Cannabis use during pregnancy and hemodynamic responses to infant cues in pregnancy: an exploratory study.

Introduction: Cannabis is one of the most commonly used substances during pregnancy and has the potential to negatively impact parent-infant relationships. The prefrontal cortex (PFC) response to infant cues during pregnancy has been associated with subsequent positive parenting behaviors. However, PFC activation is altered in individuals who use cannabis. As the potency of cannabis has changed over the years, little is known about the specific role of cannabis use on gestational parent brain responses to infant cues. Materials and methods: Using functional Near-Infrared Spectroscopy (fNIRS) in the second trimester of pregnancy, we measured hemodynamic responses to an infant cry task and an infant faces task among individuals who were using cannabis (N = 14) and compared them with those who were not using cannabis (N = 45). For the infant cry task, pregnant individuals listened to cry sounds and matched white noise. For the infant faces task, they viewed happy, sad, and neutral faces. Results: There was no significant difference between the two groups after adjusting for multiple comparisons. Without adjusting for multiple comparisons, we found preliminary evidence for the differences in the dorsomedial PFC associated with heightened response to infant cry among individuals who use cannabis. The groups were also different in the dorsolateral PFC associated with decreased response to infant sad faces among individuals who use cannabis. Discussion: Our preliminary data suggests that cannabis use during pregnancy was associated with brain activation in the regions involved in the emotional regulation and information processes. However, the results did not survive after adjustment for multiple comparisons, thus future research with larger sample sizes is needed to confirm potential differences in brain function among cannabis-using pregnant individuals.

Interdisciplinary views of fNIRS: Current advancements, equity challenges, and an agenda for future needs of a diverse fNIRS research community.

Functional Near-Infrared Spectroscopy (fNIRS) is an innovative and promising neuroimaging modality for studying brain activity in real-world environments. While fNIRS has seen rapid advancements in hardware, software, and research applications since its emergence nearly 30 years ago, limitations still exist regarding all three areas, where existing practices contribute to greater bias within the neuroscience research community. We spotlight fNIRS through the lens of different end-application users, including the unique perspective of a fNIRS manufacturer, and report the challenges of using this technology across several research disciplines and populations. Through the review of different research domains where fNIRS is utilized, we identify and address the presence of bias, specifically due to the restraints of current fNIRS technology, limited diversity among sample populations, and the societal prejudice that infiltrates today's research. Finally, we provide resources for minimizing bias in neuroscience research and an application agenda for the future use of fNIRS that is equitable, diverse, and inclusive.

Quantification of Smoking Characteristics Using Smartwatch Technology: Pilot Feasibility Study of New Technology.

BACKGROUND: While there have been many technological advances in studying the neurobiological and clinical basis of tobacco use disorder and nicotine addiction, there have been relatively minor advances in technologies for monitoring, characterizing, and intervening to prevent smoking in real time. Better understanding of real-time smoking behavior can be helpful in numerous applications without the burden and recall bias associated with self-report. OBJECTIVE: The goal of this study was to test the validity of using a smartwatch to advance the study of temporal patterns and characteristics of smoking in a controlled laboratory setting prior to its implementation in situ. Specifically, the aim was to compare smoking characteristics recorded by Automated Smoking PerceptIon and REcording (ASPIRE) on a smartwatch with the pocket Clinical Research Support System (CReSS) topography device, using video observation as the gold standard. METHODS: Adult smokers (N=27) engaged in a video-recorded laboratory smoking task using the pocket CReSS while also wearing a Polar M600 smartwatch. In-house software, ASPIRE, was used to record accelerometer data to identify the duration of puffs and interpuff intervals (IPIs). The recorded sessions from CReSS and ASPIRE were manually annotated to assess smoking topography. Agreement between CReSS-recorded and ASPIRE-recorded smoking behavior was compared. RESULTS: ASPIRE produced more consistent number of puffs and IPI durations relative to CReSS, when comparing both methods to visual puff count. In addition, CReSS recordings reported many implausible measurements in the order of milliseconds. After filtering implausible data recorded from CReSS, ASPIRE and CReSS produced consistent results for puff duration (R2=.79) and IPIs (R2=.73). CONCLUSIONS: Agreement between ASPIRE and other indicators of smoking characteristics was high, suggesting that the use of ASPIRE is a viable method of passively characterizing smoking behavior. Moreover, ASPIRE was more accurate than CReSS for measuring puffs and IPIs. Results from this study provide the foundation for future utilization of ASPIRE to passively and accurately monitor and quantify smoking behavior in situ.

Impaired frontostriatal functional connectivity among chronic opioid using pain patients is associated with dysregulated affect.

