[Integration of natural sciences and basic medical subjects in the integrated dentistry program (iMED DENT) at the University of Hamburg]. / Integration naturwissenschaftlicher und medizinischer Grundlagen im Hamburger Modellstudiengang Zahnmedizin iMED DENT.

In October 2019, an integrated dentistry program (iMED DENT) was implemented at the University of Hamburg and was the first of its kind in Germany. This model curriculum builds on didactic concepts that have been applied successfully for many years in curricula for human medicine, including interdisciplinary teaching, early clinical experience, and scientific education. The first year focuses on the healthy situation ("normal function") and aims to integrate the natural sciences (biology, chemistry, physics) and the basic medical subjects (anatomy, biochemistry, physiology, medical terminology) in the context of dental health. Further, basic practical and clinical tasks are assigned to the students during the first year.From the experience of the first four cohorts, initial conclusions can be drawn about this stage of study. Generally, its modular structure results in a condensation of learning content, which students judge as demanding. However, its interdisciplinary approach is well accepted. For instance, presenting the basics of the natural sciences in the context of their dental relevance is much better evaluated in the new compared to the previous curriculum, in which this content was taught without specific references to dental health. Teaching the basics of medicine within clinical context and the inclusion of early clinical practice are similarly appreciated. Presently, the interdisciplinary approach is limited by the focus on practical competencies of the dentistry curriculum, as some practical courses offer only few opportunities for other disciplines to interconnect their teaching. The continuous evaluation of the curriculum and exchange of experiences between the disciplines will further improve the integrative concept of the curriculum.

GPER1 deficiency causes sex-specific dysregulation of hippocampal plasticity and cognitive function.

Estrogens regulate synaptic properties and influence hippocampus-related learning and memory via estrogen receptors, which include the G-protein-coupled estrogen receptor 1 (GPER1). Studying mice, in which the GPER1 gene is dysfunctional (GPER1-KO), we here provide evidence for sex-specific roles of GPER1 in these processes. GPER1-KO males showed reduced anxiety in the elevated plus maze, whereas the fear response ('freezing') was specifically increased in GPER1-KO females in a contextual fear conditioning paradigm. In the Morris water maze, spatial learning and memory consolidation was impaired by GPER1 deficiency in both sexes. Notably, in the females, spatial learning deficits and the fear response were more pronounced if mice were in a stage of the estrous cycle, in which E2 serum levels are high (proestrus) or rising (diestrus). On the physiological level, excitability at Schaffer collateral synapses in CA1 increased in GPER1-deficient males and in proestrus/diestrus ('E2 high') females, concordant with an increased hippocampal expression of the AMPA-receptor subunit GluA1 in GPER1-KO males and females as compared to wildtype males. Further changes included an augmented early long-term potentiation (E-LTP) maintenance specifically in GPER1-KO females and an increased hippocampal expression of spinophilin in metestrus/estrus ('E2 low') GPER1-KO females. Our findings suggest modulatory and sex-specific functions of GPER1 in the hippocampal network, which reduce rather than increase neuronal excitability. Dysregulation of these functions may underlie sex-specific cognitive deficits or mood disorders.

Sex-specific Regulation of Spine Density and Synaptic Proteins by G-protein-coupled Estrogen Receptor (GPER)1 in Developing Hippocampus.

G-protein-coupled-estrogen-receptor 1 (GPER1) is a membrane-bound receptor that mediates estrogen signaling via intracellular signaling cascades. We recently showed that GPER1 promotes the distal dendritic enrichment of hyperpolarization activated and cyclic nucleotide-gated (HCN)1 channels in CA1 stratum lacunosum-moleculare (SLM), suggesting a role of GPER1-mediated signaling in neuronal plasticity. Here we studied whether this role involves processes of structural plasticity, such as the regulation of spine and synapse density in SLM. In organotypic entorhino-hippocampal cultures from mice expressing eGFP, we analyzed spine densities in SLM after treatment with GPER1 agonist G1 (20 nM). G1 significantly increased the density of "non-stubby" spines (maturing spines with a spine head and a neck), but did so only in cultures from female mice. In support of this finding, the expression of synaptic proteins was sex-specifically altered in the cultures: G1 increased the protein (but not mRNA) expression of PSD95 and reduced the p-/n-cofilin ratio only in cultures from females. Application of E2 (2 nM) reproduced the sex-specific effect on spine density in SLM, but only partially on the expression of synaptic proteins. Spine synapse density was, however, not altered after G1-treatment, suggesting that the increased spine density did not translate into an increased spine synapse density in the culture model. Taken together, our results support a role of GPER1 in mediating structural plasticity in CA1 SLM, but suggest that in developing hippocampus, this role is sex-specific.

