Quantitative analysis and subcellular distribution of mRNA and protein expression of the hyperpolarization-activated cyclic nucleotide-gated channels throughout development in rat hippocampus.

The properties of the hyperpolarization-activated current (I(h)) and its roles in hippocampal network function evolve radically during development. Because I(h) is conducted by the hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels, we tested the hypothesis that understanding the quantitative developmental profiles of HCN1, HCN2, and HCN4 expression, and the isoform- and age-specific progression of their subcellular distribution, should shed light on the established modifications of the properties of I(h) throughout development. Combined quantitative in situ hybridization, regional western blots, and high-resolution, dual-label immunocytochemistry revealed striking and novel information about the expression and distribution of the HCN channel isoforms in the developing hippocampal formation. In cornus ammon 1 (CA) pyramidal cell layer, a robust increase of HCN1 mRNA and protein expression occurred with age, with reciprocal reduction of HCN4 and relatively stable HCN2 levels. These distinct expression patterns raised the contribution of HCN1 to the total HCN channel pool from 33% to 65% consonant with acceleration and reduced cyclic adenosine mono phosphate (cAMP) sensitivity of I(h) in this region with age. In CA3, strong expression of HCN1 already neonatally supports the recently established role of this conductance in neonatal, age-specific, hippocampal oscillations (giant depolarizing potentials). Notably, HCN1 channels were present and probably transported to dendritic compartments already on postnatal day (P) 2, whereas HCN2 channel protein was not evident in dendrites for the first 2 weeks of life. HCN2 mRNA and protein expression remained fairly constant subsequent to the first week of life in all hippocampal subfields examined, whereas HCN4 mRNA and protein expression declined after maximal neonatal expression, so that the contribution of this isoform to the total HCN channel pool dropped from 43% (CA1) and 34% (CA3) on P11 to 8% (CA1) and 19% (CA3) on P90. Interneuronal expression of all HCN channel isoforms in stratum pyramidale was robust in parvalbumin-but not in cholecystokinin-expressing populations and with a subunit-specific subcellular distribution. Taken together, these data suggest that early in life, HCN4 may contribute significantly to the functions of I(h) in specific hippocampal regions. In addition, these evolving, differential quantitative, and subcellular expression patterns of the HCN channel isoforms support age-specific properties and functions of I(h) within the developing hippocampal formation

Fever, febrile seizures and epilepsy.

Seizures induced by fever (febrile seizures) are the most common type of pathological brain activity in infants and children. These febrile seizures and their potential contribution to the mechanisms of limbic (temporal lobe) epilepsy have been a topic of major clinical and scientific interest. Key questions include the mechanisms by which fever generates seizures, the effects of long febrile seizures on neuronal function and the potential contribution of these seizures to epilepsy. This review builds on recent advances derived from animal models and summarizes our current knowledge of the mechanisms underlying febrile seizures and of changes in neuronal gene expression and function that facilitate the enduring effects of prolonged febrile seizures on neuronal and network excitability. The review also discusses the relevance of these findings to the general mechanisms of epileptogenesis during development and points out gaps in our knowledge, including the relationship of animal models to human febrile seizures and epilepsy

Regulated expression of HCN channels and cAMP levels shape the properties of the h current in developing rat hippocampus.

The hyperpolarization-activated current (I(h)) contributes to intrinsic properties and network responses of neurons. Its biophysical properties depend on the expression profiles of the underlying hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and the presence of cyclic AMP (cAMP) that potently and differentially modulates I(h) conducted by HCN1, HCN2 and/or HCN4. Here, we studied the properties of I(h) in hippocampal CA1 pyramidal cells, the developmental evolution of the HCN-subunit isoforms that contribute to this current, and their interplay with age-dependent free cAMP concentrations, using electrophysiological, molecular and biochemical methods. I(h) amplitude increased progressively during the first four postnatal weeks, consistent with the observed overall increased expression of HCN channels. Activation kinetics of the current accelerated during this period, consonant with the quantitative reduction of mRNA and protein expression of the slow-kinetics HCN4 isoform and increased levels of HCN1. The sensitivity of I(h) to cAMP, and the contribution of the slow component to the overall I(h), decreased with age. These are likely a result of the developmentally regulated transition of the complement of HCN channel isoforms from cAMP sensitive to relatively cAMP insensitive. Thus, although hippocampal cAMP concentrations increased over twofold during the developmental period studied, the coordinated changes in expression of three HCN channel isoforms resulted in reduced effects of this signalling molecule on neuronal h currents

Repeated Use of the Psychoactive Substance Ethylphenidate Impacts Neurochemistry and Reward Learning in Adolescent Male and Female Mice

