Choice impulsivity after repeated social stress is associated with increased perineuronal nets in the medial prefrontal cortex.

Repeated stress can predispose to substance abuse. However, behavioral and neurobiological adaptations that link stress to substance abuse remain unclear. This study investigates whether intermittent social defeat (ISD), a stress protocol that promotes drug-seeking behavior, alters intertemporal decision-making and cortical inhibitory function in the medial prefrontal cortex (mPFC). Male long evans rats were trained in a delay discounting task (DDT) where rats make a choice between a fast (1 s) small reward (1 sugar pellet) and a large reward (3 sugar pellets) that comes with a time delay (10 s or 20 s). A decreased preference for delayed rewards was used as an index of choice impulsivity. Rats were exposed to ISD and tested in the DDT 24 h after each stress episode, and one- and two-weeks after the last stress episode. Immunohistochemistry was performed in rat's brains to evaluate perineuronal nets (PNNs) and parvalbumin GABA interneurons (PV) labeling as markers of inhibitory function in mPFC. ISD significantly decreased the preference for delayed large rewards in low impulsive, but not high impulsive, animals. ISD also increased the density of PNNs in the mPFC. These results suggest that increased choice impulsivity and cortical inhibition predispose animals to seek out rewards after stress.

Ginger alleviates mechanical hypersensitivity and anxio-depressive behavior in rats with diabetic neuropathy through beneficial actions on gut microbiome composition, mitochondria, and neuroimmune cells of colon and spinal cord.

The relationship among gut microbiota, mitochondrial dysfunction/neuroinflammation, and diabetic neuropathic pain (DNP) has received increased attention. Ginger has antidiabetic and analgesic effects because of its anti-inflammatory property. We examined the effects of gingerols-enriched ginger (GEG) supplementation on pain-associated behaviors, gut microbiome composition, and mitochondrial function and neuroinflammation of colon and spinal cord in DNP rats. Thirty-three male rats were randomly divided into 3 groups: control group, DNP group (high-fat diet plus single dose of streptozotocin at 35 mg/kg body weight, and GEG group (DNP+GEG at 0.75% in the diet for 8 weeks). Von Frey and open field tests were used to assess pain sensitivity and anxio-depressive behaviors, respectively. Colon and spinal cord were collected for gene expression analysis. 16S rRNA gene sequencing was done from cecal samples and microbiome data analysis was performed using QIIME 2. GEG supplementation mitigated mechanical hypersensitivity and anxio-depressive behavior in DNP animals. GEG supplementation suppressed the dynamin-related protein 1 protein expression (colon) and gene expression (spinal cord), astrocytic marker GFAP gene expression (colon and spinal cord), and tumor necrosis factor-α gene expression (colon, P < .05; spinal cord, P = .0974) in DNP rats. GEG supplementation increased microglia/macrophage marker CD11b gene expression in colon and spinal cord of DNP rats. GEG treatment increased abundance of Acinetobacter, Azospirillum, Colidextribacter, and Fournierella but decreased abundance of Muribaculum intestinale in cecal feces of rats. This study demonstrates that GEG supplementation decreased pain, anxio-depression, and neuroimmune cells, and improved the composition of gut microbiomes and mitochondrial function in rats with diabetic neuropathy.

Utility of Repetitive Transcranial Magnetic Stimulation for Chronic Daily Headache Prophylaxis: A Systematic Review and Meta-Analysis.

