Viscerosensory signalling to the nucleus accumbens via the solitary tract nucleus.

The nucleus of the solitary tract (NTS) receives direct viscerosensory vagal afferent input that drives autonomic reflexes, neuroendocrine function and modulates behaviour. A subpopulation of NTS neurons project to the nucleus accumbens (NAc); however, the function of this NTS-NAc pathway remains unknown. A combination of neuroanatomical tracing, slice electrophysiology and fibre photometry was used in mice and/or rats to determine how NTS-NAc neurons fit within the viscerosensory network. NTS-NAc projection neurons are predominantly located in the medial and caudal portions of the NTS with 54 ± 7% (mice) and 65 ± 3% (rat) being TH-positive, representing the A2 NTS cell group. In horizontal brainstem slices, solitary tract (ST) stimulation evoked excitatory post-synaptic currents (EPSCs) in NTS-NAc projection neurons. The majority (75%) received low-jitter, zero-failure EPSCs characteristic of monosynaptic ST afferent input that identifies them as second order to primary sensory neurons. We then examined whether NTS-NAc neurons respond to cholecystokinin (CCK, 20 µg/kg ip) in vivo in both mice and rats. Surprisingly, there was no difference in the number of activated NTS-NAc cells between CCK and saline-treated mice. In rats, just 6% of NTS-NAc cells were recruited by CCK. As NTS TH neurons are the primary source for NAc noradrenaline, we measured noradrenaline release in the NAc and showed that NAc noradrenaline levels declined in response to cue-induced reward retrieval but not foot shock. Combined, these findings suggest that high-fidelity afferent information from viscerosensory afferents reaches the NAc. These signals are likely unrelated to CCK-sensitive vagal afferents but could interact with other sensory and higher order inputs to modulate learned appetitive behaviours.

The challenges for implementing Good Clinical Practices in the bioanalytical laboratory: a discussion paper from the European Bioanalysis Forum.

This manuscript reports back from the discussion in the European Bioanalysis Forum community on the challenges observed when implementing Good Clinical Practices in the bioanalytical laboratory. It is not intended to challenge any regulatory requirements but to open a discussion on where the bioanalytical community sees ambiguities on implementing Good Clinical Practices or areas where expectations are either felt not being owned by Bioanalysis or where Good Clinical Practices requirements are at risk of getting contaminated with requirements originating from Good Laboratory Practices. In addition to this, the discussions focused on three additional main challenges: the informed consent withdrawal, expedited reporting of unexpected results and the risk-based approach to quality management, The European Bioanalysis Forum community is continuing discussions, but already this manuscript should help to appreciate the challenges and to try and resolve them, involving all stakeholders.

Recommendations and feedback from the European Bioanalysis Forum Workshop: 1 year into ICH M10 - keeping our finger on the pulse.

The ICH M10 guideline on bioanalytical method validation and sample analysis is being adopted since 2023. However, and inevitably, some paragraphs or requirements remain ambiguous and are open for different interpretations. In support of a harmonized interpretation by the industry and health authorities, the European Bioanalysis Forum organized a workshop on 14 November 2023 in Barcelona, Spain, to discuss unclear and/or ambiguous paragraphs which were identified by the European Bioanalysis Forum community and delegates of the workshop prior to the workshop. This manuscript reports back from the workshop with recommendations and aims at continuing an open scientific discussion within the industry and with regulators in support of a science-driven guideline for the bioanalytical community and in line with the ICH mission - that is, achieve greater harmonization worldwide to ensure that safe, effective and high-quality medicines are developed and registered in the most resource-efficient manner.

Feeding signals inhibit fluid-satiation signals in the mouse lateral parabrachial nucleus to increase intake of highly palatable, caloric solutions.