Sex hormones modulate pathogenic processes in experimental traumatic brain injury.

Clinical and animal studies have revealed sex-specific differences in histopathological and neurological outcome after traumatic brain injury (TBI). The impact of perioperative administration of sex steroid inhibitors on TBI is still elusive. Here, we subjected male and female C57Bl/6N mice to the controlled cortical impact (CCI) model of TBI and applied pharmacological inhibitors of steroid hormone synthesis, that is, letrozole (LET, inhibiting estradiol synthesis by aromatase) and finasteride (FIN, inhibiting dihydrotestosterone synthesis by 5α-reductase), respectively, starting 72 h prior CCI, and continuing for a further 48 h after CCI. Initial gene expression analyses showed that androgen (Ar) and estrogen receptors (Esr1) were sex-specifically altered 72 h after CCI. When examining brain lesion size, we found larger lesions in male than in female mice, but did not observe effects of FIN or LET treatment. However, LET treatment exacerbated neurological deficits 24 and 72 h after CCI. On the molecular level, FIN administration reduced calpain-dependent spectrin breakdown products, a proxy of excitotoxicity and disturbed Ca2+ homeostasis, specifically in males, whereas LET increased the reactive astrocyte marker glial fibrillary acid protein specifically in females. Examination of neurotrophins (brain-derived neurotrophic factor, neuronal growth factor, NT-3) and their receptors (p75NTR , TrkA, TrkB, TrkC) revealed CCI-induced down-regulation of TrkB and TrkC protein expression, which was reduced by LET in both sexes. Interestingly, FIN decreased neuronal growth factor mRNA expression and protein levels of its receptor TrkA only in males. Taken together, our data suggest a sex-specific impact on pathogenic processes in the injured brain after TBI. Sex hormones may thus modulate pathogenic processes in experimental TBI.

Distal Dendritic Enrichment of HCN1 Channels in Hippocampal CA1 Is Promoted by Estrogen, but Does Not Require Reelin.

HCN1 compartmentalization in CA1 pyramidal cells, essential for hippocampal information processing, is believed to be controlled by the extracellular matrix protein Reelin. Expression of Reelin, in turn, is stimulated by 17ß-estradiol (E2). In this study, we therefore tested whether E2 regulates the compartmentalization of HCN1 in CA1 via Reelin. In organotypic entorhino-hippocampal cultures, we found that E2 promotes HCN1 distal dendritic enrichment via the G protein-coupled estrogen receptor GPER1, but apparently independent of Reelin, because GST-RAP, known to reduce Reelin signaling, did not prevent E2-induced HCN1 enrichment in distal CA1. We therefore re-examined the role of Reelin for the regulation of HCN1 compartmentalization and could not detect effects of reduced Reelin signaling on HCN1 distribution in CA1, either in the (developmental) slice culture model or in tamoxifen-inducible conditional reelin knockout mice during adulthood. We conclude that for HCN1 channel compartmentalization in CA1 pyramidal cells, Reelin is not as essential as previously proposed, and E2 effects on HCN1 distribution in CA1 are mediated by mechanisms that do not involve Reelin. Because HCN1 localization was not altered at different phases of the estrous cycle, gonadally derived estradiol is unlikely to regulate HCN1 channel compartmentalization, while the pattern of immunoreactivity of aromatase, the final enzyme of estradiol synthesis, argues for a role of local hippocampal E2 synthesis.

Reelin and aromatase cooperate in ovarian follicle development.

