The role of school-based health centers in providing long-active reversible contraceptive care to adolescents in New York City.

Long-acting reversible contraceptives (LARCs) are effective contraceptive methods for adolescents. This study describes the initiation and continuation of LARC care to adolescents at school-based health centers (SBHCs) during the COVID-19 pandemic. Participants received contraceptive care in New York City SBHCs from April 2021-June 2022. LARC initiation, LARC discontinuation, and total contraceptive visits were measured monthly. During the study period, the SBHCs provided 1,303 contraceptive visits, including 77 LARC initiations. Among LARC initiations, six-month continuation probability was 79.3 % (95 %CI: 69.0-91.1). SBHCs play an important role in providing adolescents contraceptive services, particularly LARC care, when other health care systems are disrupted.

Family members' perceptions of older person discharge from emergency departments.

BACKGROUND: People aged over 64 years account for approximately 20% of adult emergency presentations, with up to 60% of people discharged home from emergency departments (EDs). Many older people discharged home are supported by family. OBJECTIVES: The objective of this study was to explore the family members' perspectives of older people's discharge from ED to inform new alternative or innovative models of care. METHODS: The design was a descriptive exploratory study. A convenience sample of family members was recruited from three EDs across Sydney, New South Wales. Telephone interviews were conducted over a six-month period and data were analysed using statistics or thematic analysis. RESULTS: Interviews were conducted with 133 family members of whom the majority were female (n = 80, 60%) with a median age of 70 years (IQR 91-35). Over 87% of family members were satisfied with ED care and discharge processes that were provided to the older person. The majority (n = 129, 97%) of family members reported that they understood the treatment and perceived that the older person's condition was well managed (n = 119, 86%). The majority (n = 114, 86%) of family members reported being informed of the medical diagnosis and were confident (87%, n = 115) to continue care of the older person at home. Three themes emerged from qualitative data: (a) a sense of time-moving through ED; (b) giving voice to the impact of clinician communication; and (c) the delivery of comfort and basic care. DISCUSSION: Family members reported that they were engaged in and satisfied with the older person's ED treatment and discharge. However, family members suggested that there was opportunity to improve communication consistency for ED discharge and managing the wait. CONCLUSION: Clinicians need to engage with family members to optimise quality and safety. Clinicians need to understand that family members considered comfort and fundamentals of care to be an important dimension of the older person's ED management plan.

Hybridization Liquid Chromatography-Tandem Mass Spectrometry: An Alternative Bioanalytical Method for Antisense Oligonucleotide Quantitation in Plasma and Tissue Samples.

Quantitative bioanalysis in plasma and tissues samples is required to study the pharmacokinetic and pharmacodynamic properties of antisense oligonucleotides (ASOs). To overcome intrinsic drawbacks in specificity, sensitivity, and throughput of traditional ligand-binding assay (LBA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, an alternative bioanalytical method was developed by combining oligonucleotide hybridization and LC-MS/MS technologies. Target ASOs were extracted from biological samples by hybridization with biotinylated sense-strand oligonucleotides coupled to streptavidin magnetic beads. Using ion-pairing chromatography and tandem mass spectrometry, this method demonstrated high sensitivity (0.5 ng/mL using 100 µL of plasma), high specificity, wide linear range, complete automation, and generic applications in tests with multiple ASOs. The typical challenge of sensitivity drop in traditional ion-pairing LC-MS/MS was for the first time overcome by the introduction of a ternary pump system. Due to the high specificity, quantitation in various biological matrixes was achieved using calibration standards in plasma, largely improving efficiency and consistency. Another major advantage was the capability of simultaneous quantitation of ASO metabolites. The hybridization LC-MS/MS was considered an improved alternative for quantitation of ASOs and metabolites in plasma and tissue samples, showing a great potential to replace traditional LBA and LC-MS/MS methods.

γCaMKII shuttles Ca²âº/CaM to the nucleus to trigger CREB phosphorylation and gene expression.

Activity-dependent CREB phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at CaV1 channels triggers these events, but how information is relayed onward to the nucleus remains unclear. Here, we report a mechanism that mediates long-distance communication within cells: a shuttle that transports Ca(2+)/calmodulin from the surface membrane to the nucleus. We show that the shuttle protein is γCaMKII, its phosphorylation at Thr287 by ßCaMKII protects the Ca(2+)/CaM signal, and CaN triggers its nuclear translocation. Both ßCaMKII and CaN act in close proximity to CaV1 channels, supporting their dominance, whereas γCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca(2+)/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves long-standing puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of CaV1, γCaMKII, ßCaMKII, and CaN in multiple neuropsychiatric disorders.

CaV1.2 calcium channels: just cut out to be regulated?

Tight regulation of calcium entry through the L-type calcium channel CaV1.2 ensures optimal excitation-response coupling. In this issue of Neuron, Michailidis et al. (2014) demonstrate that CaV1.2 activity triggers negative feedback regulation through proteolytic cleavage of the channel within the core of the pore-forming subunit.

Ca(V)1 and Ca(V)2 channels engage distinct modes of Ca(2+) 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.

