Cortical RORß is required for layer 4 transcriptional identity and barrel integrity.

Retinoic acid-related orphan receptor beta (RORß) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find RORß is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age in wildtype mice. Loss of RORß delays excitatory input and disrupts gene expression and chromatin accessibility, with down-regulation of L4 and up-regulation of L5 genes, suggesting a disruption in cellular specification. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of RORß, Thsd7a, is down-regulated without RORß, and Thsd7a knock-out alone disrupts TCA organization in adult barrels.

A Mammalian enhancer trap resource for discovering and manipulating neuronal cell types.

There is a continuing need for driver strains to enable cell-type-specific manipulation in the nervous system. Each cell type expresses a unique set of genes, and recapitulating expression of marker genes by BAC transgenesis or knock-in has generated useful transgenic mouse lines. However, since genes are often expressed in many cell types, many of these lines have relatively broad expression patterns. We report an alternative transgenic approach capturing distal enhancers for more focused expression. We identified an enhancer trap probe often producing restricted reporter expression and developed efficient enhancer trap screening with the PiggyBac transposon. We established more than 200 lines and found many lines that label small subsets of neurons in brain substructures, including known and novel cell types. Images and other information about each line are available online (enhancertrap.bio.brandeis.edu).

Downregulation of dendritic HCN channel gating in epilepsy is mediated by altered phosphorylation signaling.

The onset of spontaneous seizures in the pilocarpine model of epilepsy causes a hyperpolarized shift in the voltage-dependent activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated current (Ih) in CA1 hippocampal pyramidal neuron dendrites, contributing to neuronal hyperexcitability and possibly to epileptogenesis. However, the specific mechanisms by which spontaneous seizures cause downregulation of HCN channel gating are yet unknown. We asked whether the seizure-dependent downregulation of HCN channel gating was due to altered phosphorylation signaling mediated by the phosphatase calcineurin (CaN) or the kinase p38 mitogen-activated protein kinase (p38 MAPK). We first found that CaN inhibition upregulated HCN channel gating and reduced neuronal excitability under normal conditions, showing that CaN is a strong modulator of HCN channels. We then found that an in vitro model of seizures (1 h in 0 Mg2+ and 50 microM bicuculline at 35-37 degrees C) reproduced the HCN channel gating change seen in vivo. Pharmacological inhibition of CaN or activation of p38 MAPK partially reversed the in vitro seizure-induced hyperpolarized shift in HCN channel gating, and the shift was fully reversed by the combination of CaN inhibition and p38 MAPK activation. We then demonstrated enhanced CaN activity as well as reduced p38 MAPK activity in vivo in the CA1 hippocampal area of chronically epileptic animals. Pharmacological reversal of these phosphorylation changes restored HCN channel gating downregulation and neuronal hyperexcitability in epileptic tissue to control levels. Together, these results suggest that alteration of two different phosphorylation pathways in epilepsy contributes to the downregulation of HCN channel gating, which consequently produces neuronal hyperexcitability and thus may be a target for novel antiepileptic therapies.

Reversed somatodendritic I(h) gradient in a class of rat hippocampal neurons with pyramidal morphology.

In CA1 and neocortical pyramidal neurons, I(h) is present primarily in the dendrites. We asked if all neurons of a pyramidal morphology have a similar density of I(h). We characterized a novel class of hippocampal neurons with pyramidal morphology found in the stratum radiatum, which we termed the 'pyramidal-like principal' (PLP) neuron. Morphological similarities to pyramidal neurons were verified by filling the neurons with biocytin. PLPs did not stain for markers associated with interneurons, and projected to both the septum and olfactory bulb. By using cell-attached patch-clamp recordings, we found that these neurons expressed a high density of I(h) in the soma that declined to a lower density in the dendrites, a pattern that is reversed compared to pyramidal neurons. The voltage-dependent activation and activation time constants of I(h) in the PLPs were similar to pyramidal neurons. Whole-cell patch-clamp recordings from the soma and dendrites of PLP neurons showed no significant differences in input resistance and local temporal summation between the two locations. Blockade of I(h) by ZD7288 increased the input resistance and temporal summation of simulated EPSPs, as in pyramidal neurons. When NMDA receptors were blocked, temporal summation at the soma of distal synaptic potentials was similar to that seen with current injections at the soma, suggesting a 'normalization' of temporal summation similar to that observed in pyramidal neurons. Thus, we have characterized a principal neuronal subtype in the hippocampus with a similar morphology but reversed I(h) somatodendritic gradient to that previously observed in CA1 hippocampal and neocortical pyramidal neurons.

Modulation of h-channels in hippocampal pyramidal neurons by p38 mitogen-activated protein kinase.

Hyperpolarization-activated cyclic nucleotide-gated ion channels (h-channels; I(h); HCN) modulate intrinsic excitability in hippocampal and neocortical pyramidal neurons, among others. Whereas I(h) mediated by the HCN2 isoform is regulated by cAMP, there is little known about kinase modulation of I(h), especially for the HCN1 isoform predominant in pyramidal neurons. We used a computational method to identify a novel kinase modulator of h-channels, p38 mitogen-activated protein kinase (p38 MAPK). Inhibition of p38 MAPK in hippocampal pyramidal neurons caused a approximately 25 mV hyperpolarization of I(h) voltage-dependent activation. This downregulation of I(h) produced hyperpolarization of resting potential, along with increased input resistance and temporal summation of excitatory inputs. Activation of p38 MAPK caused a approximately 11 mV depolarizing shift in I(h) activation, along with depolarized resting potential, and decreased input resistance and temporal summation. Inhibition of related MAPKs, ERK1/2 (extracellular signal-related kinase 1/2) and JNK (c-Jun N-terminal kinase), produced no effect on I(h). These results show that p38 MAPK is a strong modulator of h-channel biophysical properties and may deserve additional exploration as a link between altered I(h) and pathological conditions such as epilepsy.