Preclinical studies have shown effects of chronic exposure to addictive drugs on glutamatergic-mediated neuroplasticity in frontostriatal circuitry. These initial findings have been paralleled by human functional magnetic resonance imaging (fMRI) research demonstrating weaker frontostriatal resting-state functional connectivity (rsFC) among individuals with psychostimulant use disorders. However, there is a dearth of human imaging literature describing associations between long-term prescription opioid use, frontostriatal rsFC, and brain morphology among chronic pain patients. We hypothesized that prescription opioid users with chronic pain, as compared with healthy control subjects, would evidence weaker frontostriatal rsFC coupled with less frontostriatal gray matter volume (GMV). Further, those opioid use-related deficits in frontostriatal circuitry would be associated with negative affect and drug misuse. Prescription opioid users with chronic pain (n = 31) and drug-free healthy controls (n = 30) underwent a high-resolution anatomical and an eyes-closed resting-state functional scan. The opioid group, relative to controls, exhibited weaker frontostriatal rsFC, and less frontostriatal GMV in both L.NAc and L.vmPFC. Frontostriatal rsFC partially mediated group differences in negative affect. Within opioid users, L.NAc GMV predicted opioid misuse severity. The current study revealed that prescription opioid use in the context of chronic pain is associated with functional and structural abnormalities in frontostriatal circuitry. These results suggest that opioid use-related abnormalities in frontostriatal circuitry may undergird disturbances in affect that may contribute to the ongoing maintenance of opioid use and misuse. These findings warrant further examination of interventions to treat opioid pathophysiology in frontostriatal circuitry over the course of treatment.

A rapid strategy to detect the recombined allele in LSL-TßRICA transgenic mice.

We have previously generated a transgenic mouse strain (LSL-TßRI(CA)) containing a Cre-inducible constitutively active TGFß type I receptor (Bartholin et al., 2008, Genesis 46: 724-731). Transgene expression depends on the excision of a floxed-transcriptional STOP (LSL, Lox-STOP-Lox) located upstream the TßRI(CA) coding sequence. To evaluate the correct excision of the STOP signal in the presence of Cre-recombinase, we developed a rapid screening based on an original PCR genotyping strategy. More precisely, we designed a set of primers flanking the LSL containing region. The size of the amplified products will differ according to recombination status of the LSL-TßRI(CA) allele. Indeed, the size of the STOP containing PCR product is 1.93 kb, but is reduced to 0.35 kb when the STOP signal is removed after Cre-mediated recombination. We validated excision in several compartments, including pancreas, liver, T lymphocytes, and embryos using different Cre expressing transgenic mouse strains. This represents a simple and efficient way of monitoring the tissue specific recombination of the LSL-TßRI(CA) allele.

Inhibition of CtBP1 activity by Akt-mediated phosphorylation.

Pc2 (Cbx4) is a member of the chromobox family of polycomb proteins, and is a SUMO E3 ligase for the transcriptional corepressor CtBP1. Here, we show that both CtBP1 and Pc2 are phosphorylated by the kinase Akt1, which is activated by growth factor signaling via the PI3-kinase pathway. In the presence of Pc2, phosphorylation of CtBP1 is increased, and this requires interaction of both CtBP1 and Akt1 with Pc2. Pc2 promotes CtBP1 phosphorylation by recruiting Akt1 and, in part, by preventing de-phosphorylation of activated Akt1. Alteration of the Akt-phosphorylated residue in CtBP1 to a phosphomimetic results in decreased CtBP1 dimerization, but does not prevent interaction with other transcriptional regulators. The phosphomimetic mutant of CtBP1 is expressed at a lower level than the wild type protein, resulting in decreased transcriptional repression. We show that this CtBP1 mutant is targeted for poly-ubiquitylation and is less stable than the wild type protein. Co-expression of Pc2 and Akt1 results in both phosphorylation and ubiquitylation of CtBP1, thereby targeting CtBP1 for degradation. This work suggests that Pc2 might coordinate multiple enzymatic activities to regulate CtBP1 function.

Tgif1 and Tgif2 regulate Nodal signaling and are required for gastrulation.

Tgif1 and Tgif2 are transcriptional co-repressors that limit the response to TGFbeta signaling and play a role in regulating retinoic-acid-mediated gene expression. Mutations in human TGIF1 are associated with holoprosencephaly, but it is unclear whether this is a result of deregulation of TGFbeta/Nodal signaling, or of effects on other pathways. Surprisingly, mutation of Tgif1 in mice results in only relatively mild developmental phenotypes in most strain backgrounds. Here, we show that loss-of-function mutations in both Tgif1 and Tgif2 result in a failure of gastrulation. By conditionally deleting Tgif1 in the epiblast, we demonstrate that a single wild-type allele of Tgif1 in the extra-embryonic tissue allows the double null embryos to gastrulate and begin organogenesis, suggesting that extra-embryonic Tgif function is required for patterning the epiblast. Genetically reducing the dose of Nodal in embryos lacking all Tgif function results in partial rescue of the gastrulation defects. Conditional double null embryos have defects in left-right asymmetry, which are also alleviated by reducing the dose of Nodal. Together, these data show that Tgif function is required for gastrulation, and provide the first clear evidence that Tgifs limit the transcriptional response to Nodal signaling during early embryogenesis.

Maternal Tgif is required for vascularization of the embryonic placenta.