PURPOSE OF REVIEW: Management of chronic daily headaches (CDH) remains challenging due to the limited efficacy of standard prophylactic pharmacological measures. Several studies have reported that repetitive transcranial magnetic stimulation (rTMS) can effectively treat chronic headaches. The objective was to determine the utility of rTMS for immediate post-treatment and sustained CDH prophylaxis. RECENT FINDINGS: All procedures were conducted per PRISMA guidelines. PubMed, Scopus, Web of Science, and ProQuest databases were searched for controlled clinical trials that have tested the efficacy of rTMS on populations with CDH. DerSimonian-Laird random-effects meta-analyses were performed using the 'meta' package in R to examine the post- vs. pre-rTMS changes in standardized headache intensity and frequency compared to sham-control conditions. Thirteen trials were included with a combined study population of N = 538 patients with CDH (rTMS, N = 284; Sham, N = 254). Patients exposed to rTMS had significantly reduced standardized CDH intensity and frequency in the immediate post-treatment period (Hedges' g = -1.16 [-1.89, -0.43], p = 0.002 and Δ = -5.07 [-10.05, -0.11], p = 0.045 respectively). However, these effects were sustained marginally in the follow-up period (Hedges' g = -0.43 [-0.76, -0.09], p = 0.012 and Δ = -3.33 [-5.52, -1.14], p = 0.003). Significant between-study heterogeneity was observed, at least partially driven by variations in rTMS protocols. Despite the observed clinically meaningful and statistically significant benefits in the immediate post-treatment period, the prophylactic effects of rTMS on CDH do not seem to sustain with discontinuation. Thus, the cost-effectiveness of the routine use of rTMS for CDH prophylaxis remains questionable. REGISTRATION: Protocol preregistered in PROSPERO International Prospective Register of Systematic Reviews (CRD42021250100).

Experiences and preferences for learning about neonatal research: insights from parent interviews.

INTRODUCTION: Parents struggle with being asked to participate in neonatal research. Past work has largely failed to include views of minoritized parents, low-socioeconomic status parents, and those who declined research. We aimed to describe parents' preferences related to learning about eligibility for neonatal research. METHODS: Qualitative interviews of parents who were asked to enroll their infant in neonatal research. Themes related to parental experiences and preferences for learning about neonatal research were identified using content analysis. RESULTS: Many parents desired greater involvement of their clinical team. Emotions at the time of recruitment were critically important to parents' experience, where were deeply impacted by interpersonal relationships with research staff. DISCUSSION: Increased involvement of the clinical team and greater sensitivity to the stressors around parent and infant conditions at the time of recruitment for neonatal research should be considered by those attempting to improve recruitment for neonatal research.

Hemopexin reverses activation of lung eIF2α and decreases mitochondrial injury in chlorine-exposed mice.

We assessed the mechanisms by which nonencapsulated heme, released in the plasma of mice after exposure to chlorine (Cl2) gas, resulted in the initiation and propagation of acute lung injury. We exposed adult male and female C57BL/6 mice to Cl2 (500 ppm for 30 min), returned them to room air, and injected them intramuscularly with either human hemopexin (hHPX; 5 µg/g BW in 50-µL saline) or vehicle at 1 h post-exposure. Upon return to room air, Cl2-exposed mice, injected with vehicle, developed respiratory acidosis, increased concentrations of plasma proteins in the alveolar space, lung mitochondrial DNA injury, increased levels of free plasma heme, and major alterations of their lung proteome. hHPX injection mice mitigated the onset and development of lung and mitochondrial injury and the increase of plasma heme, reversed the Cl2-induced changes in 83 of 237 proteins in the lung proteome at 24 h post-exposure, and improved survival at 15 days post-exposure. Systems biology analysis of the lung global proteomics data showed that hHPX reversed changes in a number of key pathways including elF2 signaling, verified by Western blotting measurements. Recombinant human hemopexin, generated in tobacco plants, injected at 1 h post-Cl2 exposure, was equally effective in reversing acute lung and mtDNA injury. The results of this study offer new insights as to the mechanisms by which exposure to Cl2 results in acute lung injury and the therapeutic effects of hemopexin.NEW & NOTEWORTHY Herein, we demonstrate that exposure of mice to chlorine gas causes significant changes in the lung proteome 24 h post-exposure. Systems biology analysis of the proteomic data is consistent with damage to mitochondria and activation of eIF2, the master regulator of transcription and protein translation. Post-exposure injection of hemopexin, which scavenges free heme, attenuated mtDNA injury, eIF2α phosphorylation, decreased lung injury, and increased survival.

Use of term reference infants in assessing the developmental outcome of extremely preterm infants: lessons learned in a multicenter study.