Reelin plays an important role in cerebral cortex development and synaptogenesis. In the hippocampus, the neurosteroid estrogen affects reelin expression. In this study we tested a potential crosstalk between estradiol and reelin, thus the possibility of a reelin-induced activation of the estradiol synthesizing enzyme aromatase. As a model system, we used ovaries, which express reelin and are a major source of estradiol. We found that in wild-type mice, reelin and aromatase are expressed in granulosa cells of growing follicles. The expression of reelin varies with the estrus cycle and is highest shortly before ovulation, when estradiol serum levels are at their maximum. In ovaries of reelin-deficient reeler mice, aromatase mRNA and protein are significantly reduced, as evidenced by real-time PCR, western blot analysis, and quantitative immunohistochemistry in granulosa cells of preovulatory follicles. In line with reduced estradiol synthesis, ovarian estrus cycle length is prolonged in reeler mice. Most importantly, treating cultured granulosa cells with recombinant reelin results in significant upregulation of aromatase mRNA and protein and increased secretion of estradiol into the supernatant. Our data provide evidence of a local increase of aromatase expression by reelin. Regarding reproduction, this crosstalk may contribute to follicular stability and counteract luteinization in ovaries.

Sex-Dependent Regulation of Aromatase-Mediated Synaptic Plasticity in the Basolateral Amygdala.

The basolateral amygdala (BLA) integrates sensory input from cortical and subcortical regions, a function that requires marked synaptic plasticity. Here we provide evidence that cytochrome P450 aromatase (AROM), the enzyme converting testosterone to 17ß-estradiol (E2), contributes to the regulation of this plasticity in a sex-specific manner. We show that AROM is expressed in the BLA, particularly in the basolateral nucleus (BL), in male and female rodents. Systemic administration of the AROM inhibitor letrozole reduced spine synapse density in the BL of adult female mice but not in the BL of male mice. Similarly, in organotypic corticoamygdalar slice cultures from immature rats, treatment with letrozole significantly reduced spine synapses in the BL only in cultures derived from females. In addition, letrozole sex-specifically altered synaptic properties in the BL: in acute slices from juvenile (prepubertal) female rats, wash-in of letrozole virtually abolished long-term potentiation (LTP), whereas it did not prevent the generation of LTP in the slices from males. Together, these data indicate that neuron-derived E2 modulates synaptic plasticity in rodent BLA sex-dependently. As protein expression levels of AROM, estrogen and androgen receptors did not differ between males and females and were not sex-specifically altered by letrozole, the findings suggest sex-specific mechanisms of E2 signaling.SIGNIFICANCE STATEMENT The basolateral amygdala (BLA) is a key structure of the fear circuit. This research reveals a sexually dimorphic regulation of synaptic plasticity in the BLA involving neuronal aromatase, which produces the neurosteroid 17ß-estradiol (E2). As male and female neurons in rodent BLA responded differently to aromatase inhibition both in vivo and in vitro, our findings suggest that E2 signaling in BLA neurons is regulated sex-dependently, presumably via mechanisms that have been established during sexual determination. These findings could be relevant for the understanding of sex differences in mood disorders and of the side effects of cytochrome P450 aromatase inhibitors, which are frequently used for breast cancer therapy.

Nedd4-2 regulates surface expression and may affect N-glycosylation of hyperpolarization-activated cyclic nucleotide-gated (HCN)-1 channels.

HCN channels are important regulators of neuronal excitability. The proper function of these channels is governed by various mechanisms, including post-translational modifications of channel subunits. Here, we provide evidence that ubiquitination via a ubiquitin ligase, neuronal precursor cell expressed developmentally downregulated (Nedd)-4-2, is involved in the regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We identified a PY motif (L/PPxY), the characteristic binding motif for Nedd4-2 in the C terminus of the HCN1 subunit, and showed that HCN1 and Nedd4-2 interacted both in vivo (rat hippocampus, neocortex, and cerebellum) and in vitro [human embryonic kidney 293 (HEK293) cells], resulting in increased HCN1 ubiquitination. Elimination of the PY motif reduced, but did not abolish, Nedd4-2 binding, which further involved a stretch of ∼100 aa downstream in the HCN1 C terminus. Coexpression of Nedd4-2 and HCN1 drastically reduced the HCN1-mediated h-current amplitude (85-92%) in Xenopus laevis oocytes and reduced surface expression (34%) of HCN1 channels in HEK293 cells, thereby opposing effects of tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b)-(1a-4), an auxiliary subunit that promotes HCN1 surface expression. Regulation may further include N-glycosylation of HCN1 channels, which is significantly enhanced by TRIP8b(1a-4), but may be reduced by Nedd4-2. Taken together, our data indicate that Nedd4-2 plays an important role in the regulation of HCN1 trafficking and may compete with TRIP8b(1a-4) in this process.