Schedule II prescription psychostimulants, such as methylphenidate (MPH), can be misused as nootropic drugs, i.e., drugs that enhance focus and cognition. When users are unable to obtain these prescribed medications, they may seek out novel psychoactive substances (NPSs) that are not yet scheduled. An example of a NPS reportedly being abused is ethylphenidate (EPH), a close analog of MPH but with a higher preference for the dopamine transporter compared with the norepinephrine transporter. Therefore, based upon this pharmacological profile and user self-reports, we hypothesized that repeated EPH exposure in adolescent mice may be rewarding and alter cognition. Here, we report that repeated exposure to 15 mg/kg EPH decreased spatial cognitive performance as assessed by the Barnes maze spatial learning task in adolescent male C57Bl/6 mice; however, male mice did not show alterations in the expression of mature BDNF – a protein associated with increased cognitive function – in key brain regions. Acute EPH exposure induced hyperlocomotion at a high dose (15 mg/kg, i.p.), but not a low dose (5 mg/kg, i.p.). Interestingly, mice exhibited significant conditioned place preference at the low EPH dose, suggesting that even non-stimulating doses of EPH are rewarding. In both males and females, repeated EPH exposure increased expression of deltaFosB – a marker associated with increased risk of drug abuse – in the dorsal striatum, nucleus accumbens, and prefrontal cortex. Overall, our results suggest that repeated EPH use in adolescence is psychostimulatory, rewarding, increases crucial brain markers of reward-related behaviors, and may negatively impact spatial performance

Enhanced expression of a specific hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN) in surviving dentate gyrus granule cells of human and experimental epileptic hippocampus.

Changes in the expression of ion channels, contributing to altered neuronal excitability, are emerging as possible mechanisms in the development of certain human epilepsies. In previous immature rodent studies of experimental prolonged febrile seizures, isoform-specific changes in the expression of hyperpolarization-activated cyclic nucleotide-gated cation channels (HCNs) correlated with long-lasting hippocampal hyperexcitability and enhanced seizure susceptibility. Prolonged early-life seizures commonly precede human temporal lobe epilepsy (TLE), suggesting that transcriptional dysregulation of HCNs might contribute to the epileptogenic process. Therefore, we determined whether HCN isoform expression was modified in hippocampi of individuals with TLE. HCN1 and HCN2 expression were measured using in situ hybridization and immunocytochemistry in hippocampi from three groups: TLE with hippocampal sclerosis (HS; n = 17), epileptic hippocampi without HS, or non-HS (NHS; n = 10), and autopsy material (n = 10). The results obtained in chronic human epilepsy were validated by examining hippocampi from the pilocarpine model of chronic TLE. In autopsy and most NHS hippocampi, HCN1 mRNA expression was substantial in pyramidal cell layers and lower in dentate gyrus granule cells (GCs). In contrast, HCN1 mRNA expression over the GC layer and in individual GCs from epileptic hippocampus was markedly increased once GC neuronal density was reduced by >50%. HCN1 mRNA changes were accompanied by enhanced immunoreactivity in the GC dendritic fields and more modest changes in HCN2 mRNA expression. Furthermore, similar robust and isoform-selective augmentation of HCN1 mRNA expression was evident also in the pilocarpine animal model of TLE. These findings indicate that the expression of HCN isoforms is dynamically regulated in human as well as in experimental hippocampal epilepsy. After experimental febrile seizures (i.e., early in the epileptogenic process), the preserved and augmented inhibition onto principal cells may lead to reduced HCN1 expression. In contrast, in chronic epileptic HS hippocampus studied here, the profound loss of interneuronal and principal cell populations and consequent reduced inhibition, coupled with increased dendritic excitation of surviving GCs, might provoke a "compensatory" enhancement of HCN1 mRNA and protein expression

Localization of HCN1 channels to presynaptic compartments: novel plasticity that may contribute to hippocampal maturation.

Increasing evidence supports roles for the current mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, I(h), in hippocampal maturation and specifically in the evolving changes of intrinsic properties as well as network responses of hippocampal neurons. Here, we describe a novel developmental plasticity of HCN channel expression in axonal and presynaptic compartments: HCN1 channels were localized to axon terminals of the perforant path (the major hippocampal afferent pathway) of immature rats, where they modulated synaptic efficacy. However, presynaptic expression and functions of the channels disappeared with maturation. This was a result of altered channel transport to the axons, because HCN1 mRNA and protein levels in entorhinal cortex neurons, where the perforant path axons originate, were stable through adulthood. Blocking action potential firing in vitro increased presynaptic expression of HCN1 channels in the perforant path, suggesting that network activity contributed to regulating this expression. These findings support a novel developmentally regulated axonal transport of functional ion channels and suggest a role for HCN1 channel-mediated presynaptic I(h) in hippocampal maturation