OBJECTIVE: Extremely preterm (EP) impairment rates are likely underestimated using the Bayley III norm-based thresholds scores and may be better assessed relative to concurrent healthy term reference (TR) infants born in the same hospital. STUDY DESIGN: Blinded, certified examiners in the Neonatal Research Network (NRN) evaluated EP survivors and a sample of healthy TR infants recruited near the 2-year assessment age. RESULTS: We assessed 1452 EP infants and 183 TR infants. TR-based thresholds showed higher overall EP impairment than Bayley norm-based thresholds (O.R. = 1.86; [95% CI 1.56-2.23], especially for severe impairment (36% vs. 24%; p ≤ 0.001). Difficulty recruiting TR patients at 2 years extended the study by 14 months and affected their demographics. CONCLUSION: Impairment rates among EP infants appear to be substantially underestimated from Bayley III norms. These rates may be best assessed by comparison with healthy term infants followed with minimal attrition from birth in the same centers. GOV ID: Term Reference (under the Generic Database Study): NCT00063063.

Hemopexin Reverses Activation of Lung eIF2a and Decreases Mitochondrial Injury in Chlorine Exposed Mice.

We assessed the mechanisms by which non-encapsulated heme, released in the plasma of mice post exposure to chlorine (Cl 2 ) gas, resulted in the initiation and propagation of acute lung injury. We exposed adult C57BL/6 male and female to Cl 2 (500 ppm for 30 min) in environmental chambers and returned them to room air and injected them intramuscularly with a single dose of human hemopexin (hHPX; 5 µg/ g BW), the most efficient scavenger of heme, 30-60 min post exposure. Concentrations of hHPX in plasma of air and Cl 2 exposed mice were 9081±900 vs. 1879± 293 at 6 h and 2966±463 vs. 1555±250 at 50 h post injection (ng/ml; X±1 SEM=3; p<0.01). Cl 2 exposed mice developed progressive acute lung injury post exposure characterized by increased concentrations of plasma heme, marked inflammatory response, respiratory acidosis and increased concentrations of plasma proteins in the alveolar space. Injection of hHPX decreased the onset of acute lung injury at 24 h post exposure; mean survival, for the saline and hHPX groups were 40 vs. 80% (P<0.001) at 15 d post exposure. Non-supervised global proteomics analysis of mouse lungs at 24 h post exposure, revealed the upregulation of 92 and downregulation of 145 lung proteins. Injection of hHPX at one h post exposure moderated the Cl 2 induced changes in eighty-three of these 237 lung proteins. System biology analysis of the global proteomics data showed that hHPX reversed changes in mitochondrial dysfunction and elF2 and integrin signaling. Western blot analysis of lung tissue showed significant increase of phosphorylated elF2 at 24 h post exposure in vehicle treated mice but normal levels in those injected with hHPX. Similarly, RT-PCR analysis of lung tissue showed that hHPX reversed the onset of mtDNA lesions. A form of recombinant human hemopexin generated in tobacco plants was equally effective in reversing acute lung and mtDNA injury. The results of this study offer new insights as to the mechanisms by which exposure to Cl 2 results in acute lung injury and to the therapeutic effects of hemopexin.

Effect of reduced versus usual lipid emulsion dosing on bilirubin neurotoxicity and neurodevelopmental impairment in extremely preterm infants: study protocol for a randomized controlled trial.

