Mir324 knockout regulates the structure of dendritic spines and impairs hippocampal long-term potentiation.

MicroRNAs are an emerging class of synaptic regulators. These small noncoding RNAs post-transcriptionally regulate gene expression, thereby altering neuronal pathways and shaping cell-to-cell communication. Their ability to rapidly alter gene expression and target multiple pathways makes them interesting candidates in the study of synaptic plasticity. Here, we demonstrate that the proconvulsive microRNA miR-324-5p regulates excitatory synapse structure and function in the hippocampus of mice. Both Mir324 knockout (KO) and miR-324-5p antagomir treatment significantly reduce dendritic spine density in the hippocampal CA1 subregion, and Mir324 KO, but not miR-324-5p antagomir treatment, shift dendritic spine morphology, reducing the proportion of thin, "unstable" spines. Western blot and quantitative Real-Time PCR revealed changes in protein and mRNA levels for potassium channels, cytoskeletal components, and synaptic markers, including MAP2 and Kv4.2, which are important for long-term potentiation (LTP). In line with these findings, slice electrophysiology revealed that LTP is severely impaired in Mir324 KO mice, while neurotransmitter release probability remains unchanged. Overall, this study demonstrates that miR-324-5p regulates dendritic spine density, morphology, and plasticity in the hippocampus, potentially via multiple cytoskeletal and synaptic modulators.

Impaired dendritic spike generation in the Fragile X prefrontal cortex is due to loss of dendritic sodium channels.

Patients with Fragile X syndrome, the leading monogenetic cause of autism, suffer from impairments related to the prefrontal cortex, including working memory and attention. Synaptic inputs to the distal dendrites of layer 5 pyramidal neurons in the prefrontal cortex have a weak influence on the somatic membrane potential. To overcome this filtering, distal inputs are transformed into local dendritic Na+ spikes, which propagate to the soma and trigger action potential output. Layer 5 extratelencephalic (ET) prefrontal cortex (PFC) neurons project to the brainstem and various thalamic nuclei and are therefore well positioned to integrate task-relevant sensory signals and guide motor actions. We used current clamp and outside-out patch clamp recording to investigate dendritic spike generation in ET neurons from male wild-type and Fmr1 knockout (FX) mice. The threshold for dendritic spikes was more depolarized in FX neurons compared to wild-type. Analysis of voltage responses to simulated in vivo 'noisy' current injections showed that a larger dendritic input stimulus was required to elicit dendritic spikes in FX ET dendrites compared to wild-type. Patch clamp recordings revealed that the dendritic Na+ conductance was significantly smaller in FX ET dendrites. Taken together, our results suggest that the generation of Na+ -dependent dendritic spikes is impaired in ET neurons of the PFC in FX mice. Considering our prior findings that somatic D-type K+ and dendritic hyperpolarization-activated cyclic nucleotide-gated-channel function is reduced in ET neurons, we suggest that dendritic integration by PFC circuits is fundamentally altered in Fragile X syndrome. KEY POINTS: Dendritic spike threshold is depolarized in layer 5 prefrontal cortex neurons in Fmr1 knockout (FX) mice. Simultaneous somatic and dendritic recording with white noise current injections revealed that larger dendritic stimuli were required to elicit dendritic spikes in FX extratelencephalic (ET) neurons. Outside-out patch clamp recording revealed that dendritic sodium conductance density was lower in FX ET neurons.

Ionic and morphological contributions to the variable gain of membrane responses in layer 2/3 pyramidal neurons of mouse primary visual cortex.