Chemogenetic activation of oxytocin receptor-expressing neurons in the parabrachial nucleus (OxtrPBN neurons) acts as a satiation signal for water. In this research, we investigated the effect of activating OxtrPBN neurons on satiation for different types of fluids. Chemogenetic activation of OxtrPBN neurons in male and female transgenic OxtrCre mice robustly suppressed the rapid, initial (15-min) intake of several solutions after dehydration: water, sucrose, ethanol and saccharin, but only slightly decreased intake of Ensure®, a highly caloric solution (1 kcal/mL; containing 3.72 g protein, 3.27 g fat, 13.42 g carbohydrates, and 1.01 g dietary fibre per 100 mL). OxtrPBN neuron activation also suppressed cumulative, longer-term (2-h) intake of lower caloric, less palatable solutions, but not highly caloric, palatable solutions. These results suggest that OxtrPBN neurons predominantly control initial fluid-satiation responses after rehydration, but not longer-term intake of highly caloric, palatable solutions. The suppression of fluid intake was not because of anxiogenesis, but because OxtrPBN neuron activation decreased anxiety-like behaviour. To investigate the role of different PBN subdivisions on the intake of different solutions, we examined FOS as a proxy marker of PBN neuron activation. Different PBN subdivisions were activated by different solutions: the dorsolateral PBN similarly by all fluids; the external lateral PBN by caloric but not non-caloric solutions; and the central lateral PBN primarily by highly palatable solutions, suggesting PBN subdivisions regulate different aspects of fluid intake. To explore the possible mechanisms underlying the minimal suppression of Ensure® after OxtrPBN neuron activation, we demonstrated in in vitro slice recordings that the feeding-associated agouti-related peptide (AgRP) inhibited OxtrPBN neuron firing in a concentration-related manner, suggesting possible inhibition by feeding-related neurocircuitry of fluid satiation neurocircuitry. Overall, this research suggests that although palatable beverages like sucrose- and ethanol-containing beverages activate fluid satiation signals encoded by OxtrPBN neurons, these neurons can be inhibited by hunger-related signals (agouti-related peptide, AgRP), which may explain why these fluids are often consumed in excess of what is required for fluid satiation.

Sites and mechanisms of action of colokinetics at dopamine, ghrelin and serotonin receptors in the rodent lumbosacral defecation centre.

Agonists of dopamine D2 receptors (D2R), 5-hydroxytryptamine (5-HT, serotonin) receptors (5-HTR) and ghrelin receptors (GHSR) activate neurons in the lumbosacral defecation centre, and act as 'colokinetics', leading to increased propulsive colonic motility, in vivo. In the present study, we investigated which neurons in the lumbosacral defecation centre express the receptors and whether dopamine, serotonin and ghrelin receptor agonists act on the same lumbosacral preganglionic neurons (PGNs). We used whole cell electrophysiology to record responses from neurons in the lumbosacral defecation centre, following colokinetic application, and investigated their expression profiles and the chemistries of their neural inputs. Fluorescence in situ hybridisation revealed Drd2, Ghsr and Htr2C transcripts were colocalised in lumbosacral PGNs of mice, and immunohistochemistry showed that these neurons have closely associated tyrosine hydroxylase and 5-HT boutons. Previous studies showed that they do not receive ghrelin inputs. Whole cell electrophysiology in adult mice spinal cord revealed that dopamine, serotonin, α-methylserotonin and capromorelin each caused inward, excitatory currents in overlapping populations of lumbosacral PGNs. Furthermore, dopamine caused increased frequency of both IPSCs and EPSCs in a cohort of D2R neurons. Tetrodotoxin blocked the IPSCs and EPSCs, revealing a post-synaptic excitatory action of dopamine. In lumbosacral PGNs of postnatal day 7-14 rats, only dopamine's postsynaptic effects were observed. Furthermore, inward, excitatory currents evoked by dopamine were reduced by the GHSR antagonist, YIL781. We conclude that lumbosacral PGNs are the site where the action of endogenous ligands of D2R and 5-HT2R converge, and that GHSR act as a cis-modulator of D2R expressed by the same neurons. KEY POINTS: Dopamine, 5-hydroxytryptamine (5-HT, serotonin) and ghrelin (GHSR) receptor agonists increase colorectal motility and have been postulated to act at receptors on parasympathetic preganglionic neurons (PGNs) in the lumbosacral spinal cord. We aimed to determine which neurons in the lumbosacral spinal cord express dopamine, serotonin and GHSR receptors, their neural inputs, and whether agonists at these receptors excite them. We show that dopamine, serotonin and ghrelin receptor transcripts are contained in the same PGNs and that these neurons have closely associated tyrosine hydroxylase and serotonin boutons. Whole cell electrophysiology revealed that dopamine, serotonin and GHSR receptor agonists induce an inward excitatory current in overlapping populations of lumbosacral PGNs. Dopamine-induced excitation was reversed by GHSR antagonism. The present study demonstrates that lumbosacral PGNs are the site at which actions of endogenous ligands of dopamine D2 receptors and 5-HT type 2 receptors converge. Ghrelin receptors are functional, but their role appears to be as modulators of dopamine effects at D2 receptors.