BACKGROUND: Bilirubin neurotoxicity (BN) occurs in premature infants at lower total serum bilirubin levels than term infants and causes neurodevelopmental impairment. Usual dose lipid infusions in preterm infants may increase free fatty acids sufficiently to cause bilirubin displacement from albumin, increasing passage of unbound bilirubin (UB) into the brain leading to BN and neurodevelopmental impairment not reliably identifiable in infancy. These risks may be influenced by whether cycled or continuous phototherapy is used to control bilirubin levels. OBJECTIVE: To assess differences in wave V latency measured by brainstem auditory evoked responses (BAER) at 34-36 weeks gestational age in infants born ≤ 750 g or < 27 weeks' gestational age randomized to receive usual or reduced dose lipid emulsion (half of the usual dose) irrespective of whether cycled or continuous phototherapy is administered. METHODS: Pilot factorial randomized controlled trial (RCT) of lipid dosing (usual and reduced) with treatment groups balanced between cycled or continuous phototherapy assignment. Eligible infants are born at ≤ 750 g or < 27 weeks' gestational age enrolled in the NICHD Neonatal Research Network RCT of cycled or continuous phototherapy. Infants will randomize 1:1 to reduced or usual dose lipid assignment during the first 2 weeks after birth and stratified by phototherapy assignment. Free fatty acids and UB will be measured daily using a novel probe. BAER testing will be performed at 34-36 weeks postmenstrual age or prior to discharge. Blinded neurodevelopmental assessments will be performed at 22-26 months. Intention-to-treat analyses will be performed with generalized linear mixed models with lipid dose and phototherapy assignments as random effects covariates, and assessment for interactions. Bayesian analyses will be performed as a secondary analysis. DISCUSSION: Pragmatic trials are needed to evaluate whether lipid emulsion dosing modifies the effect of phototherapy on BN. This factorial design presents a unique opportunity to evaluate both therapies and their interaction. This study aims to address basic controversial questions about the relationships between lipid administration, free fatty acids, UB, and BN. Findings suggesting a reduced lipid dose can diminish the risk of BN would support the need for a large multicenter RCT of reduced versus usual lipid dosing. TRIAL REGISTRATION: Clinical Trials.gov, NCT04584983, Registered 14 October 2020, https://clinicaltrials.gov/ct2/show/NCT04584983 Protocol version: Version 3.2 (10/5/2022).

Loss of insulin signaling in astrocytes exacerbates Alzheimer-like phenotypes in a 5xFAD mouse model.

Brain insulin signaling controls peripheral energy metabolism and plays a key role in the regulation of mood and cognition. Epidemiological studies have indicated a strong connection between type 2 diabetes (T2D) and neurodegenerative disorders, especially Alzheimer's disease (AD), linked via dysregulation of insulin signaling, i.e., insulin resistance. While most studies have focused on neurons, here, we aim to understand the role of insulin signaling in astrocytes, a glial cell type highly implicated in AD pathology and AD progression. To this end, we created a mouse model by crossing 5xFAD transgenic mice, a well-recognized AD mouse model that expresses five familial AD mutations, with mice carrying a selective, inducible insulin receptor (IR) knockout in astrocytes (iGIRKO). We show that by age 6 mo, iGIRKO/5xFAD mice exhibited greater alterations in nesting, Y-maze performance, and fear response than those of mice with the 5xFAD transgenes alone. This was associated with increased Tau (T231) phosphorylation, increased Aß plaque size, and increased association of astrocytes with plaques in the cerebral cortex as assessed using tissue CLARITY of the brain in the iGIRKO/5xFAD mice. Mechanistically, in vitro knockout of IR in primary astrocytes resulted in loss of insulin signaling, reduced ATP production and glycolic capacity, and impaired Aß uptake both in the basal and insulin-stimulated states. Thus, insulin signaling in astrocytes plays an important role in the control of Aß uptake, thereby contributing to AD pathology, and highlighting the potential importance of targeting insulin signaling in astrocytes as a site for therapeutics for patients with T2D and AD.

Effect of Reduced Versus Usual Lipid Emulsion Dosing on Bilirubin Neurotoxicity and Neurodevelopmental Impairment in Extremely Preterm Infants: Study Protocol for a Randomized Controlled Trial.

