Rachel D. Groth

Estradiol Activates Group I and II Metabotropic Glutamate Receptor Signaling, Leading to Opposing Influences on cAMP Response Element-Binding Protein

In addition to mediating sexual maturation and reproduction through stimulation of classical intracellular receptors that bind DNA and regulate gene expression, estradiol is also thought to influence various brain functions by acting on receptors localized to the neuronal membrane surface. Many intracellular signaling pathways and modulatory proteins are affected by estradiol via this unconventional route, including regulation of the transcription factor cAMP response element-binding protein (CREB). However, the mechanisms by which estradiol acts at the membrane surface are poorly understood. Because both estradiol and CREB have been implicated in regulating learning and memory, we characterized the effects of estradiol on this transcription factor in cultured rat hippocampal neurons. Within minutes of administration, estradiol triggered mitogen-activated protein kinase (MAPK)-dependent CREB phosphorylation in unstimulated neurons. Furthermore, after brief depolarization, estradiol attenuated L-type calcium channel-mediated CREB phosphorylation. Thus, estradiol exhibited both positive and negative influences on CREB activity. These effects of estradiol were sex specific and traced to membrane-localized estrogen receptors that stimulated group I and II metabotropic glutamate receptor (mGluR) signaling. Activation of estrogen receptor α (ERα) led to mGluR1a signaling, triggering CREB phosphorylation through phospholipase C regulation of MAPK. In addition, estradiol stimulation of ERα or ERβ triggered mGluR2/3 signaling, decreasing L-type calcium channel-mediated CREB phosphorylation. These results not only characterize estradiol regulation of CREB but also provide two putative signaling mechanisms that may account for many of the unexplained observations regarding the influence of estradiol on nervous system function.

CaV1 and CaV2 Channels Engage Distinct Modes of Ca2+ Signaling to Control CREB-Dependent Gene Expression

Activity-dependent gene expression triggered by Ca(2+) entry into neurons is critical for learning and memory, but whether specific sources of Ca(2+) act distinctly or merely supply Ca(2+) to a common pool remains uncertain. Here, we report that both signaling modes coexist and pertain to Ca(V)1 and Ca(V)2 channels, respectively, coupling membrane depolarization to CREB phosphorylation and gene expression. Ca(V)1 channels are advantaged in their voltage-dependent gating and use nanodomain Ca(2+) to drive local CaMKII aggregation and trigger communication with the nucleus. In contrast, Ca(V)2 channels must elevate [Ca(2+)](i) microns away and promote CaMKII aggregation at Ca(V)1 channels. Consequently, Ca(V)2 channels are ~10-fold less effective in signaling to the nucleus than are Ca(V)1 channels for the same bulk [Ca(2+)](i) increase. Furthermore, Ca(V)2-mediated Ca(2+) rises are preferentially curbed by uptake into the endoplasmic reticulum and mitochondria. This source-biased buffering limits the spatial spread of Ca(2+), further attenuating Ca(V)2-mediated gene expression.

Neurotrophin activation of NFAT‐dependent transcription contributes to the regulation of pro‐nociceptive genes

Nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) play key roles in the development of inflammation‐induced hyperalgesia by triggering the expression of pro‐nociceptive genes within primary afferent and spinal neurons. However, the mechanisms by which neurotrophins elicit gene expression remain largely unknown. Recently, neurotrophins have been shown to activate members of the calcineurin (CaN)‐regulated, nuclear factor of activated T‐cells (NFATc) family of transcription factors within brain. Thus, we hypothesized that NFATc transcription factors couple neurotrophin signaling to gene expression within primary afferent and spinal neurons. In situ hybridization revealed NFATc4 mRNA within the dorsal root ganglion and spinal cord. In cultured dorsal root ganglion cells, NGF triggered NFAT‐dependent transcription in a CaN‐sensitive manner. Further, increased BDNF expression following NGF treatment relied on CaN, thereby suggesting that NGF regulates BDNF transcription via activation of NFATc4. Within cultured spinal cells, BDNF also activated CaN‐dependent, NFAT‐regulated gene expression. Interestingly, BDNF stimulation increased the expression of the pro‐nociceptive genes cyclooxygenase‐2, neurokinin‐1 receptor, inositol trisphosphates receptor type 1, and BDNF itself, through both NFAT‐dependent and NFAT‐independent transcriptional mechanisms. Our results suggest that regulation of pro‐nociceptive genes through activation of NFAT‐dependent transcription is one mechanism by which NGF and BDNF signaling contributes to the development of persistent pain states.