HCN1 subunits contribute to the kinetics of I(h) in neonatal cortical plate neurons.

The distribution of ion channels in neurons regulates neuronal activity and proper formation of neuronal networks during neuronal development. One of the channels is the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel constituting the molecular substrate of hyperpolarization-activated current (I(h)). Our previous study implied a role for the fastest activating subunit HCN1 in the generation of Ih in rat neonatal cortical plate neurons. To better understand the impact of HCN1 in early neocortical development, we here performed biochemical analysis and whole-cell recordings in neonatal cortical plate and juvenile layer 5 somatosensory neurons of HCN1(-/-) and control HCN1(+/+) mice. Western Blot analysis revealed that HCN1 protein expression in neonatal cortical plate tissue of HCN(+/+) mice amounted to only 3% of the HCN1 in young adult cortex and suggested that in HCN1(-/-) mice other isoforms (particularly HCN4) might be compensatory up-regulated. At the first day after birth, functional ablation of the HCN1 subunit did not affect the proportion of Ih expressing pyramidal cortical plate neurons. Although the contribution of individual subunit proteins remains open, the lack of HCN1 markedly slowed the current activation and deactivation in individual I(h) expressing neurons. However, it did not impair maximal amplitude/density, voltage dependence of activation, and cAMP sensitivity. In conclusion, our data imply that, although expression is relatively low, HCN1 contributes substantially to I(h) properties in individual cortical plate neurons. These properties are significantly changed in HCN1(-/-), either due to the lack of HCN1 itself or due to compensatory mechanisms.

ZD7288 enhances long-term depression at early postnatal medial perforant path-granule cell synapses.

Hyperpolarization-activated, cyclic nucleotide-gated nonselective (HCN) channels modulate both membrane potential and resistance and play a significant role in synaptic plasticity. We compared the influence of HCN channels on long-term depression (LTD) at the medial perforant path-granule cell synapse in early postnatal (P9-15) and adult (P30-60) rats. LTD was elicited in P9-15 slices using low-frequency stimulation (LFS, 900 pulses, 1 Hz; 80 ± 4% of baseline). Application of the specific HCN channel blocker ZD7288 (10 µM) before LFS significantly enhanced LTD (62 ± 4%; P < 0.01), showing HCN channels restrain LTD induction. However, when ZD7288 was applied after LFS, LTD was similar to control values and significantly different from the values obtained with ZD7288 application before LFS (81 ± 5%; P < 0.01), indicating that HCN channels do not modulate LTD expression. LTD in slices from adult rats were only marginally lower compared to those in P9-15 slices (85 ± 6%), but bath application of ZD7288 prior to LFS resulted in the same amount of LTD (85 ± 5%). HCN channels in adult tissue hence lose their modulatory effect. In conclusion, we found that HCN channels at the medial perforant path-granule cell synapse compromise LFS-associated induction, but not expression of LTD in early postnatal, but not in adult, rats.

Aromatase inhibition abolishes LTP generation in female but not in male mice.

Inhibitors of aromatase, the final enzyme of estradiol synthesis, are suspected of inducing memory deficits in women. In previous experiments, we found hippocampal spine synapse loss in female mice that had been treated with letrozole, a potent aromatase inhibitor. In this study, we therefore focused on the effects of letrozole on long-term potentiation (LTP), which is an electrophysiological parameter of memory and is known to induce spines, and on phosphorylation of cofilin, which stabilizes the spine cytoskeleton and is required for LTP in mice. In acute slices of letrozole-treated female mice with reduced estradiol serum concentrations, impairment of LTP started as early as after 6 h of treatment and progressed further, together with dephosphorylation of cofilin in the same slices. Theta-burst stimulation failed to induce LTP after 1 week of treatment. Impairment of LTP was followed by spine and spine synapse loss. The effects were confirmed in vitro by using hippocampal slice cultures of female mice. The sequence of effects in response to letrozole were similar in ovariectomized female and male mice, with, however, differences as to the degree of downregulation. Our data strongly suggest that impairment of LTP, followed by loss of mushroom spines and spine synapses in females, may have implications for memory deficits in women treated with letrozole.

