Glucagon-like peptide-1 receptor differentially controls mossy cell activity across the dentate gyrus longitudinal axis.

Understanding the role of dentate gyrus (DG) mossy cells (MCs) in learning and memory has rapidly evolved due to increasingly precise methods for targeting MCs and for in vivo recording and activity manipulation in rodents. These studies have shown MCs are highly active in vivo, strongly remap to contextual manipulation, and that their inhibition or hyperactivation impairs pattern separation and location or context discrimination. Less well understood is how MC activity is modulated by neurohormonal mechanisms, which might differentially control the participation of MCs in cognitive functions during discrete states, such as hunger or satiety. In this study, we demonstrate that glucagon-like peptide-1 (GLP-1), a neuropeptide produced in the gut and the brain that regulates food consumption and hippocampal-dependent mnemonic function, might regulate MC function through expression of its receptor, GLP-1R. RNA-seq demonstrated that most, though not all, Glp1r in hippocampal principal neurons is expressed in MCs, and in situ hybridization revealed strong expression of Glp1r in hilar neurons. Glp1r-ires-Cre mice crossed with Ai14D reporter mice followed by co-labeling for the MC marker GluR2/3 revealed that almost all MCs in the ventral DG expressed Glp1r and that almost all Glp1r-expressing hilar neurons were MCs. However, only ~60% of dorsal DG MCs expressed Glp1r, and Glp1r was also expressed in small hilar neurons that were not MCs. Consistent with this expression pattern, peripheral administration of the GLP-1R agonist exendin-4 (5 µg/kg) increased cFos expression in ventral but not dorsal DG hilar neurons. Finally, whole-cell patch-clamp recordings from ventral MCs showed that bath application of exendin-4 (200 nM) depolarized MCs and increased action potential firing. Taken together, this study adds to known MC activity modulators a neurohormonal mechanism that may preferentially affect ventral DG physiology and may potentially be targetable by several GLP-1R pharmacotherapies already in clinical use.

Hippocampal volume and hippocampal neuron density, number and size in schizophrenia: a systematic review and meta-analysis of postmortem studies.

Reduced hippocampal volume is a consistent finding in neuroimaging studies of individuals with schizophrenia. While these studies have the advantage of large-sample sizes, they are unable to quantify the cellular basis of structural or functional changes. In contrast, postmortem studies are well suited to explore subfield and cellular alterations, but low sample sizes and subject heterogeneity impede establishment of statistically significant differences. Here we use a meta-analytic approach to synthesize the extant literature of hippocampal subfield volume and cellular composition in schizophrenia patients and healthy control subjects. Following pre-registration (PROSPERO CRD42019138280), PubMed, Web of Science, and PsycINFO were searched using the term: (schizophrenia OR schizoaffective) AND (post-mortem OR postmortem) AND hippocampus. Subjects were adult men and women with schizophrenia or schizoaffective disorder or non-psychiatric control subjects, and key outcomes, stratified by hippocampal hemisphere and subfield, were volume, neuron number, neuron density, and neuron size. A random effects meta-analysis was performed. Thirty-two studies were included (413 patients, 415 controls). In patients, volume and neuron number were significantly reduced in multiple hippocampal subfields in left, but not right hippocampus, whereas neuron density was not significantly different in any hippocampal subfield. Neuron size, averaged bilaterally, was also significantly reduced in all calculated subfields. Heterogeneity was minimal to moderate, with rare evidence of publication bias. Meta-regression of age and illness duration did not explain heterogeneity of total hippocampal volume effect sizes. These results extend neuroimaging findings of smaller hippocampal volume in schizophrenia patients and further our understanding of regional and cellular neuropathology in schizophrenia.

Reduction of thalamic and cortical Ih by deletion of TRIP8b produces a mouse model of human absence epilepsy.

