±4), which are potentially able to modulate membrane excitability, are abundantly expressed by neurons in spinal dorsal root ganglia (DRG). In the present experiment, we investigated whether HCN2 protein is con®ned exclusively to the perikarya of DRG neurons or is transported from the somata to the central axons of DRG neurons that terminate in the spinal dorsal horn. Using immunohistochemical methods, we have demonstrated that laminae I-IIo of the super®cial spinal dorsal horn of the adult rat spinal cord show a strong punctate immunoreactivity for HCN2. Dorsal rhizotomy resulted in a complete loss of immunostaining in the dorsal horn, suggesting that HCN2 is con®ned to axon terminals of primary afferents. In double labelling immunohistochemical studies, we have also shown that HCN2 widely co-localizes with calcitonin gene-related peptide, but is almost completely segregated from isolectin-B4 binding, indicating that HCN2 is primarily expressed in peptidergic nociceptive primary afferents. The expression of HCN2 in central terminals of peptidergic primary afferents was also veri®ed with electron microscopy. Utilizing the pre-embedding nanogold method, we found that HCN2 is largelycon®ned to axon terminals with dense-core vesicles. Within these terminals, some of the silver grains marking the accurate location of HCN2 molecules were associated with the cell membrane, and others were scattered in the axoplasm. Within the cell membrane, HCN2 was found almost exclusively in extrasynaptic locations. The results suggest that HCN2 may contribute to the modulation of membrane excitability of nociceptive primary afferent terminals in the spinal dorsal horn. Introduction Therichvarietyandcomplexityoffunctionsofthenervoussystemare largely attained by a ®ne-tuned interplay between intrinsic neuronal properties and synaptic interconnections. Intrinsic electrophysiologi- calcharacteristicsre¯ectthetype,locationanddensityofvoltage-and ligand-gatedionchannelsthatregulatethe¯owofioniccurrentsacross the plasma membrane. In accordance with their highly complex electroresponsiveness, neurons possess a rich repertoire of ion chan- nels, including hyperpolarization-activated and cyclic nucleotide- gated channels (HCN). HCN channels are usually activated during membrane hyperpolarization following the termination of action potentialsandprovideamixedinwardNa  /K  current(I h )thatslowly depolarizes the plasma membrane. One of the most interesting char- acteristicsof I h isitsregulationbycAMP.HCNchannelsdirectlybind cAMP that enhances I h , shifting the activation curve to more positive potentials(Pape,1996;Robinson&Siegelbaum,2003).Byproviding aninwardcationcurrentatmembranepotentialsnegativeto 60mV, I h regulates the resting membrane potential and the responses of neurons to hyperpolarizing currents. It has also been proposed that I h contributes to the generation of `pacemaker' potentials that control the rate of rhythmic oscillatory behaviours of neurons (Pape, 1996; Robinson & Siegelbaum, 2003). One of the most interesting and controversial roles of I h is its contribution to the control of synaptic transmission. I h has been demonstrated in a number of presynaptic terminals, including cray®sh neuromuscular junction (Beaumont & Zucker, 2000), presynaptic terminals in chick ciliary ganglion (Fletcher & Chiappinelli, 1992) and axon terminals of cerebellar basket cells (Southan et al., 2000), and it was suggested that I h may play a regulatory role in the long-term facilitation of synaptic transmission in a cAMP-dependent manner. Four genes encoding HCN channel proteins have recently been cloned (Ludwig etal., 1998), and the detailed distribution of the four identi®edproteins(HCN1±4)andtheirmRNAsinthenervoussystem hasbeenreported(Moosmang etal.,1999).HCNproteinsaswellas I h currentshavebeenfoundinadiversegroupofexcitablecellsincluding primary sensory neurons in spinal dorsal root ganglia (DRG). In situ hybridizationstudiesrevealedthatallneuronsintheratDRGexpress mRNA for HCN1, HCN2 and HCN3. Signals for HCN4 could not be distinguished from background (Chaplan etal., 2003). Con®rming the anatomical observations, I h has been recorded in the membranes of cell bodies and peripheral nerve ®bres of DRG neurons (Grafe et al., 1997; Yao et al., 2003). However, there is no available evi- dence concerning the presence of I h in the central axon terminals of DRG neurons in the spinal grey matter. The failure to obtain such European Journal of Neuroscience, Vol. 19, pp. 1336±1342, 2004 ß Federation of European Neuroscience Societies doi:10.1111/j.1460-9568.2004.03235.x Correspondence: Dr M. Antal, as above. E-mail: antal@chondron.anat.dote.hu Received 18 September 2003, revised 16 December 2003, accepted 18 December 2003