Postsynaptic GluA1 enables acute retrograde enhancement of presynaptic function to coordinate adaptation to synaptic inactivity

Prolonged blockade of AMPA-type glutamate receptors in hippocampal neuron cultures leads to homeostatic enhancements of pre- and postsynaptic function that appear correlated at individual synapses, suggesting some form of transsynaptic coordination. The respective modifications are important for overall synaptic strength but their interrelationship, dynamics, and molecular underpinnings are unclear. Here we demonstrate that adaptation begins postsynaptically but is ultimately communicated to presynaptic terminals and expressed as an accelerated turnover of synaptic vesicles. Critical postsynaptic modifications occur over hours, but enable retrograde communication within minutes once AMPA receptor (AMPAR) blockade is removed, causing elevation of both spontaneous and evoked vesicle fusion. The retrograde signaling does not require spiking activity and can be interrupted by NBQX, philanthotoxin, postsynaptic BAPTA, or external sequestration of BDNF, consistent with the acute release of retrograde messenger, triggered by postsynaptic Ca2+ elevation via Ca2+-permeable AMPARs.

β Ca2+/CaM-dependent kinase type II triggers upregulation of GluA1 to coordinate adaptation to synaptic inactivity in hippocampal neurons

Prolonged AMPA-receptor blockade in hippocampal neuron cultures leads to both an increased expression of GluA1 postsynaptically and an increase in vesicle pool size and turnover rate presynaptically, adaptive changes that extend beyond simple synaptic scaling. As a molecular correlate, expression of the β Ca2+/CaM-dependent kinase type II (βCaMKII) is increased in response to synaptic inactivity. Here we set out to clarify the role of βCaMKII in the various manifestations of adaptation. Knockdown of βCaMKII by lentiviral-mediated expression of shRNA prevented the synaptic inactivity-induced increase in GluA1, as did treatment with the CaM kinase inhibitor KN-93, but not the inactive analog KN-92. These results demonstrate that, spurred by AMPA-receptor blockade, up-regulation of βCaMKII promotes increased GluA1 expression. Indeed, transfection of βCaMKII, but not a kinase-dead mutant, increased GluA1 expression on dendrites and elevated vesicle turnover (Syt-Ab uptake), mimicking the effect of synaptic inactivity on both sides of the synapse. In cells with elevated βCaMKII, relief of synaptic-activity blockade uncovered an increase in the frequency of miniature excitatory postsynaptic currents that could be rapidly and fully suppressed by PhTx blockade of GluA1 receptors. This increased mini frequency involved a genuine presynaptic enhancement, not merely an increased abundance of synapses. This finding suggests that Ca2+ flux through GluA1 receptors may trigger the acute release of a retrograde messenger. Taken together, our results indicate that synaptic inactivity-induced increases in βCaMKII expression set in motion a series of events that culminate in coordinated pre- and postsynaptic adaptations in synaptic transmission.

Excitation–transcription coupling in sympathetic neurons and the molecular mechanism of its initiation

In excitable cells, membrane depolarization and activation of voltage-gated Ca²+ (Ca(V)) channels trigger numerous cellular responses, including muscle contraction, secretion, and gene expression. Yet, while the mechanisms underlying excitation-contraction and excitation-secretion coupling have been extensively characterized, how neuronal activity is coupled to gene expression has remained more elusive. In this article, we will discuss recent progress toward understanding the relationship between patterns of channel activity driven by membrane depolarization and activation of the nuclear transcription factor CREB. We show that signaling strength is steeply dependent on membrane depolarization and is more sensitive to the open probability of Ca(V) channels than the Ca²+ entry itself. Furthermore, our data indicate that by decoding Ca(V) channel activity, CaMKII (a Ca²+/calmodulin-dependent protein kinase) links membrane excitation to activation of CREB in the nucleus. Together, these results revealed some interesting and unexpected similarities between excitation-transcription coupling and other forms of excitation-response coupling.