Absence seizures occur in several types of human epilepsy and result from widespread, synchronous feedback between the cortex and thalamus that produces brief episodes of loss of consciousness. Genetic rodent models have been invaluable for investigating the pathophysiological basis of these seizures. Here, we identify tetratricopeptide-containing Rab8b-interacting protein (TRIP8b) knockout mice as a new model of absence epilepsy, featuring spontaneous spike-wave discharges on electroencephalography (EEG) that are the electrographic hallmark of absence seizures. TRIP8b is an auxiliary subunit of the hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels, which have previously been implicated in the pathogenesis of absence seizures. In contrast to mice lacking the pore-forming HCN channel subunit HCN2, TRIP8b knockout mice exhibited normal cardiac and motor function and a less severe seizure phenotype. Evaluating the circuit that underlies absence seizures, we found that TRIP8b knockout mice had significantly reduced HCN channel expression and function in thalamic-projecting cortical layer 5b neurons and thalamic relay neurons, but preserved function in inhibitory neurons of the reticular thalamic nucleus. Our results expand the known roles of TRIP8b and provide new insight into the region-specific functions of TRIP8b and HCN channels in constraining cortico-thalamo-cortical excitability.

Association of Cigarette Smoking With Interpersonal and Self-Directed Violence in a Large Community-Based Sample.

INTRODUCTION: Substance use is a major risk factor for various forms of violence, yet how cigarette smoking influences violence outcomes is incompletely understood. We investigated associations between cigarette smoking and three types of violence in a large, nationally representative, community-based sample. METHODS: Adult subjects participating in both Wave 1 (2001-2002; N = 43 093) and Wave 2 (2004-2005; N = 34 653) of the National Epidemiological Survey on Alcohol and Related Conditions (NESARC) were stratified by daily cigarette smoking status at Wave 1, and individuals with unchanged smoking status between waves were analyzed (nonsmokers [consisting of never and former daily smokers]: N = 22 529; daily smokers: N = 7442). We created composites of other- and self-directed violence and victimization occurring between Waves 1 and 2, and performed logistic regression models, controlling for psychiatric diagnoses, alcohol and substance use, and relevant demographic covariates. RESULTS: Daily smokers at Wave 1 were 2.1 (95% CI: 1.5-3.0), 2.5 (2.1-2.9), and 1.7 (1.5-2.1) times more likely than nonsmokers to report self-directed violence, other-directed violence, or victimization between Waves 1 and 2, respectively. Former daily smokers were significantly less likely to report other-directed violence than individuals who were never daily smokers. CONCLUSIONS: Daily cigarette smoking is temporally associated with multiple forms of violence compared to never and former cigarette smokers, even when common covariates associated with violence are controlled. Smoking status should be carefully controlled for in studies designed to identify risk factors for violence, and may be a useful component of violence risk assessment. IMPLICATIONS: The findings suggest that cigarette smoking status should be carefully and systematically controlled for in studies of violence risk factors. The findings also support further investigation of the utility of cigarette smoking status for violence risk assessment, and whether smoking cessation strategies mitigate violence risk.

Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels.

Ion channels operate in intact tissues as part of large macromolecular complexes that can include cytoskeletal proteins, scaffolding proteins, signaling molecules, and a litany of other molecules. The proteins that make up these complexes can influence the trafficking, localization, and biophysical properties of the channel. TRIP8b (tetratricopetide repeat-containing Rab8b-interacting protein) is a recently discovered accessory subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that contributes to the substantial dendritic localization of HCN channels in many types of neurons. TRIP8b interacts with the carboxyl-terminal region of HCN channels and regulates their cell-surface expression level and cyclic nucleotide dependence. Here we examine the molecular determinants of TRIP8b binding to HCN2 channels. Using a single-molecule fluorescence bleaching method, we found that TRIP8b and HCN2 form an obligate 4:4 complex in intact channels. Fluorescence-detection size-exclusion chromatography and fluorescence anisotropy allowed us to confirm that two different domains in the carboxyl-terminal portion of TRIP8b--the tetratricopepide repeat region and the TRIP8b conserved region--interact with two different regions of the HCN carboxyl-terminal region: the carboxyl-terminal three amino acids (SNL) and the cyclic nucleotide-binding domain, respectively. And finally, using X-ray crystallography, we determined the atomic structure of the tetratricopepide region of TRIP8b in complex with a peptide of the carboxy-terminus of HCN2. Together, these experiments begin to uncover the mechanism for TRIP8b binding and regulation of HCN channels.