Background : Bilirubin neurotoxicity ( BN ) occurs in premature infants at lower total serum bilirubin levels than term infants and causes neurodevelopmental impairment. Usual dose lipid infusions in preterm infants may increase free fatty acids sufficiently to cause bilirubin displacement from albumin, increasing passage of unbound bilirubin ( UB ) into the brain leading to BN and neurodevelopmental impairment not reliably identifiable in infancy. These risks may be influenced by whether cycled or continuous phototherapy is used to control bilirubin levels. Objective : To assess differences in wave V latency measured by brainstem auditory evoked responses ( BAER ) at 34-36 weeks gestational age in infants born ≤750 g or <27 weeks' gestational age randomized to receive usual or reduced dose lipid emulsion (half of the usual dose) irrespective of whether cycled or continuous phototherapy is administered. Methods : Pilot factorial randomized controlled trial ( RCT ) of lipid dosing (usual and reduced) with treatment groups balanced between cycled or continuous phototherapy assignment. Eligible infants are born at ≤750 g or <27 weeks' gestational age enrolled in the NICHD Neonatal Research Network RCT of cycled or continuous phototherapy. Infants will randomize 1:1 to reduced or usual dose lipid assignment during the first 2 weeks after birth and stratified by phototherapy assignment. Free fatty acids and UB will be measured daily using a novel probe. BAER testing will be performed at 34-36 weeks postmenstrual age or prior to discharge. Blinded neurodevelopmental assessments will be performed at 22-26 months. Intention-to-treat analyses will be performed with generalized linear mixed models with lipid dose and phototherapy assignments as random effects covariates, and assessment for interactions. Bayesian analyses will be performed as a secondary analysis. Discussion : Pragmatic trials are needed to evaluate whether lipid emulsion dosing modifies the effect of phototherapy on BN. This factorial design presents a unique opportunity to evaluate both therapies and their interaction. This study aims to address basic controversial questions about the relationships between lipid administration, free fatty acids, UB, and BN. Findings suggesting a reduced lipid dose can diminish the risk of BN would support the need for a large multicenter RCT of reduced versus usual lipid dosing. Trial Registration : Clinical Trials.gov, NCT04584983, Registered 14 October 2020, https://clinicaltrials.gov/ct2/show/NCT04584983 Protocol Version : Version 3.2 (10/5/2022).

Ginger Root Extract Improves GI Health in Diabetic Rats by Improving Intestinal Integrity and Mitochondrial Function.

Background Emerging research suggests hyperglycemia can increase intestinal permeability. Ginger and its bioactive compounds have been reported to benefit diabetic animals due to their anti-inflammatory and antioxidant properties. In this study, we revealed the beneficial effect of gingerol-enriched ginger (GEG) on intestinal health (i.e., barrier function, mitochondrial function, and anti-inflammation) in diabetic rats. Methods Thirty-three male Sprague Dawley rats were assigned to three groups: low-fat diet (control group), high-fat-diet (HFD) + streptozotocin (single low dose 35 mg/kg body weight (BW) after 2 weeks of HFD feeding) (DM group), and HFD + streptozotocin + 0.75% GEG in diet (GEG group) for 42 days. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were conducted at baseline and prior to sample collection. Total pancreatic insulin content was determined by ELISA. Total RNA of intestinal tissues was extracted for mRNA expression using qRT-PCR. Results Compared to the DM group, the GEG group had improved glucose tolerance and increased pancreatic insulin content. Compared to those without GEG (DM group), GEG supplementation (GEG group) increased the gene expression of tight junction (Claudin-3) and antioxidant capacity (SOD1), while it decreased the gene expression for mitochondrial fusion (MFN1), fission (FIS1), biogenesis (PGC-1α, TFAM), mitophagy (LC3B, P62, PINK1), and inflammation (NF-κB). Conclusions Ginger root extract improved glucose homeostasis in diabetic rats, in part, via improving intestinal integrity and mitochondrial dysfunction of GI health.

Challenges and opportunities for chiral covalent organic frameworks.

As highly versatile crystalline porous materials, covalent organic frameworks (COFs) have emerged as an ideal platform for developing novel functional materials, attributed to their precise tunability of structure and functionality. Introducing chiral functional units into frameworks produces chiral COFs (CCOFs) with chiral superiorities through chirality conservation and conversion processes. This review summarises recent research progress in CCOFs, including synthetic methods, chiroptical characterisations, and their applications in asymmetric catalysis, chiral separation, and enantioselective recognition and sensing. Challenges and limitations are discussed to uncover future opportunities in CCOF research.

Short sleep duration and dementia: a narrative review.

Short sleep duration has been linked to the development of neurocognitive disorders. Still, current evidence for this relationship is conflicting. In this review, we summarize evidence regarding the relationship between short sleep duration and neurocognitive disorders, which shows that short sleep duration increases the risk of incident major neurocognitive disorders beginning as early as midlife. The pathological brain changes attributed to poor sleep may be related to changes in brain microstructure and accumulation of debris in the brain. More evidence is needed to fully understand the relationship between sleep duration and cognitive decline and the molecular changes that link the two. Measures of sleep quality such as sleep duration represent a potentially modifiable risk factor for the prevention of cognitive decline and neurocognitive disorders.