Regulation of axonal HCN1 trafficking in perforant path involves expression of specific TRIP8b isoforms.

The functions of HCN channels in neurons depend critically on their subcellular localization, requiring fine-tuned machinery that regulates subcellular channel trafficking. Here we provide evidence that regulatory mechanisms governing axonal HCN channel trafficking involve association of the channels with specific isoforms of the auxiliary subunit TRIP8b. In the medial perforant path, which normally contains HCN1 channels in axon terminals in immature but not in adult rodents, we found axonal HCN1 significantly increased in adult mice lacking TRIP8b (TRIP8b(-/-)). Interestingly, adult mice harboring a mutation that results in expression of only the two most abundant TRIP8b isoforms (TRIP8b[1b/2](-/-)) exhibited an HCN1 expression pattern similar to wildtype mice, suggesting that presence of one or both of these isoforms (TRIP8b(1a), TRIP8b(1a-4)) prevents HCN1 from being transported to medial perforant path axons in adult mice. Concordantly, expression analyses demonstrated a strong increase of expression of both TRIP8b isoforms in rat entorhinal cortex with age. However, when overexpressed in cultured entorhinal neurons of rats, TRIP8b(1a), but not TRIP8b(1a-4), altered substantially the subcellular distribution of HCN1 by promoting somatodendritic and reducing axonal expression of the channels. Taken together, we conclude that TRIP8b isoforms are important regulators of HCN1 trafficking in entorhinal neurons and that the alternatively-spliced isoform TRIP8b(1a) could be responsible for the age-dependent redistribution of HCN channels out of perforant path axon terminals.

The 27-kDa heat shock protein (HSP27) is a reliable hippocampal marker of full development of pilocarpine-induced status epilepticus.

PURPOSE: The pilocarpine-induced status epilepticus (SE) in rodents provides a valuable animal model of temporal lobe epilepsy. Since not all animals enter SE following pilocarpine injection, we aimed to find a biomarker for full development of pilocarpine-induced SE using a proteomic approach. METHODS: Two-dimensional gel electrophoresis and Western blot analysis were performed with protein extracts from hippocampal tissue taken from four different groups of animals: pilocarpine-treated rats with full development of SE, pilocarpine-treated rats without seizures, pilocarpine-treated rats with seizures but without SE, and saline-injected rats. RESULTS: 2D gel electrophoresis revealed two protein spots which were only present after full development of pilocarpine-induced SE, but neither in gels from pilocarpine-treated rats without seizures nor in those of saline-injected animals. The protein in both spots was identified as the small 27-kDa heat shock protein (HSP27) by MALDI-TOF mass spectrometry. Subsequent Western blot analyses confirmed that HSP27 immunoreactivity was only observed with the hippocampal protein extracts in the status epilepticus group, but in none of the other groups. Furthermore, immunocytochemistry showed that HSP27 expression following pilocarpine-induced SE was localized in astrocytes. CONCLUSION: We propose that HSP27 is a highly sensitive and specific hippocampal marker for full development of pilocarpine-induced status epilepticus.

Upregulation of presynaptic mGluR2, but not mGluR3 in the epileptic medial perforant path.

Presynaptic metabotropic glutamate receptors (mGluRs) at glutamatergic synapses play a major role in governing release probability. Previous reports indicated a downregulation of group III mGluRs at the lateral perforant path-granule cell synapse in the chronically epileptic hippocampus. Here, we investigated the mGluR-dependent presynaptic inhibition at the medial perforant path-granule cell synapse in the pilocarpine-treated chronically epileptic rat. The specific group II mGluR agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV, 10µM) significantly depressed medial perforant path-evoked responses in control slices, but significantly more so in epileptic tissue. This depression was accompanied by a significant increase of the paired-pulse ratio in both animal groups indicating a presynaptic mechanism. Moreover, we also found that this significantly enhanced DCG-IV effect in the medial perforant path recorded in slices from pilocarpine-treated rats was due to a significant increase of mGluR2, but not mGluR3 transcripts in the entorhinal cortex using quantitative real-time reverse transcriptase-PCR. Immunohistochemistry confirmed the increased expression of group II mGluRs in the epileptic medial molecular layer. These results demonstrate that chronic epilepsy not only causes downregulation of mGluRs in the hippocampus, but may also lead to enhanced expression of these receptors - at least in the medial perforant path.