TRIP8b-independent trafficking and plasticity of adult cortical presynaptic HCN1 channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are subthreshold activated voltage-gated ion channels. In the cortex, these channels are predominantly expressed in dendrites where they significantly modify dendritic intrinsic excitability as well synaptic potential shapes and integration. HCN channel trafficking to dendrites is regulated by the protein, TRIP8b. Additionally, altered TRIP8b expression may be one mechanism underlying seizure-induced dendritic HCN channel plasticity. HCN channels, though, are also located in certain mature cortical synaptic terminals, where they play a vital role in modulating synaptic transmission. In this study, using electrophysiological recordings as well as electron microscopy we show that presynaptic, but not dendritic, cortical HCN channel expression and function is comparable in adult TRIP8b-null mice and wild-type littermates. We further investigated whether presynaptic HCN channels undergo seizure-dependent plasticity. We found that, like dendritic channels, wild-type presynaptic HCN channel function was persistently decreased following induction of kainic acid-induced seizures. Since TRIP8b does not affect presynaptic HCN subunit trafficking, seizure-dependent plasticity of these cortical HCN channels is not conditional upon TRIP8b. Our results, thus, suggest that the molecular mechanisms underlying HCN subunit targeting, expression and plasticity in adult neurons is compartment selective, providing a means by which pre- and postsynaptic processes that are critically dependent upon HCN channel function may be distinctly influenced.

An essential role for modulation of hyperpolarization-activated current in the development of binaural temporal precision.

In sensory circuits of the brain, developmental changes in the expression and modulation of voltage-gated ion channels are a common occurrence, but such changes are often difficult to assign to clear functional roles. We have explored this issue in the binaural neurons of the medial superior olive (MSO), whose temporal precision in detecting the coincidence of binaural inputs dictates the resolution of azimuthal sound localization. We show that in MSO principal neurons of gerbils during the first week of hearing, a hyperpolarization-activated current (I(h)) progressively undergoes a 13-fold increase in maximal conductance, a >10-fold acceleration of kinetics, and, most surprisingly, a 30 mV depolarizing shift in the voltage dependence of activation. This period is associated with an upregulation of the hyperpolarization-activated and cyclic nucleotide-gated (HCN) channel subunits HCN1, HCN2, and HCN4 in the MSO, but only HCN1 and HCN4 were expressed strongly in principal neurons. I(h) recorded in nucleated patches from electrophysiologically mature MSO neurons (>P18) exhibited kinetics and an activation range nearly identical to the I(h) found in whole-cell recordings before hearing onset. These results indicate that the developmental changes in I(h) in MSO neurons can be explained predominantly by modulation from diffusible intracellular factors, and not changes in channel subunit composition. The exceptionally large modulatory changes in I(h), together with refinements in synaptic properties transform the coding strategy from one of summation and integration to the submillisecond coincidence detection known to be required for transmission of sound localization cues.

Dorsoventral differences in intrinsic properties in developing CA1 pyramidal cells.

The dorsoventral and developmental gradients of entorhinal layer II cell grid properties correlate with their resonance properties and with their hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel current characteristics. We investigated whether such correlation existed in rat hippocampal CA1 pyramidal cells, where place fields also show spatial and temporal gradients. Resonance was absent during the first postnatal week, and emerged during the second week. Resonance was stronger in dorsal than ventral cells, in accord with HCN current properties. Resonance responded to cAMP in ventral but not in dorsal cells. The dorsoventral distribution of HCN1 and HCN2 subunits and of the auxiliary protein tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) could account for these differences between dorsal and ventral cells. The analogous distribution of the intrinsic properties of entorhinal stellate and hippocampal cells suggests the existence of general rules of organization among structures that process complementary features of the environment.

Differential dorso-ventral distributions of Kv4.2 and HCN proteins confer distinct integrative properties to hippocampal CA1 pyramidal cell distal dendrites.