Selective transduction and photoinhibition of pre-Bötzinger complex neurons that project to the facial nucleus in rats affects nasofacial activity.

The pre-Bötzinger complex (preBötC), a key primary generator of the inspiratory breathing rhythm, contains neurons that project directly to facial nucleus (7n) motoneurons to coordinate orofacial and nasofacial activity. To further understand the identity of 7n-projecting preBötC neurons, we used a combination of optogenetic viral transgenic approaches to demonstrate that selective photoinhibition of these neurons affects mystacial pad activity, with minimal effects on breathing. These effects are altered by the type of anesthetic employed and also between anesthetized and conscious states. The population of 7n-projecting preBötC neurons we transduced consisted of both excitatory and inhibitory neurons that also send collaterals to multiple brainstem nuclei involved with the regulation of autonomic activity. We show that modulation of subgroups of preBötC neurons, based on their axonal projections, is a useful strategy to improve our understanding of the mechanisms that coordinate and integrate breathing with different motor and physiological behaviors. This is of fundamental importance, given that abnormal respiratory modulation of autonomic activity and orofacial behaviors have been associated with the development and progression of diseases.

While breathing seems to come easy, it is a complex process in which many muscles coordinate to allow air to flow into the lungs. These muscles also control the flow of air we breathe out to allow us to talk, sing, eat, or drink. The brain circuits that control these muscles, can also influence other parts of the brain. The preBötzinger Complex, which is a key region of brainstem circuits that generate and control breathing, contains neurons that also project widely, connecting to other regions of the brain. This helps to modulate the sense of smell, emotional state, heart rate, and even blood pressure. Understanding how the preBötzinger Complex is organized can untangle how breathing can influence these other processes. Melo et al. wanted to learn whether they could manipulate the activity of a subgroup of preBötzinger Complex neurons that project into the facial nucleus ­ a region of the brain that controls the muscles of the face when we breathe ­ without affecting breathing. If this can be done, it might also be possible to affect blood pressure by manipulating selective preBötzinger neurons, and thus the development of hypertension, without having any impact on breathing. To test this hypothesis, Melo et al. used rats in which the activation of preBötzinger Complex neurons that project into the facial nucleus was blocked. This decreased the activity of the muscles around the nose with hardly any effect on breathing. Melo et al. also found that the state of consciousness of the rat (anesthetized or conscious) could affect how preBötzinger Complex neurons control these muscles. Melo et al. also observed that preBötzinger Complex neurons projecting into the facial nucleus had projections into many other regions in the brainstem. This might help to the coordinate respiratory, cardiovascular, orofacial, and potentially other physiological functions. The findings of Melo et al. set a technical foundation for exploring the influence of specific subgroups of preBötzinger Complex neurons on respiratory modulation of other physiological activities, including blood pressure and heart rate and in conditions, such as hypertension and heart failure. More broadly, most brain regions contain complex and heterogeneous groups of neurons and the strategy validated by Melo et. al. could be applied to unravel other brain-function relationships.

Analysis of the distribution of vagal afferent projections from different peripheral organs to the nucleus of the solitary tract in rats.