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.

Beta 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 ß Ca(2+)/CaM-dependent kinase type II (ßCaMKII) is increased in response to synaptic inactivity. Here we set out to clarify the role of ßCaMKII in the various manifestations of adaptation. Knockdown of ßCaMKII 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 ßCaMKII promotes increased GluA1 expression. Indeed, transfection of ßCaMKII, 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 ßCaMKII, 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 Ca(2+) flux through GluA1 receptors may trigger the acute release of a retrograde messenger. Taken together, our results indicate that synaptic inactivity-induced increases in ßCaMKII expression set in motion a series of events that culminate in coordinated pre- and postsynaptic adaptations in synaptic transmission.

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 Ca(2+) elevation via Ca(2+)-permeable AMPARs.

CaMKII locally encodes L-type channel activity to signal to nuclear CREB in excitation-transcription coupling.

Communication between cell surface proteins and the nucleus is integral to many cellular adaptations. In the case of ion channels in excitable cells, the dynamics of signaling to the nucleus are particularly important because the natural stimulus, surface membrane depolarization, is rapidly pulsatile. To better understand excitation-transcription coupling we characterized the dependence of cAMP response element-binding protein phosphorylation, a critical step in neuronal plasticity, on the level and duration of membrane depolarization. We find that signaling strength is steeply dependent on depolarization, with sensitivity far greater than hitherto recognized. In contrast, graded blockade of the Ca(2+) channel pore has a remarkably mild effect, although some Ca(2+) entry is absolutely required. Our data indicate that Ca(2+)/CaM-dependent protein kinase II acting near the channel couples local Ca(2+) rises to signal transduction, encoding the frequency of Ca(2+) channel openings rather than integrated Ca(2+) flux-a form of digital logic.

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.

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.

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.

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 NFATc4's widespread expression, these results are consistent with the notion that NFATc4 may underlie activity-dependent neuronal plasticity throughout the adult brain.

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 alpha (ERalpha) led to mGluR1a signaling, triggering CREB phosphorylation through phospholipase C regulation of MAPK. In addition, estradiol stimulation of ERalpha or ERbeta 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.

Calcineurin regulation of neuronal plasticity.

From the most basic of nervous systems to the intricate circuits found within the human brain, a fundamental requirement of neuronal function is that it be malleable, altering its output based upon experience. A host of cellular proteins are recruited for this purpose, which themselves are regulated by protein phosphorylation. Over the past several decades, research has demonstrated that the Ca(2+) and calmodulin-dependent protein phosphatase calcineurin (protein phosphatase 2B) is a critical regulator of a diverse array of proteins, leading to both short- and long-term effects on neuronal excitability and function. This review describes many of the influences of calcineurin on a variety of proteins, including ion channels, neurotransmitter receptors, enzymes, and transcription factors. Intriguingly, due to the bi-directional influences of Ca(2+) and calmodulin on calcineurin activity, the strength and duration of particular stimulations may cause apparently antagonistic functions of calcineurin to work in concert.

Brain-derived neurotrophic factor activation of NFAT (nuclear factor of activated T-cells)-dependent transcription: a role for the transcription factor NFATc4 in neurotrophin-mediated gene expression.

A member of the neurotrophin family, brain-derived neurotrophic factor (BDNF) regulates neuronal survival and differentiation during development. Within the adult brain, BDNF is also important in neuronal adaptive processes, such as the activity-dependent plasticity that underlies learning and memory. These long-term changes in synaptic strength are mediated through alterations in gene expression. However, many of the mechanisms by which BDNF is linked to transcriptional and translational regulation remain unknown. Recently, the transcription factor NFATc4 (nuclear factor of activated T-cells isoform 4) was discovered in neurons, where it is believed to play an important role in long-term changes in neuronal function. Interestingly, NFATc4 is particularly sensitive to the second messenger systems activated by BDNF. Thus, we hypothesized that NFAT-dependent transcription may be an important mediator of BDNF-induced plasticity. In cultured rat CA3-CA1 hippocampal neurons, BDNF activated NFAT-dependent transcription via TrkB receptors. Inhibition of calcineurin blocked BDNF-induced nuclear translocation of NFATc4, thus preventing transcription. Further, phospholipase C was a critical signaling intermediate between BDNF activation of TrkB and the initiation of NFAT-dependent transcription. Both inositol 1,4,5-triphosphate (IP3)-mediated release of calcium from intracellular stores and activation of protein kinase C were required for BDNF-induced NFAT-dependent transcription. Finally, increased expression of IP3 receptor 1 and BDNF after neuronal exposure to BDNF was linked to NFAT-dependent transcription. These results suggest that NFATc4 plays a crucial role in neurotrophin-mediated synaptic plasticity.

Interactions with PDZ proteins are required for L-type calcium channels to activate cAMP response element-binding protein-dependent gene expression.