Many neuronal cell types exhibit a sliding scale of neuronal excitability in the subthreshold voltage range. This is due to a variable contribution of different voltage-gated ion channels, leading to scaling of input resistance (RN) as a function of membrane potential (Vm) and a voltage-dependent dynamic gain of neuronal responsiveness. In layer 2/3 pyramidal neurons within the primary visual cortex (V1), this response influences sensory processing by tightening neuronal tuning to preferred orientations, but the identity of the ionic conductances involved remains unknown. Here, we used in vitro physiological recordings in acute slices to identify the contributions of several voltage-dependent conductances to the dynamic gain of membrane responses in layer 2/3 pyramidal neurons in mouse primary visual cortex. We found that the steep voltage dependence of input resistance in these cells was mediated in part by a combination of persistent sodium, inwardly rectifying potassium, and hyperpolarization-activated nonselective cation channels. In addition, the steepness of the slope of the RN/Vm relationship was inversely correlated with the number of branches on the proximal apical dendrite. These data have uncovered physiological and morphological factors that underlie the scaling of membrane responses in L2/3 neurons of rodent V1. Regulation of these channels would serve as a mechanism of real-time neuromodulation of neuronal processing of sensory information.NEW & NOTEWORTHY Layer 2/3 pyramidal neurons in primary visual cortex scale subthreshold voltage responses with resting membrane potential because RN increases as Vm is depolarized. Here, we uncovered the voltage-dependent contributions of NaP, Kir, and HCN conductances toward this behavior, and we additionally demonstrated that the strength of the RN/Vm relationship is inversely correlated with proximal branching along the apical dendrite.

Altered A-type potassium channel function impairs dendritic spike initiation and temporoammonic long-term potentiation in Fragile X syndrome.

Fragile X syndrome (FXS) is the leading monogenetic cause of cognitive impairment and autism spectrum disorder. Area CA1 of the hippocampus receives current information about the external world from the entorhinal cortex via the temporoammonic (TA) pathway. Given its role in learning and memory, it is surprising that little is known about TA long-term potentiation (TA-LTP) in FXS. We found that TA-LTP was impaired in male fmr1 KO mice. Although there were no significant differences in basal synaptic transmission, synaptically evoked dendritic calcium signals were smaller in KO neurons. Using dendritic recording, we found no difference in complex spikes or pharmacologically isolated Ca2+ spikes; however, the threshold for fast, Na+ dependent dendritic spikes was depolarized in fmr1 KO mice. Cell-attached patch clamp recordings found no difference in Na+ channels between wild type and fmr1 KO CA1 dendrites. Dendritic spike threshold and TA-LTP were restored by block of A-type K+ channels with either 150 µM Ba2+ or the more specific toxin AmmTx3. The impairment of TA-LTP shown here, coupled with previously described enhanced Schaffer collateral LTP, may contribute to spatial memory alterations in FXS. Furthermore, as both of these LTP phenotypes are attributed to changes in A-type K+ channels in FXS, our findings provide a potential therapeutic target to treat cognitive impairments in FXS.SIGNIFICANCE STATEMENTAlterations in synaptic function and plasticity are likely contributors to learning and memory impairments in many neurological disorders. Fragile X syndrome is marked by dysfunctional learning and memory and changes in synaptic structure and function. This study shows a lack of LTP at temporoammonic synapses in CA1 neurons associated with biophysical differences in A-type K+ channels in fmr1 KO CA1 neurons. Our results, along with previous findings on A-type K+ channel effects on Schaffer collateral LTP, reveal differential effects of a single ion channelopathy on LTP at the two major excitatory pathways of CA1 pyramidal neurons. These findings expand our understanding of memory deficits in FXS and provide a potential therapeutic target for the treatment of memory dysfunction in FXS.

High and low expression of the hyperpolarization activated current (Ih ) in mouse CA1 stratum oriens interneurons.

Inhibitory interneurons are among the most diverse cell types in the brain; the hippocampus itself contains more than 28 different inhibitory interneurons. Interneurons are typically classified using a combination of physiological, morphological, and biochemical observations. One broad separator is action potential firing: low threshold, regular spiking versus higher threshold, fast spiking. We found that spike frequency adaptation (SFA) was highly heterogeneous in low threshold interneurons in the mouse stratum oriens region of area CA1. Analysis with a k-means clustering algorithm parsed the data set into two distinct clusters based on a constellation of physiological parameters and reliably sorted strong and weak SFA cells into different groups. Interneurons with strong SFA fired fewer action potentials across a range of current inputs and had lower input resistance compared to cells with weak SFA. Strong SFA cells also had higher sag and rebound in response to hyperpolarizing current injections. Morphological analysis shows no difference between the two cell types and the cell types did not segregate along the dorsal-ventral axis of the hippocampus. Strong and weak SFA cells were labeled in hippocampal slices from SST:cre Ai14 mice suggesting both cells express somatostatin. Voltage-clamp recordings showed hyperpolarization activated current Ih was significantly larger in strong SFA cells compared to weak SFA cells. We suggest that the strong SFA cell represents a previously uncharacterized type of CA1 stratum oriens interneuron. Due to the combination of physiological parameters of these cells, we will refer to them as Low Threshold High Ih (LTH) cells.