The dorsal and ventral regions of the hippocampus perform different functions. Whether the integrative properties of hippocampal cells reflect this heterogeneity is unknown. We focused on dendrites where most synaptic input integration takes place. We report enhanced backpropagation and theta resonance and decreased summation of synaptic inputs in ventral versus dorsal CA1 pyramidal cell distal dendrites. Transcriptional Kv4.2 down-regulation and post-transcriptional hyperpolarization-activated cyclic AMP-gated channel (HCN1/2) up-regulation may underlie these differences, respectively. Our results reveal differential dendritic integrative properties along the dorso-ventral axis, reflecting diverse computational needs.

Short- and long-term plasticity in CA1 neurons from mice lacking h-channel auxiliary subunit TRIP8b.

Hyperpolarization-activated cyclic nucleotide-gated nonselective cation channels (HCN or h-channels) are important regulators of neuronal physiology contributing to passive membrane properties, such as resting membrane potential and input resistance (R(N)), and to intrinsic oscillatory activity and synaptic integration. The correct membrane targeting of h-channels is regulated in part by the auxiliary h-channel protein TRIP8b. The genetic deletion of TRIP8b results in a loss of functional h-channels, which affects the postsynaptic integrative properties of neurons. We investigated the impact of TRIP8b deletion on long-term potentiation (LTP) at the two major excitatory inputs to CA1 pyramidal neurons: Schaffer collateral (SC) and perforant path (PP). We found that SC LTP was not significantly different between neurons from wild-type and TRIP8b-knockout mice. There was, however, significantly more short-term potentiation in knockout neurons. We also found that the persistent increase in h-current (I(h)) that normally occurs after LTP induction was absent in knockout neurons. The lack of I(h) plasticity was not restricted to activity-dependent induction, because the depletion of intracellular calcium stores also failed to produce the expected increase in I(h). Interestingly, pairing of SC and PP inputs resulted in a form of LTP in knockout neurons that did not occur in wild-type neurons. These results suggest that the physiological impact of TRIP8b deletion is not restricted to the integrative properties of neurons but also includes both synaptic and intrinsic plasticity.

Narrowing the translational research gap by aligning replication concepts in basic and clinical neuroscience.

Replicability and reproducibility are widely considered to be cornerstones of valid scientific research. Yet, the elements of replication in fundamental neuroscience studies do not fully overlap with the process of replication in clinical neuroscience involving patients. Here we discuss how better aligning the concept of replication across this translational spectrum might enhance the rate at which basic findings in the organization and function of the nervous system are leveraged to develop new treatments for psychiatric and neurological disorders.

Effects of repeated developmental GLP-1R agonist exposure on young adult behavior and hippocampal structure in mice.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are common type 2 diabetes medications that have been repurposed for adult chronic weight management. Clinical trials suggest this class may also be beneficial for obesity in pediatric populations. Since several GLP-1R agonists cross the blood-brain barrier, it is important to understand how postnatal developmental exposure to GLP-1R agonists might affect brain structure and function later in life. Toward that end, we systemically treated male and female C57BL/6 mice with the GLP-1R agonist exendin-4 (0.5 mg/kg, twice daily) or saline from postnatal day 14 to 21, then allowed uninterrupted development to young adulthood. Beginning at 7 weeks of age, we performed open field and marble burying tests to assess motor behavior and the spontaneous location recognition (SLR) task to assess hippocampal-dependent pattern separation and memory. Mice were sacrificed, and we counted ventral hippocampal mossy cells, as we have recently shown that most murine hippocampal neuronal GLP-1R is expressed in this cell population. We found that GLP-1R agonist treatment did not alter P14-P21 weight gain, but modestly reduced young adult open field distance traveled and marble burying. Despite these motor changes, there was no effect on SLR memory performance or time spent investigating objects. Finally, we did not detect any changes in ventral mossy cell number using two different markers. These data suggest developmental exposure to GLP-1R agonists might have specific rather than global effects on behavior later in life and that extensive additional study is necessary to clarify how drug timing and dose affect distinct constellations of behavior in young adulthood.