Outbreaks of Adenovirus-associated Respiratory Illness on 5 College Campuses in the United States, 2018-2019.

BACKGROUND: Human adenoviruses (HAdVs) are commonly associated with acute respiratory illness. HAdV outbreaks are well documented in congregate military training settings, but less is known about outbreaks on college campuses. During fall 2018 and spring 2019, 5 United States (US) colleges reported increases in HAdV-associated respiratory illness. Investigations were performed to better understand HAdV epidemiology in this setting. METHODS: A case was defined as a student at one of the 5 colleges, with acute respiratory illness and laboratory-confirmed HAdV infection during October 2018-December 2018 or March-May 2019. Available respiratory specimens were typed by HAdV type-specific real-time polymerase chain reaction assays, and for a subset, whole genome sequencing was performed. We reviewed available medical records and cases were invited to complete a questionnaire, which included questions on symptom presentation, social history, and absenteeism. RESULTS: We identified 168 HAdV cases. Median age was 19 (range, 17-22) years and 102 cases (61%) were male. Eleven cases were hospitalized, 10 with pneumonia; 2 cases died. Among questionnaire respondents, 80% (75/94) missed ≥ 1 day of class because of their illness. Among those with a type identified (79%), HAdV types 4 and 7 were equally detected, with frequency of each varying by site. Genome types 4a1 and 7d were identified, respectively, by whole genome sequence analysis. CONCLUSIONS: HAdV respiratory illness was associated with substantial morbidity and missed class time among young, generally healthy adults on 5 US college campuses. HAdVs should be considered a cause of respiratory illness outbreaks in congregate settings such as college campuses.

Current Status of Healthy Aging and Dementia Research: A Symposium Summary.

The purpose of the 'First Regional Healthy Aging and Dementia Research Symposium' was to discuss the latest research in healthy aging and dementia research, public health trends related to neurodegenerative diseases of aging, and community-based programs and research studying health, nutrition, and cognition. This symposium was organized by the Garrison Institute on Aging (GIA) of the Texas Tech University Health Sciences Center (TTUHSC), and was held in Lubbock, Texas, October 24-25, 2018. The Symposium joined experts from educational and research institutions across the United States. The two-day Symposium included all GIA staff and researchers. Students, postdoctoral fellows, and faculty members involved in dementia research presented at the Symposium. Healthcare professionals, from geriatricians to social workers working with patients with neurodegenerative diseases, also presented. In addition, experts traveled from across the United States to participate. This event was comprised of multiple sessions, each with several oral presentations, followed by questions and answers, and discussion.

Corrigendum: Mechanisms Underlying Serotonergic Excitation of Callosal Projection Neurons in the Mouse Medial Prefrontal Cortex.

[This corrects the article DOI: 10.3389/fncir.2018.00002.].

Serotonergic Regulation of Corticoamygdalar Neurons in the Mouse Prelimbic Cortex.

Neuromodulatory transmitters, such as serotonin (5-HT), selectively regulate the excitability of subpopulations of cortical projection neurons to gate cortical output to specific target regions. For instance, in the mouse prelimbic cortex, 5-HT selectively excites commissurally projecting (COM) intratelencephalic neurons via activation of 5-HT2A (2A) receptors, while simultaneously inhibiting, via 5-HT1A (1A) receptors, corticofugally projecting pyramidal neurons targeting the pons. Here we characterize the physiology, morphology, and serotonergic regulation of corticoamygdalar (CAm) projection neurons in the mouse prelimbic cortex. Layer 5 CAm neurons shared a number of physiological and morphological characteristics with COM neurons, including higher input resistances, smaller HCN-channel mediated responses, and sparser dendritic arbors than corticopontine neurons. Across cortical lamina, CAm neurons also resembled COM neurons in their serotonergic modulation; focally applied 5-HT (100 µM; 1 s) generated 2A-receptor-mediated excitation, or 1A- and 2A-dependent biphasic responses, in ipsilaterally and contralaterally projecting CAm neurons. Serotonergic excitation depended on extrinsic excitatory drive, as 5-HT failed to depolarize CAm neurons from rest, but could enhance the number of action potentials generated by simulated barrages of synaptic input. Finally, using dual tracer injections, we identified double-labeled CAm/COM neurons that displayed primarily excitatory or biphasic responses to 5-HT. Overall, our findings reveal that prelimbic CAm neurons in layer 5 overlap, at least partially, with COM neurons, and that neurons projecting to either, or both targets, exhibit 2A-dependent serotonergic excitation. These results suggest that 5-HT, acting at 2A receptors, may promote cortical output to the amygdala.