Fragile X mental retardation protein modulates somatic D-type K+ channels and action potential threshold in the mouse prefrontal cortex.

Axo-somatic K+ channels control action potential output in part by acting in concert with voltage-gated Na+ channels to set action potential threshold. Slowly inactivating, D-type K+ channels are enriched at the axo-somatic region of cortical pyramidal neurons of the prefrontal cortex, where they regulate action potential firing. We previously demonstrated that D-type K+ channels are downregulated in extratelencephalic-projecting (ET) L5 neurons in the medial prefrontal cortex (mPFC) of the Fmr1-knockout mouse model of fragile X syndrome (FX mice), resulting in a hyperpolarized action potential threshold. To test whether K+ channel alterations are regulated in a cell-autonomous manner in FXS, we used a virus-mediated approach to restore expression of fragile X mental retardation protein (FMRP) in a small population of prefrontal neurons in male FX mice. Outside-out voltage-clamp recordings revealed a higher D-type K+ conductance in FMRP-positive ET neurons compared with nearby FMRP-negative ET neurons. FMRP did not affect either rapidly inactivating A-type or noninactivating K+ conductance. ET neuron patches recorded with FMRP1-298, a truncated form of FMRP that lacks mRNA binding domains, included in the pipette solution had larger D-type K+ conductance compared with heat-inactivated controls. Viral expression of FMRP in FX mice depolarized action potential threshold to near-wild-type levels in ET neurons. These results suggest that FMRP influences the excitability of ET neurons in the mPFC by regulating somatic D-type K+ channels in a cell-autonomous, protein-protein-dependent manner.NEW & NOTEWORTHY We demonstrate that fragile X mental retardation protein (FMRP), which is absent in fragile X syndrome (FXS), regulates D-type potassium channels in prefrontal cortex L5 pyramidal neurons with subcerebral projections but not in neighboring pyramidal neurons without subcerebral projections. FMRP regulates D-type potassium channels in a protein-protein-dependent manner and rescues action potential threshold in a mouse model of FXS. These findings have implications for how changes in voltage-gated channels contribute to neurodevelopmental disorders.

Fragile X Mental Retardation Protein Bidirectionally Controls Dendritic Ih in a Cell Type-Specific Manner between Mouse Hippocampus and Prefrontal Cortex.

Channelopathies are implicated in Fragile X syndrome (FXS), yet the dysfunction of a particular ion channel varies with cell type. We previously showed that HCN channel function is elevated in CA1 dendrites of the fmr1-/y mouse model of FXS, but reduced in L5 PFC dendrites. Using male mice, we tested whether Fragile X Mental Retardation Protein (FMRPO), the protein whose absence causes FXS, differentially modulates HCN channels in CA1 versus L5 PFC dendrites. Using a combination of viral tools, intracellular peptide, and dendritic electrophysiology, we found that FMRP regulates HCN channels via a cell-autonomous protein-protein interaction. Virally expressed FMRP restored WT HCN channel-related dendritic properties in both CA1 and L5 neurons. Rapid intracellular perfusion of the non-mRNA binding N-terminal fragment, FMRP1-298, similarly restored dendritic function. In support of a protein-protein interaction, we found that FMRP associated with HCN-TRIP8b complexes in both hippocampus and PFC. Finally, voltage-clamp recordings showed that FMRP modulated Ih by regulating the number of functional dendritic HCN channels rather than individual channel properties. Together, these represent three novel findings as to the nature of the changes in dendritic function in CA1 and PFC neurons based on the presence or absence of FMRP. Moreover, our findings provide evidence that FMRP can regulate its targets in opposite directions depending upon the cellular milieu.SIGNIFICANCE STATEMENT Changes in dendritic function, and voltage-gated ion channels in particular, are increasingly the focus of neurological disorders. We, and others, previously identified cell type-specific channelopathies in a mouse of model of Fragile X syndrome. The present study shows that replacing Fragile X Mental Retardation Protein, which is absent in Fragile X syndrome, in adult CA1 and L5 PFC neurons regulates the number of functional dendritic HCN channels in a cell type-specific manner. These results suggest that Fragile X Mental Retardation Protein regulates dendritic HCN channels via a cell-autonomous protein--protein mechanism.