Effects of repeated developmental GLP-1R agonist exposure on adult behavior and hippocampal structure in mice.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are common type 2 diabetes medications that have been repurposed for adult chronic weight management. Clinical trials suggest this class may also be beneficial for obesity in pediatric populations. Since several GLP-1R agonists cross the blood-brain barrier, it is important to understand how postnatal developmental exposure to GLP-1R agonists might affect brain structure and function in adulthood. Toward that end, we systemically treated male and female C57BL/6 mice with the GLP-1R agonist exendin-4 (0.5 mg/kg, twice daily) or saline from postnatal day 14 to 21, then allowed uninterrupted development to adulthood. Beginning at 7 weeks of age, we performed open field and marble burying tests to assess motor behavior and the spontaneous location recognition (SLR) task to assess hippocampal-dependent pattern separation and memory. Mice were sacrificed, and we counted ventral hippocampal mossy cells, as we have recently shown that most murine hippocampal neuronal GLP-1R is expressed in this cell population. We found that GLP-1R agonist treatment did not alter P14-P21 weight gain, but modestly reduced adult open field distance traveled and marble burying. Despite these motor changes, there was no effect on SLR memory performance or time spent investigating objects. Finally, we did not detect any changes in ventral mossy cell number using two different markers. These data suggest developmental exposure to GLP-1R agonists might have specific rather than global effects on behavior later in life and that extensive additional study is necessary to clarify how drug timing and dose affect distinct constellations of behavior in adulthood.

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.

Trafficking and gating of hyperpolarization-activated cyclic nucleotide-gated channels are regulated by interaction with tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) and cyclic AMP at distinct sites.

Ion channel trafficking and gating are often influenced by interactions with auxiliary subunits. Tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) is an auxiliary subunit for neuronal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. TRIP8b interacts directly with two distinct sites of HCN channel pore-forming subunits to control channel trafficking and gating. Here we use mutagenesis combined with electrophysiological studies to define and distinguish the functional importance of the HCN/TRIP8b interaction sites. Interaction with the last three amino acids of the HCN1 C terminus governed the effect of TRIP8b on channel trafficking, whereas TRIP8b interaction with the HCN1 cyclic nucleotide binding domain (CNBD) affected trafficking and gating. Biochemical studies revealed that direct interaction between TRIP8b and the HCN1 CNBD was disrupted by cAMP and that TRIP8b binding to the CNBD required an arginine residue also necessary for cAMP binding. In accord, increasing cAMP levels in cells antagonized the up-regulation of HCN1 channels mediated by a TRIP8b construct binding the CNBD exclusively. These data illustrate the distinct roles of the two TRIP8b-HCN interaction domains and suggest that TRIP8b and cAMP may directly compete for binding the HCN CNBD to control HCN channel gating, kinetics, and trafficking.

HCN channels in behavior and neurological disease: too hyper or not active enough?

The roles of cells within the nervous system are based on their properties of excitability, which are in part governed by voltage-gated ion channels. HCN channels underlie the hyperpolarization-activated current, I(h), an important regulator of excitability and rhythmicity through control of basic membrane properties. I(h) is present in multiple neuronal types and regions of the central nervous system, and changes in I(h) alter cellular input-output properties and neuronal circuitry important for behavior such as learning and memory. Furthermore, the pathophysiology of neurological diseases of both the central and peripheral nervous system involves defects in excitability, rhythmicity, and signaling, and animal models of many of these disorders have implicated changes in HCN channels and I(h) as critical for pathogenesis. In this review, we focus on recent research elucidating the role of HCN channels and I(h) in behavior and disease. These studies have utilized knockout mice as well as animal models of disease to examine how I(h) may be important in regulating learning and memory, sleep, and consciousness, as well as how misregulation of I(h) may contribute to epilepsy, chronic pain, and other neurological disorders. This review will help guide future studies aimed at further understanding the function of this unique conductance in both health and disease of the mammalian brain.

Toward Standardized Guidelines for Investigating Neural Circuit Control of Behavior in Animal Research.