The mammalian placenta is the site of exchange of nutrients and waste between mother and embryo. In humans, placental insufficiency can result in intrauterine growth retardation, perinatal death and spontaneous abortion. We show that in C57BL/6J mice a null mutation in the gene encoding the transcriptional corepressor, Tgif, causes placental defects. The major defects are decreased vascularization of the placenta, due to a decrease in the fetal blood vessels, and decreased expression of the gap junction protein Gjb2 (Cx26). These defects result in severe growth retardation in a proportion of Tgif null embryos in Tgif heterozygous mothers, and an overall growth delay in Tgif null animals. Placental defects are much more severe if the mother also completely lacks Tgif function, and placentas from heterozygous Tgif embryos are defective in a Tgif null mother. Embryo transfer experiments show that even the placenta from a wild type embryo is compromised in the absence of maternal Tgif. These results demonstrate that Tgif functions in the normal development of the placenta, and suggest a role for maternal factors in regulating the morphogenesis of embryonically-derived placental tissues.

Functional analysis of mutations in TGIF associated with holoprosencephaly.

Holoprosencephaly (HPE) is the most common structural malformation of the forebrain and face in humans. Our current understanding of the pathogenesis of HPE attempts to integrate genetic susceptibility, evidenced by mutations in the known HPE genes, with the epigenetic influence of environmental factors. Mutations or deletions of the human TGIF gene have been associated with HPE in multiple population cohorts. Here we examine the functional effects of all previously reported mutations, and describe four additional variants. Of the eleven sequence variations in TGIF, all but four can be demonstrated to be functionally abnormal. In contrast, no potentially pathogenic sequence alterations were detected in the related gene TGIF2. These results provide further evidence of a role for TGIF in HPE and demonstrate the importance of functional analysis of putative disease-associated alleles.

TGIF inhibits retinoid signaling.

TGIF (TG-interacting factor) represses transforming growth factor beta (TGF-beta)-activated gene expression and can repress transcription via a specific retinoid response element. Mutations in human TGIF are associated with holoprosencephaly, a severe defect of craniofacial development with both genetic and environmental causes. Both TGF-beta and retinoic acid signaling are implicated in craniofacial development. Here, we analyze the role of TGIF in regulating retinoid responsive gene expression. We demonstrate that TGIF interacts with the ligand binding domain of the RXRalpha retinoid receptor and represses transcription from retinoid response elements. TGIF recruits the general corepressor, CtBP, to RXRalpha, and this recruitment is required for full repression by TGIF. Interaction between TGIF and RXRalpha is reduced by the addition of retinoic acid, consistent with a role for TGIF as an RXRalpha transcriptional corepressor. We created a Tgif null mutation in mice and tested the sensitivity of mutant mice to increased levels of retinoic acid. Tgif mutant embryos are more sensitive to retinoic acid-induced teratogenesis, and retinoid target genes are expressed at a higher level in tissues from Tgif null mice. These results demonstrate an important role for TGIF as a transcriptional corepressor, which regulates developmental signaling by retinoic acid, and raises the possibility that TGIF may repress other RXR-dependent transcriptional responses.

Multiple activities contribute to Pc2 E3 function.

Pc2 is a polycomb protein, which has SUMO E3 activity for the corepressors CtBP and CtBP2. Here we demonstrate that, in vivo, Pc2 adapter function contributes to enhancement of CtBP sumoylation. Mutation of the CtBP binding site on Pc2 abolishes E3 activity toward CtBP. However, a carboxyl-terminal fragment of Pc2 that recruits both Ubc9 and CtBP lacks E3 activity. We identify a second domain, which, when coexpressed with the carboxyl-terminal adapter region, restores E3 function. In vitro, this domain has E3 activity in isolation, suggesting that it is a functional domain, and that adapter function is required to selectively corecruit E2 and substrate in vivo. These results demonstrate the presence of two domains in Pc2 that contribute to full in vivo E3 activity, and suggest that SUMO E3s are more than simple platforms to which E2 and substrate bind.

Aurora B kinase exists in a complex with survivin and INCENP and its kinase activity is stimulated by survivin binding and phosphorylation.

Aurora B regulates chromosome segregation and cytokinesis and is the first protein to be implicated as a regulator of bipolar attachment of spindle microtubules to kinetochores. Evidence from several systems suggests that Aurora B is physically associated with inner centromere protein (INCENP) in mitosis and has genetic interactions with Survivin. It is unclear whether the Aurora B and INCENP interaction is cell cycle regulated and if Survivin physically interacts in this complex. In this study, we cloned the Xenopus Survivin gene, examined its association with Aurora B and INCENP, and determined the effect of its binding on Aurora B kinase activity. We demonstrate that in the Xenopus early embryo, all of the detectable Survivin is in a complex with both Aurora B and INCENP throughout the cell cycle. Survivin and Aurora B bind different domains on INCENP. Aurora B activity is stimulated >10-fold in mitotic extracts; this activation is phosphatase sensitive, and the binding of Survivin is required for full Aurora B activity. We also find the hydrodynamic properties of the Aurora B/Survivin/INCENP complex are cell cycle regulated. Our data indicate that Aurora B kinase activity is regulated by both Survivin binding and cell cycle-dependent phosphorylation.