MicroRNA inhibition upregulates hippocampal A-type potassium current and reduces seizure frequency in a mouse model of epilepsy.

Epilepsy is often associated with altered expression or function of ion channels. One example of such a channelopathy is the reduction of A-type potassium currents in the hippocampal CA1 region. The underlying mechanisms of reduced A-type channel function in epilepsy are unclear. Here, we show that inhibiting a single microRNA, miR-324-5p, which targets the pore-forming A-type potassium channel subunit Kv4.2, selectively increased A-type potassium currents in hippocampal CA1 pyramidal neurons in mice. Resting membrane potential, input resistance and other potassium currents were not altered. In a mouse model of acquired chronic epilepsy, inhibition of miR-324-5p reduced the frequency of spontaneous seizures and interictal epileptiform spikes supporting the physiological relevance of miR-324-5p-mediated control of A-type currents in regulating neuronal excitability. Mechanistic analyses demonstrated that microRNA-induced silencing of Kv4.2 mRNA is increased in epileptic mice leading to reduced Kv4.2 protein levels, which is mitigated by miR-324-5p inhibition. By contrast, other targets of miR-324-5p were unchanged. These results suggest a selective miR-324-5p-dependent mechanism in epilepsy regulating potassium channel function, hyperexcitability and seizures.

D-type potassium channels normalize action potential firing between dorsal and ventral CA1 neurons of the mouse hippocampus.

Specific memory processes and neurological disorders can be ascribed to different dorsoventral regions of the hippocampus. Recently, differences in the anatomical and physiological properties between dorsal and ventral hippocampal CA1 neurons were described for both the rat and mouse hippocampus and have greatly contributed to our understanding of these processes. While differences in the subthreshold properties were similar between rat and mouse neurons, differences in action potential output between dorsal and ventral neurons were strikingly less divergent in mouse compared with rat CA1 neurons. Here, we investigate the mechanism underlying the lack of difference in action potential firing between dorsal and ventral CA1 pyramidal neurons in mouse hippocampus. Consistent with rat, we found that ventral CA1 neurons had a more depolarized resting membrane potential and higher input resistance than dorsal CA1 neurons in the mouse hippocampus. Despite these differences, action potential output in response to current injection was not significantly different. We found that ventral neurons have a more depolarized action potential threshold compared with dorsal neurons and that threshold in ventral neurons was more sensitive to block of KV1 channels compared with dorsal neurons. Outside-out voltage-clamp recordings found that slowly inactivating K+ currents were larger in ventral CA1 neurons. These results suggest that, despite differences in subthreshold properties between dorsal and ventral CA1 neurons, action potential output is normalized by the differential functional expression of D-type K+ channels. NEW & NOTEWORTHY Understanding differences in neurons within a brain region is integral in the reliable interpretation of comparative studies. Our findings identify a novel mechanism by which D-type potassium channels normalize action potential firing between dorsal and ventral CA1 neurons of mouse hippocampus despite differences in subthreshold intrinsic properties. Action potential threshold in ventral neurons is influenced by a greater functional expression of D-type potassium channels resulting in a depolarized action potential threshold compared with dorsal hippocampus.

Increased transient Na+ conductance and action potential output in layer 2/3 prefrontal cortex neurons of the fmr1-/y mouse.