Anatomical tracing studies examining the vagal system can conflate details of sensory afferent and motor efferent neurons. Here, we used a serotype of adeno-associated virus that transports retrogradely and exhibits selective tropism for vagal afferents, to map their soma location and central termination sites within the nucleus of the solitary tract (NTS). We examined the vagal sensory afferents innervating the trachea, duodenum, stomach, or heart, and in some animals, from two organs concurrently. We observed no obvious somatotopy in the somata distribution within the nodose ganglion. The central termination patterns of afferents from different organs within the NTS overlap substantially. Convergence of vagal afferent inputs from different organs onto single NTS neurons is observed. Abdominal and thoracic afferents terminate throughout the NTS, including in the rostral NTS, where the 7th cranial nerve inputs are known to synapse. To address whether the axonal labeling produced by viral transduction is so widespread because it fills axons traveling to their targets, and not just terminal fields, we labeled pre and postsynaptic elements of vagal afferents in the NTS . Vagal afferents form multiple putative synapses as they course through the NTS, with each vagal afferent neuron distributing sensory signals to multiple second-order NTS neurons. We observe little selectivity between vagal afferents from different visceral targets and NTS neurons with common neurochemical phenotypes, with afferents from different organs making close appositions with the same NTS neuron. We conclude that specific viscerosensory information is distributed widely within the NTS and that the coding of this input is probably determined by the intrinsic properties and projections of the second-order neuron.

Conference Report from the European Bioanalysis Forum Workshop: toward harmonized implementation of the ICH M10 guideline.

In this report, the European Bioanalysis Forum shares the proposals for harmonized implementation of the ICH M10 guideline on bioanalytical method validation and study sample analysis from the ICH M10 workshop. The focus of the discussions was to understand new, changed or still ambiguous regulatory expectations in the guideline, as identified in feedback from the pre-workshop surveys or during the workshop. The proposals from the workshop aim at stimulating and helping a harmonized implementation of the guideline, and using our community as a sounding board during and after implementation to highlight areas of misalignment and to create a platform for continued sharing with the regulatory authorities in an effort to contribute to industry and regulators developing similar interpretations on guideline expectations.

Inductively coupled plasma mass spectrometry method for plasma and intracellular antimony quantification applied to pharmacokinetics of meglumine antimoniate.

Background: A high-throughput method using inductively coupled plasma mass spectrometry (ICP-MS) was developed and validated for the quantitative analysis of antimony in human plasma and peripheral blood mononuclear cells from patients with cutaneous leishmaniasis undergoing treatment with meglumine antimoniate. Materials & methods: Antimony was digested in clinical samples with 1% tetramethylammonium hydroxide/1% EDTA and indium was used as internal standard. Accuracy, precision and stability were evaluated. Conclusion: Taking the lower limit of quantitation to be the lowest validation concentration with precision and accuracy within 20%, the current assay was successfully validated from 25 to 10000 ng/ml for antimony in human plasma and peripheral blood mononuclear cells. This protocol will serve as a baseline for future analytical designs, aiming to provide a reference method to allow inter-study comparisons.

Lay abstract Cutaneous leishmaniasis is a disease caused by single-cell parasites in the genus Leishmania which results in painful skin ulcers and is spread by insect bites. Drugs containing antimony are the mainstay therapy for cutaneous leishmaniasis, but if and how the amount of these compounds in the cells can affect the success of the treatment, remains unknown. Validated methods to reliably measure these amounts in human cells are limited. Here we have developed a validated method that allows quantifying antimony in human plasma and peripheral blood cells from patients undergoing antileishmanial treatment. This protocol will serve as a baseline for future studies aiming to understand how antimonials work to treat leishmaniasis infections and how this therapy can be improved.

FGF-MAPK signaling regulates human deep-layer corticogenesis.