Deletion of the hyperpolarization-activated cyclic nucleotide-gated channel auxiliary subunit TRIP8b impairs hippocampal Ih localization and function and promotes antidepressant behavior in mice.

Output properties of neurons are greatly shaped by voltage-gated ion channels, whose biophysical properties and localization within axodendritic compartments serve to significantly transform the original input. The hyperpolarization-activated current, I(h), is mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and plays a fundamental role in influencing neuronal excitability by regulating both membrane potential and input resistance. In neurons such as cortical and hippocampal pyramidal neurons, the subcellular localization of HCN channels plays a critical functional role, yet mechanisms controlling HCN channel trafficking are not fully understood. Because ion channel function and localization are often influenced by interacting proteins, we generated a knock-out mouse lacking the HCN channel auxiliary subunit, tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Eliminating expression of TRIP8b dramatically reduced I(h) expression in hippocampal pyramidal neurons. Loss of I(h)-dependent membrane voltage properties was attributable to reduction of HCN channels on the neuronal surface, and there was a striking disruption of the normal expression pattern of HCN channels in pyramidal neuron dendrites. In heterologous cells and neurons, absence of TRIP8b increased HCN subunit targeting to and degradation by lysosomes. Mice lacking TRIP8b demonstrated motor learning deficits and enhanced resistance to multiple tasks of behavioral despair with high predictive validity for antidepressant efficacy. We observed similar resistance to behavioral despair in distinct mutant mice lacking HCN1 or HCN2. These data demonstrate that interaction with the auxiliary subunit TRIP8b is a major mechanism underlying proper expression of HCN channels and I(h) in vivo, and suggest that targeting I(h) may provide a novel approach to treatment of depression.

Roles of 17ß-estradiol involve regulation of reelin expression and synaptogenesis in the dentate gyrus.

Studies on the role of 17ß-estradiol (E2) in the hippocampus have mainly focused on CA1 and CA3 regions, whereas in dentate gyrus (DG), its role is largely unknown. Here, we examined potential functions of E2 in DG, particularly during development. Immunohistochemistry and in situ hybridization revealed abundance of estrogen receptor (ER)α, but not ERß, expression in DG. Similar to CA1, analysis of synapse densities revealed a reduction in spine synapse number in DG molecular layer of immature rats and adult mice after inhibition of estradiol synthesis using letrozole. Interestingly, strong expression of ERα was found in Cajal-Retzius (CR) cells, which regulate neuronal migration and synaptogenesis via the extracellular matrix protein reelin. Immunoreactivity of aromatase, the final enzyme of estradiol synthesis, was strongest in mature granule cells. In hippocampal slice cultures, exogenous application of E2 caused an increase in reelin expression in CR cells, which was abolished after blockade of ERs using ICI182,780. Vice versa, inhibition of aromatase activity by letrozole resulted in reduced reelin expression, suggesting that E2 deriving from hippocampal sources contributes to the regulation of reelin as well as to the maintenance of spine synapses in DG. E2 further regulated Notch1, a signaling protein involved in neuronal differentiation.

HCN1 channels constrain DHPG-induced LTD at hippocampal Schaffer collateral-CA1 synapses.

HCN channels play a fundamental role in determining resting membrane potential and regulating synaptic function. Here, we investigated the involvement of HCN channels in basal synaptic transmission and long-term depression (LTD) at the Schaffer collateral-CA1 synapse. Bath application of the HCN channel blocker ZD7288 (10 microM) caused a significant increase in synaptic transmission that was due to an enhancement in AMPA receptor-mediated excitatory postsynaptic potentials. This enhancement was accompanied by a significant decrease in the paired-pulse ratio (PPR), suggesting a presynaptic mechanism. Experiments with the irreversible use-dependent NMDA receptor blocker MK-801 showed that ZD7288 led to an increase in glutamate release probability. LTD induced by brief application of (RS)-3,5-dihydroxyphenylglycine (DHPG, 100 microM, 10 min) was significantly enhanced when HCN channels were blocked by ZD7288 (10 microM) prior to DHPG application. Moreover, the concomitant increase in PPR after DHPG-induced LTD was significantly larger than without ZD7288 bath application. Conversely, ZD7288 application after DHPG washout did not alter DHPG-LTD. A significant enhancement of DHPG-LTD was also observed in HCN1-deficient mice as compared with wild types. However, LTD induced by low-frequency stimulation (LFS) remained unaltered in HCN1-deficient mice, suggesting a differential effect of HCN1 channels on synaptic plasticity constraining DHPG-LTD, but not LFS-LTD.