Mechanisms Underlying Serotonergic Excitation of Callosal Projection Neurons in the Mouse Medial Prefrontal Cortex.

Serotonin (5-HT) selectively excites subpopulations of pyramidal neurons in the neocortex via activation of 5-HT2A (2A) receptors coupled to Gq subtype G-protein alpha subunits. Gq-mediated excitatory responses have been attributed primarily to suppression of potassium conductances, including those mediated by KV7 potassium channels (i.e., the M-current), or activation of non-specific cation conductances that underlie calcium-dependent afterdepolarizations (ADPs). However, 2A-dependent excitation of cortical neurons has not been extensively studied, and no consensus exists regarding the underlying ionic effector(s) involved. In layer 5 of the mouse medial prefrontal cortex, we tested potential mechanisms of serotonergic excitation in commissural/callosal (COM) projection neurons, a subpopulation of pyramidal neurons that exhibits 2A-dependent excitation in response to 5-HT. In baseline conditions, 5-HT enhanced the rate of action potential generation in COM neurons experiencing suprathreshold somatic current injection. This serotonergic excitation was occluded by activation of muscarinic acetylcholine (ACh) receptors, confirming that 5-HT acts via the same Gq-signaling cascades engaged by ACh. Like ACh, 5-HT promoted the generation of calcium-dependent ADPs following spike trains. However, calcium was not necessary for serotonergic excitation, as responses to 5-HT were enhanced (by >100%), rather than reduced, by chelation of intracellular calcium with 10 mM BAPTA. This suggests intracellular calcium negatively regulates additional ionic conductances gated by 2A receptors. Removal of extracellular calcium had no effect when intracellular calcium signaling was intact, but suppressed 5-HT response amplitudes, by about 50%, when BAPTA was included in patch pipettes. This suggests that 2A excitation involves activation of a non-specific cation conductance that is both calcium-sensitive and calcium-permeable. M-current suppression was found to be a third ionic effector, as blockade of KV7 channels with XE991 (10 µM) reduced serotonergic excitation by ∼50% in control conditions, and by ∼30% with intracellular BAPTA present. Together, these findings demonstrate a role for at least three distinct ionic effectors, including KV7 channels, a calcium-sensitive and calcium-permeable non-specific cation conductance, and the calcium-dependent ADP conductance, in mediating serotonergic excitation of COM neurons.

Evidence for an iterative module in chain elongation on the azalomycin polyketide synthase.

The assembly-line synthases that produce bacterial polyketide natural products follow a modular paradigm in which each round of chain extension is catalysed by a different set or module of enzymes. Examples of deviation from this paradigm, in which a module catalyses either multiple extensions or none are of interest from both a mechanistic and an evolutionary viewpoint. We present evidence that in the biosynthesis of the 36-membered macrocyclic aminopolyol lactones (marginolactones) azalomycin and kanchanamycin, isolated respectively from Streptomyces malaysiensis DSM4137 and Streptomyces olivaceus Tü4018, the first extension module catalyses both the first and second cycles of polyketide chain extension. To confirm the integrity of the azl gene cluster, it was cloned intact on a bacterial artificial chromosome and transplanted into the heterologous host strain Streptomyces lividans, which does not possess the genes for marginolactone production. When furnished with 4-guanidinobutyramide, a specific precursor of the azalomycin starter unit, the recombinant S. lividans produced azalomycin, showing that the polyketide synthase genes in the sequenced cluster are sufficient to accomplish formation of the full-length polyketide chain. This provides strong support for module iteration in the azalomycin and kanchanamycin biosynthetic pathways. In contrast, re-sequencing of the gene cluster for biosynthesis of the polyketide ß-lactone ebelactone in Streptomyces aburaviensis has shown that, contrary to a recently-published proposal, the ebelactone polyketide synthase faithfully follows the colinear modular paradigm.