After brief periods of heightened stimulation, calcium entry through L-type calcium channels leads to activation of the transcription factor cAMP response element-binding protein (CREB) and CRE-dependent transcription. Many of the details surrounding the mechanism by which L-type calcium channels are privileged in signaling to CREB, to the exclusion of other calcium entry pathways, has remained unclear. We hypothesized that the PDZ interaction sequence contained within the last four amino acids of the calcium channel alpha1C (Ca(V)1.2) subunit [Val-Ser-Asn-Leu (VSNL)] is critical for L-type calcium channels (LTCs) to interact with the signaling machinery that triggers activity-dependent gene expression. To disrupt this interaction, hippocampal CA3-CA1 pyramidal neurons were transfected with DNA encoding for enhanced green fluorescent protein tethered to VSNL (EGFP-VSNL). EGFP-VSNL significantly attenuated L-type calcium channel-induced CREB phosphorylation and CRE-dependent transcription, although somatic calcium concentrations after stimulation remained unchanged. The effect of EGFP-VSNL was specific to the actions of L-type calcium channels, because CREB signaling after NMDA receptor stimulation remained intact. The importance of the PDZ interaction sequence was verified using dihydropyridine (DHP)-insensitive alpha1C subunits. Neurons transfected with alpha1C lacking the terminal five amino acids (DHP-LTCnoPDZ) exhibited attenuated CREB responses in comparison with cells expressing the full-length subunit (DHP-LTC). Collectively, these data suggest that localized calcium responses, regulated by interactions with PDZ domain proteins, are necessary for L-type calcium channels to effectively activate CREB and CRE-mediated gene expression.

Calcitonin gene-related peptide regulates expression of neurokinin1 receptors by rat spinal neurons.

Although neurokinin 1 (NK1) receptors contribute to hyperalgesia, and their expression is increased in the spinal cord during peripheral inflammation, little is known regarding the signaling molecules and the second messenger pathways that they activate in regulating the expression of the NK1 receptor gene. Because the promoter region of the NK1 receptor contains a cAMP response element (CRE), we tested the hypothesis that calcitonin gene-related peptide (CGRP) regulates the expression of NK1 receptors via a pathway involving activation of the transcription factor cAMP response element binding protein (CREB). Experiments were conducted on primary cultures of neonatal rat spinal neurons. Treatment of cultures with CGRP for 8-24 hr increased (125)I-substance P binding on spinal neurons; the increase in binding was preceded by an elevation in NK1 receptor mRNA. The CGRP-induced change in (125)I-substance P binding was concentration-dependent and was inhibited by the antagonist CGRP(8-37). CGRP increased phosphorylated CREB immunoreactivity and CRE-dependent transcription in neurons, indicating the involvement of the transcription factor CREB. Evidence that CGRP increased cAMP levels in spinal neurons and that the protein kinase A inhibitor H89 attenuated CGRP-induced CRE-dependent transcription suggests that the intracellular pathway stimulated by CGRP leads to activation of protein kinase A. Collectively these data define a role for CGRP as a signaling molecule that induces expression of NK1 receptors in spinal neurons. The data provide evidence that a neuropeptide receptor controls gene expression in the CNS and add another dimension to understanding the cotransmission of substance P and CGRP by primary afferent neurons.

Spinal brain-derived neurotrophic factor (BDNF) produces hyperalgesia in normal mice while antisense directed against either BDNF or trkB, prevent inflammation-induced hyperalgesia.

Although known primarily for its role in neuronal development, brain-derived neurotrophic factor (BDNF) has also recently been implicated in processes mediated by the adult nervous system, such as spinal nociception. Peripheral inflammation increases expression of BDNF preferentially in dorsal root ganglion cells that contain substance P and/or calcitonin gene-related peptide, known nociceptive transmitters for which synthesis is also increased during inflammatory states. Expression of the tyrosine kinase receptor that selectively binds BDNF, trkB, is increased in the spinal dorsal horn during inflammation as well. Additionally, intrathecal (i.t.) administration of the BDNF-scavenging protein trkB-IgG attenuates inflammation-induced behavioral responses. Collectively, this evidence implicates BDNF in spinal nociceptive processes. Here we show that, in normal mice, i.t. BDNF produces an acute, dose-dependent thermal hyperalgesic response. Selective inhibition of BDNF expression by i.t. antisense oligodeoxynucleotide treatment produces antinociception in normal mice and attenuates carrageenan-induced hyperalgesia. Further, we demonstrate that i.t. antisense treatment directed against the full-length trkB receptor (trkB.FL) attenuates carrageenan-induced hyperalgesia. Consistent with a trkB.FL-mediated mechanism, the i.t. administration of another trkB ligand, neurotrophin-4/5, also produces hyperalgesia while the trkC agonist neurotrophin-3, which weakly cross-reacts with trkB, has little effect. Finally, with the accumulating evidence linking BDNF to synaptic plasticity, we investigated whether BDNF-induced hyperalgesia in normal mice involves the N-methyl-D-aspartate (NMDA) receptor. Interestingly, i.t. co-administration of the NMDA receptor antagonist D(-)-2-amino-5-phosphonovaleric acid (D-APV) with BDNF dose-dependently inhibits BDNF-induced hyperalgesia, suggesting that BDNF induces acute hyperalgesic responses and affects central sensitization in a process dependent on NMDA receptor activation.