KEY POINTS: Layer 2/3 neurons of the prefrontal cortex display higher gain of somatic excitability, responding with a higher number of action potentials for a given stimulus, in fmr1-/y mice. In fmr1-/y L2/3 neurons, action potentials are taller, faster and narrower. Outside-out patch clamp recordings revealed that the maximum Na+ conductance density is higher in fmr1-/y L2/3 neurons. Measurements of three biophysically distinct K+ currents revealed a depolarizing shift in the activation of a rapidly inactivating (A-type) K+ conductance. Realistic neuronal simulations of the biophysical observations recapitulated the elevated action potential and repetitive firing phenotype. ABSTRACT: Fragile X syndrome is the most common form of inherited mental impairment and autism. The prefrontal cortex is responsible for higher order cognitive processing, and prefrontal dysfunction is believed to underlie many of the cognitive and behavioural phenotypes associated with fragile X syndrome. We recently demonstrated that somatic and dendritic excitability of layer (L) 5 pyramidal neurons in the prefrontal cortex of the fmr1-/y mouse is significantly altered due to changes in several voltage-gated ion channels. In addition to L5 pyramidal neurons, L2/3 pyramidal neurons play an important role in prefrontal circuitry, integrating inputs from both lower brain regions and the contralateral cortex. Using whole-cell current clamp recording, we found that L2/3 pyramidal neurons in prefrontal cortex of fmr1-/y mouse fired more action potentials for a given stimulus compared with wild-type neurons. In addition, action potentials in fmr1-/y neurons were significantly larger, faster and narrower. Voltage clamp of outside-out patches from L2/3 neurons revealed that the transient Na+ current was significantly larger in fmr1-/y neurons. Furthermore, the activation curve of somatic A-type K+ current was depolarized. Realistic conductance-based simulations revealed that these biophysical changes in Na+ and K+ channel function could reliably reproduce the observed increase in action potential firing and altered action potential waveform. These results, in conjunction with our prior findings on L5 neurons, suggest that principal neurons in the circuitry of the medial prefrontal cortex are altered in distinct ways in the fmr1-/y mouse and may contribute to dysfunctional prefrontal cortex processing in fragile X syndrome.

Cell-Type Specific Channelopathies in the Prefrontal Cortex of the fmr1-/y Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is caused by transcriptional silencing of the fmr1 gene resulting in the loss of fragile X mental retardation protein (FMRP) expression. FXS patients display several behavioral phenotypes associated with prefrontal cortex (PFC) dysfunction. Voltage-gated ion channels, some of which are regulated by FMRP, heavily influence PFC neuron function. Although there is evidence for brain region-specific alterations to the function a single type of ion channel in FXS, it is unclear whether subtypes of principal neurons within a brain region are affected uniformly. We tested for alterations to ion channels critical in regulating neural excitability in two subtypes of prefrontal L5 pyramidal neurons. Using somatic and dendritic patch-clamp recordings, we provide evidence that the functional expression of h-channels (Ih) is down-regulated, whereas A-type K(+) channel function is up-regulated in pyramidal tract-projecting (PT) neurons in the fmr1-/y mouse PFC. This is the opposite pattern of results from published findings from hippocampus where Ih is up-regulated and A-type K(+) channel function is down-regulated. Additionally, we find that somatic Kv1-mediated current is down-regulated, resulting in increased excitability of fmr1-/y PT neurons. Importantly, these h- and K(+) channel differences do not extend to neighboring intratelencephalic-projecting neurons. Thus, the absence of FMRP has divergent effects on the function of individual types of ion channels not only between brain regions, but also variable effects across cell types within the same brain region. Given the importance of ion channels in regulating neural circuits, these results suggest cell-type-specific phenotypes for the disease.

Rapamycin reveals an mTOR-independent repression of Kv1.1 expression during epileptogenesis.

Changes in ion channel expression are implicated in the etiology of epilepsy. However, the molecular leading to long-term aberrant expression of ion channels are not well understood. The mechanistic/mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that mediates activity-dependent protein synthesis in neurons. mTOR is overactive in epilepsy, suggesting that excessive protein synthesis may contribute to the neuronal pathology. In contrast, we found that mTOR activity and the microRNA miR-129-5p reduce the expression of the voltage-gated potassium channel Kv1.1 in an animal model of temporal lobe epilepsy (TLE). When mTOR activity is low, Kv1.1 expression is high and the frequency of behavioral seizures is low. However, as behavioral seizure activity rises, mTOR activity increases and Kv1.1 protein levels drop. In CA1 pyramidal neurons, the reduction in Kv1.1 lowers the threshold for action potential firing. Interestingly, blocking mTOR activity with rapamycin reduces behavioral seizures and temporarily keeps Kv1.1 levels elevated. Overtime, seizure activity increases and Kv1.1 protein decreases in all animals, even those treated with rapamycin. Notably, the concentration of miR-129-5p, the negative regulator of Kv1.1 mRNA translation, increases by 21days post-status epilepticus (SE), sustaining Kv1.1 mRNA translational repression. Our results suggest that following kainic-acid induced status epilepticus there are two phases of Kv1.1 repression: (1) an initial mTOR-dependent repression of Kv1.1 that is followed by (2) a miR-129-5p persistent reduction of Kv1.1.