With the advent of tools for recording and manipulating activity with high spatiotemporal resolution in defined neural circuits in behaving animals, behavioral neuroscience is now tasked with establishing field-wide standards for implementing and interpreting these powerful approaches. Theoretical frameworks for what constitute proof of fundamental neurobiological principles is an ongoing and frequently debated topic. On the other hand, standardizing interpretation of individual experimental findings to avoid spurious conclusions in practice has received less attention. Even within subfields, similar assays are often used to support widely disparate conclusions which in part has contributed to a slew of studies claiming highly specified functions for cell types and circuits which are often in direct disagreement with one another. In this opinion piece, we discuss common pitfalls in design and interpretation of approaches for recording or manipulating neural activity in animal models of motivated behavior. We emphasize the importance of integrating findings across multiple behavioral assays concomitant with tempered inference regarding specialized neuronal functions as a standardized starting point for parsing circuit control of behavior. Our aim is to stimulate an open and accessible discourse in the literature to address issues of continuity across behavioral neurosciences.

Modeling intrahippocampal effects of anterior hippocampal hyperactivity relevant to schizophrenia using chemogenetic excitation of long axis-projecting mossy cells in the mouse dentate gyrus.

BACKGROUND: The anterior hippocampus of individuals with early psychosis or schizophrenia is hyperactive, as is the ventral hippocampus in many rodent models for schizophrenia risk. Mossy cells (MCs) of the ventral dentate gyrus (DG) densely project in the hippocampal long axis, targeting both dorsal DG granule cells and inhibitory interneurons. Mossy cells are responsive to stimulation throughout hippocampal subfields, and thus may be suited to detect hyperactivity in areas where it originates such as CA1. Here we tested the hypothesis that hyperactivation of ventral MCs activates dorsal DG granule cells to influence dorsal hippocampal function. METHODS: In CD-1 mice, we targeted dorsal DG-projecting ventral MCs using an adeno-associated virus intersectional strategy. In vivo fiber photometry recording of ventral MCs was performed during exploratory behaviors. We used excitatory chemogenetic constructs to test the effects of ventral MC hyperactivation on long-term spatial memory during an object location memory task. RESULTS: Photometry revealed ventral MCs were activated during exploratory rearing. Ventral MCs made functional monosynaptic inputs to dorsal DG granule cells, and chemogenetic activation of ventral MCs modestly increased activity of dorsal DG granule cells measured by c-Fos. Finally, chemogenetic activation of ventral MCs during the training phase of an object location memory task impaired test performance 24 hours later, without effects on locomotion or object exploration. CONCLUSIONS: These data suggest that ventral MC activation can directly excite dorsal granule cells and interfere with dorsal DG function, supporting future study of their in vivo activity in animal models for schizophrenia featuring ventral hyperactivity.

Alternatively spliced isoforms of TRIP8b differentially control h channel trafficking and function.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (h channels) are the molecular basis for the current, I(h), which contributes crucially to intrinsic neuronal excitability. The subcellular localization and biophysical properties of h channels govern their function, but the mechanisms controlling these characteristics, and especially the potential role of auxiliary subunits or other binding proteins, remain unclear. We focused on TRIP8b, an h channel-interacting protein that colocalizes with HCN1 in cortical and hippocampal pyramidal neuron dendrites, and found that it exists in multiple alternative splice variants with distinct effects on h channel trafficking and function. The developmentally regulated splice variants of TRIP8b all shared dual, C terminus-located interaction sites with HCN1. When coexpressed with HCN1 in heterologous cells individual TRIP8b isoforms similarly modulated gating of I(h), causing a hyperpolarizing shift in voltage dependence of channel activation, but differentially upregulated or downregulated I(h) current density and HCN1 surface expression. In hippocampal neurons, coexpression of TRIP8b isoforms with HCN1 produced isoform-specific changes of HCN1 localization. Interestingly, the TRIP8b isoforms most abundant in the brain are those predicted to enhance h channel surface expression. Indeed, shRNA knockdown of TRIP8b in hippocampal neurons significantly reduced native I(h). Thus, although TRIP8b exists in multiple splice isoforms, our data suggest that the predominant role of this protein in brain is to promote h channel surface expression and enhance I(h). Because I(h) expression is altered in models of several diseases, including temporal lobe epilepsy, TRIP8b may play a role in both normal neuronal function and in aberrant neuronal excitability associated with neurological disease.