Postnatal expression pattern of HCN channel isoforms in thalamic neurons: relationship to maturation of thalamocortical oscillations.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are the molecular substrate of the hyperpolarization-activated inward current (I(h)). Because the developmental profile of HCN channels in the thalamus is not well understood, we combined electrophysiological, molecular, immunohistochemical, EEG recordings in vivo, and computer modeling techniques to examine HCN gene expression and I(h) properties in rat thalamocortical relay (TC) neurons in the dorsal part of the lateral geniculate nucleus and the functional consequence of this maturation. Recordings of TC neurons revealed an approximate sixfold increase in I(h) density between postnatal day 3 (P3) and P106, which was accompanied by significantly altered current kinetics, cAMP sensitivity, and steady-state activation properties. Quantification on tissue levels revealed a significant developmental decrease in cAMP. Consequently the block of basal adenylyl cyclase activity was accompanied by a hyperpolarizing shift of the I(h) activation curve in young but not adult rats. Quantitative analyses of HCN channel isoforms revealed a steady increase of mRNA and protein expression levels of HCN1, HCN2, and HCN4 with reduced relative abundance of HCN4. Computer modeling in a simplified thalamic network indicated that the occurrence of rhythmic delta activity, which was present in the EEG at P12, differentially depended on I(h) conductance and modulation by cAMP at different developmental states. These data indicate that the developmental increase in I(h) density results from increased expression of three HCN channel isoforms and that isoform composition and intracellular cAMP levels interact in determining I(h) properties to enable progressive maturation of rhythmic slow-wave sleep activity patterns.

Hyperpolarization activated cyclic-nucleotide gated (HCN) channels in developing neuronal networks.

Developing neuronal networks evolve continuously, requiring that neurons modulate both their intrinsic properties and their responses to incoming synaptic signals. Emerging evidence supports roles for the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in this neuronal plasticity. HCN channels seem particularly suited for fine-tuning neuronal properties and responses because of their remarkably large and variable repertoire of functions, enabling integration of a wide range of cellular signals. Here, we discuss the involvement of HCN channels in cortical and hippocampal network maturation, and consider potential roles of developmental HCN channel dysregulation in brain disorders such as epilepsy.

Activity-dependent heteromerization of the hyperpolarization-activated, cyclic-nucleotide gated (HCN) channels: role of N-linked glycosylation.

Formation of heteromeric complexes of ion channels via co-assembly of different subunit isoforms provides an important mechanism for enhanced channel diversity. We have previously demonstrated co-association of the hyperpolarization activated cyclic-nucleotide gated (HCN1/HCN2) channel isoforms that was regulated by network (seizure) activity in developing hippocampus. However, the mechanisms that underlie this augmented expression of heteromeric complexes have remained unknown. Glycosylation of the HCN channels has been implicated in the stabilization and membrane expression of heteromeric HCN1/HCN2 constructs in heterologous systems. Therefore, we used in vivo and in vitro systems to test the hypothesis that activity modifies HCN1/HCN2 heteromerization in neurons by modulating the glycosylation state of the channel molecules. Seizure-like activity (SA) increased HCN1/HCN2 heteromerization in hippocampus in vivo as well as in hippocampal organotypic slice cultures. This activity increased the abundance of glycosylated HCN1 but not HCN2-channel molecules. In addition, glycosylated HCN1 channels were preferentially co-immunoprecipitated with the HCN2 isoforms. Provoking SA in vitro in the presence of the N-linked glycosylation blocker tunicamycin abrogated the activity-dependent increase of HCN1/HCN2 heteromerization. Thus, hippocampal HCN1 molecules have a significantly higher probability of being glycosylated after SA, and this might promote a stable heteromerization with HCN2.