Channelopathies and dendritic dysfunction in fragile X syndrome.

Dendritic spine abnormalities and the metabotropic glutamate receptor theory put the focus squarely on synapses and protein synthesis as the cellular locus of fragile X syndrome. Synapses however, are only partly responsible for information processing in neuronal networks. Neurotransmitter triggered excitatory postsynaptic potentials (EPSPs) are shaped and integrated by dendritic voltage-gated ion channels. These EPSPs, and in some cases the resultant dendritic spikes, are further modified by dendritic voltage-gated ion channels as they propagate to the soma. If the resultant somatic depolarization is large enough, action potential(s) will be triggered and propagate both orthodromically down the axon, where it may trigger neurotransmitter release, and antidromically back into the dendritic tree, where it can activate and modify dendritic voltage-gated and receptor activated ion channels. Several channelopathies, both soma-dendritic (L-type calcium channels, Slack potassium channels, h-channels, A-type potassium channels) and axo-somatic (BK channels and delayed rectifier potassium channels) were identified in the fmr1-/y mouse model of fragile X syndrome. Pathological function of these channels will strongly influence the excitability of individual neurons as well as overall network function. In this chapter we discuss the role of voltage-gated ion channels in neuronal processing and describe how identified channelopathies in models of fragile X syndrome may play a role in dendritic pathophysiology.

Loss of functional A-type potassium channels in the dendrites of CA1 pyramidal neurons from a mouse model of fragile X syndrome.

Despite the critical importance of voltage-gated ion channels in neurons, very little is known about their functional properties in Fragile X syndrome: the most common form of inherited cognitive impairment. Using three complementary approaches, we investigated the physiological role of A-type K(+) currents (I(KA)) in hippocampal CA1 pyramidal neurons from fmr1-/y mice. Direct measurement of I(KA) using cell-attached patch-clamp recordings revealed that there was significantly less I(KA) in the dendrites of CA1 neurons from fmr1-/y mice. Interestingly, the midpoint of activation for A-type K(+) channels was hyperpolarized for fmr1-/y neurons compared with wild-type, which might partially compensate for the lower current density. Because of the rapid time course for recovery from steady-state inactivation, the dendritic A-type K(+) current in CA1 neurons from both wild-type and fmr1-/y mice is likely mediated by K(V)4 containing channels. The net effect of the differences in I(KA) was that back-propagating action potentials had larger amplitudes producing greater calcium influx in the distal dendrites of fmr1-/y neurons. Furthermore, CA1 pyramidal neurons from fmr1-/y mice had a lower threshold for LTP induction. These data suggest that loss of I(KA) in hippocampal neurons may contribute to dendritic pathophysiology in Fragile X syndrome.

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.

Impaired dendritic expression and plasticity of h-channels in the fmr1(-/y) mouse model of fragile X syndrome.

Despite extensive research into both synaptic and morphological changes, surprisingly little is known about dendritic function in fragile X syndrome (FXS). We found that the dendritic input resistance of CA1 neurons was significantly lower in fmr1(-/y) versus wild-type mice. Consistent with elevated dendritic I(h), voltage sag, rebound, and resonance frequency were significantly higher and temporal summation was lower in the dendrites of fmr1(-/y) mice. Dendritic expression of the h-channel subunit HCN1, but not HCN2, was higher in the CA1 region of fmr1(-/y) mice. Interestingly, whereas mGluR-mediated persistent decreases in I(h) occurred in both wildtype and fmr1(-/y) mice, persistent increases in I(h) that occurred after LTP induction in wild-type mice were absent in fmr1(-/y) mice. Thus, chronic upregulation of dendritic I(h) in conjunction with impairment of homeostatic h-channel plasticity represents a dendritic channelopathy in this model of mental retardation and may provide a mechanism for the cognitive impairment associated with FXS.