D1 dopamine receptor activation of NFAT-mediated striatal gene expression

Exposure to drugs of abuse activates gene expression and protein synthesis that result in long‐lasting adaptations in striatal signaling. Therefore, identification of the transcription factors that couple drug exposure to gene expression is of particular importance. Members of the nuclear factor of activated T‐cells (NFATc) family of transcription factors have recently been implicated in shaping neuronal function throughout the rodent nervous system. Here we demonstrate that regulation of NFAT‐mediated gene expression may also be a factor in drug‐induced changes to striatal functioning. In cultured rat striatal neurons, stimulation of D1 dopamine receptors induces NFAT‐dependent transcription through activation of L‐type calcium channels. Additionally, the genes encoding inositol‐1,4,5‐trisphosphate receptor type 1 and glutamate receptor subunit 2 are regulated by striatal NFATc4 activity. Consistent with these in‐vitro data, repeated exposure to cocaine triggers striatal NFATc4 nuclear translocation and the up‐regulation of inositol‐1,4,5‐trisphosphate receptor type 1 and glutamate receptor subunit 2 gene expression in vivo, suggesting that cocaine‐induced increases in gene expression may be partially mediated through activation of NFAT‐dependent transcription. Collectively, these findings reveal a novel molecular pathway that may contribute to the enduring modifications in striatal functioning that occur following the administration of drugs of abuse.

Substance P initiates NFAT‐dependent gene expression in spinal neurons

Persistent hyperalgesia is associated with increased expression of proteins that contribute to enhanced excitability of spinal neurons, however, little is known about how expression of these proteins is regulated. We tested the hypothesis that Substance P stimulation of neurokinin receptors on spinal neurons activates the transcription factor nuclear factor of activated T cells isoform 4 (NFATc4). The occurrence of NFATc4 in spinal cord was demonstrated with RT‐PCR and immunocytochemistry. Substance P activated NFAT‐dependent gene transcription in primary cultures of neonatal rat spinal cord transiently transfected with a luciferase DNA reporter construct. The effect of Substance P was mediated by neuronal neurokinin‐1 receptors that coupled to activation of protein kinase C, l‐type voltage‐dependent calcium channels, and calcineurin. Interestingly, Substance P had no effect on cyclic AMP response element (CRE)‐dependent gene expression. Conversely, calcitonin gene‐related peptide, which activated CRE‐dependent gene expression, did not activate NFAT signaling. These data provide evidence that peptides released from primary afferent neurons regulate discrete patterns of gene expression in spinal neurons. Because the release of Substance P and calcitonin gene‐related peptide from primary afferent neurons is increased following peripheral injury, these peptides may differentially regulate the expression of proteins that underlie persistent hyperalgesia.

Immunolocalization of NFATc4 in the adult mouse brain

NFATc4 has recently been identified as playing an important role in variety of activity‐dependent neuronal processes, including hippocampal plasticity, axonal growth, neuronal survival, and apoptosis. However, a systematic study examining the distribution of NFATc4 within the nervous system has yet to be conducted. With this in mind, we sought to determine the regional localization of NFATc4 within the adult mouse brain. Interestingly, NFATc4 was expressed broadly, but not uniformly, throughout various brain structures. The highest levels of NFATc4 expression were localized to the hippocampus, olfactory bulb, and various hypothalamic nuclei. Other brain regions that expressed NFATc4 included the cerebellum, striatum, globus pallidus, amygdala, neocortex, and brainstem nuclei. Given NFATc4s widespread expression, these results are consistent with the notion that NFATc4 may underlie activity‐dependent neuronal plasticity throughout the adult brain.

A Role for Retinoic Acid in Homeostatic Plasticity

Prolonged changes in neuronal activity trigger compensatory modifications in synaptic function to restore firing rates to normal levels. In this issue of Neuron, Aoto et al. demonstrate that synthesis of retinoic acid offsets chronic network inactivity by increasing synaptic strength through upregulation of GluR1 receptors.