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.

Plasticity of intrinsic excitability during long-term depression is mediated through mGluR-dependent changes in I(h) in hippocampal CA1 pyramidal neurons.

Bidirectional changes in synaptic strength are the proposed cellular correlate for information storage in the brain. Plasticity of intrinsic excitability, however, may also be critical for regulating the firing of neurons during mnemonic tasks. We demonstrated previously that the induction long-term potentiation was accompanied by a persistent decrease in CA1 pyramidal neuron excitability (Fan et al., 2005). We show here that induction of long-term depression (LTD) by 3 Hz pairing of back-propagating action potentials with Schaffer collateral EPSPs was accompanied by an overall increase in CA1 neuronal excitability. This increase was observed as an increase in the number of action potentials elicited by somatic current injection and was caused by an increase in neuronal input resistance. After LTD, voltage sag during hyperpolarizing current injections and subthreshold resonance frequency were decreased. All changes were blocked by ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride), suggesting that a physiological loss of dendritic h-channels was responsible for the increase in excitability. Furthermore, block of group 1 metabotropic glutamate receptors (mGluRs) or protein kinase C prevented the increase in excitability, whereas the group 1 mGluR agonist DHPG [(RS)-3,5-dihydroxyphenylglycine] mimicked the effects. We conclude that 3 Hz synaptic stimulation downregulates I(h) via activation of group 1 mGluRs and subsequent stimulation of protein kinase C. We propose these changes as part of a homeostatic and bidirectional control mechanism for intrinsic excitability during learning.

Activity-dependent decrease of excitability in rat hippocampal neurons through increases in I(h).

Hippocampal long-term potentiation (LTP) induced by theta-burst pairing of Schaffer collateral inputs and postsynaptic firing is associated with localized increases in synaptic strength and dendritic excitability. Using the same protocol, we now demonstrate a decrease in cellular excitability that was blocked by the h-channel blocker ZD7288. This decrease was also induced by postsynaptic theta-burst firing alone, yet it was blocked by NMDA receptor antagonists, postsynaptic Ca2+ chelation, low concentrations of tetrodotoxin, omega-conotoxin MVIIC, calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors and a protein synthesis inhibitor. Increasing network activity with high extracellular K+ caused a similar reduction of cellular excitability and an increase in h-channel HCN1 protein. We propose that backpropagating action potentials open glutamate-bound NMDA receptors, resulting in an increase in I(h) and a decrease in overall excitability. The occurrence of such a reduction in cellular excitability in parallel with synaptic potentiation would be a negative feedback mechanism to normalize neuronal output firing and thus promote network stability.

Endocannabinoid signaling dynamics probed with optical tools.

Intercellular signaling dynamics critically influence the functional roles that the signals can play. Small lipids are synthesized and released from neurons, acting as intercellular signals in regulating neurotransmitter release, modulating ion channels on target cells, and modifying synaptic plasticity. The repertoire of biological effects of lipids such as endocannabinoids (eCBs) is rapidly expanding, yet lipid signaling dynamics have not been studied. The eCB system constitutes a powerful tool for bioassaying the dynamics of lipid signaling. The eCBs are synthesized in, and released from, postsynaptic somatodendritic domains that are readily accessible to whole-cell patch electrodes. The dramatic effects of these lipid signals are detected electrophysiologically as CB1-dependent alterations in conventional synaptic transmission, which therefore serve as a sensitive reporter of eCB actions. We used electrophysiological recording, photolytic release of caged glutamate and a newly developed caged AEA (anandamide), together with rapid [Ca2+]i measurements, to investigate the dynamics of retrograde eCB signaling between CA1 pyramidal cells and GABAergic synapses in rat hippocampus in vitro. We show that, at 22 degrees C, eCB synthesis and release must occur within 75-190 ms after the initiating stimulus, almost an order of magnitude faster than previously thought. At 37 degrees C, the time could be < 50 ms. Activation of CB1 and downstream processes constitute a significant fraction of the total delay and are identified as major rate-limiting steps in retrograde signaling. Our findings imply that lipid messenger dynamics are comparable with those of metabotropic neurotransmitters and can modulate neuronal interactions on a similarly fast time scale.