Despite heterogeneity across the six layers of the mammalian cortex, all excitatory neurons are generated from a single founder population of neuroepithelial stem cells. However, how these progenitors alter their layer competence over time remains unknown. Here, we used human embryonic stem cell-derived cortical progenitors to examine the role of fibroblast growth factor (FGF) and Notch signaling in influencing cell fate, assessing their impact on progenitor phenotype, cell-cycle kinetics, and layer specificity. Forced early cell-cycle exit, via Notch inhibition, caused rapid, near-exclusive generation of deep-layer VI neurons. In contrast, prolonged FGF2 promoted proliferation and maintained progenitor identity, delaying laminar progression via MAPK-dependent mechanisms. Inhibiting MAPK extended cell-cycle length and led to generation of layer-V CTIP2+ neurons by repressing alternative laminar fates. Taken together, FGF/MAPK regulates the proliferative/neurogenic balance in deep-layer corticogenesis and provides a resource for generating layer-specific neurons for studying development and disease.

Use of a physiological reflex to standardize vagal nerve stimulation intensity improves data reproducibility in a memory extinction assay.

BACKGROUND: Modulating brainstem activity, via electrical vagus nerve stimulation (VNS), influences cognitive functions, including memory. However, controlling for changes in stimulus efficacy during chronic studies, and response variability between subjects, is problematic. OBJECTIVE/HYPOTHESIS: We hypothesized that recruitment of an autonomic reflex, the Hering-Breuer reflex, would provide robust confirmation of VNS efficacy. We compared this to measurement of electrode resistance over time. We also examined whether VNS modulates contextual memory extinction. METHODS: Electrodes for VNS and diaphragm electromyography recording were implanted into anesthetized Sprague Dawley rats. When conscious, we measured the electrode resistance as well as the minimum VNS current required to evoke the Hering-Breuer reflex, before, and after, an inhibitory avoidance assay - a two chamber, dark/light model, where the dark compartment was paired with an aversive foot shock. The extinction of this contextual memory was assessed in sham and VNS treated rats, with VNS administered for 30 s at 1.5 times the Hering-Breuer reflex threshold during extinction memory formation. RESULTS: Assessment of VNS-evoked Hering-Breuer reflex successfully identified defective electrodes. VNS accelerated extinction memory and decreased multiple physiological metrics of fear expression. We observed an inverse relationship between memory extinction and respiratory rate during the behavioural assay. Additionally, no current - response relationship between VNS and extinction memory formation was established. CONCLUSION: These data demonstrate that reliable, experimental VNS studies can be produced by verifying reflex initiation as a consequence of stimulation. Further, studies could be standardised by indexing stimulator efficacy to initiation of autonomic reflexes.

Selective optogenetic stimulation of efferent fibers in the vagus nerve of a large mammal.

BACKGROUND: Electrical stimulation applied to individual organs, peripheral nerves, or specific brain regions has been used to treat a range of medical conditions. In cardiovascular disease, autonomic dysfunction contributes to the disease progression and electrical stimulation of the vagus nerve has been pursued as a treatment for the purpose of restoring the autonomic balance. However, this approach lacks selectivity in activating function- and organ-specific vagal fibers and, despite promising results of many preclinical studies, has so far failed to translate into a clinical treatment of cardiovascular disease. OBJECTIVE: Here we report a successful application of optogenetics for selective stimulation of vagal efferent activity in a large animal model (sheep). METHODS AND RESULTS: Twelve weeks after viral transduction of a subset of vagal motoneurons, strong axonal membrane expression of the excitatory light-sensitive ion channel ChIEF was achieved in the efferent projections innervating thoracic organs and reaching beyond the level of the diaphragm. Blue laser or LED light (>10 mW mm-2; 1 ms pulses) applied to the cervical vagus triggered precisely timed, strong bursts of efferent activity with evoked action potentials propagating at speeds of ∼6 m s-1. CONCLUSIONS: These findings demonstrate that in species with a large, multi-fascicled vagus nerve, it is possible to stimulate a specific sub-population of efferent fibers using light at a site remote from the vector delivery, marking an important step towards eventual clinical use of optogenetic technology for autonomic neuromodulation.

A Chemogenetic Tool that Enables Functional Neural Circuit Analysis.

Chemogenetics enables manipulation of neuronal activity in experimental animals. While providing information about the transduced neuron expressing a ligand-activated molecule, chemogenetics does not provide understanding about the antecedent circuit that drives that neuron's activity. For current approaches, this is not feasible, because the activating molecules are not genetically encoded. The insect allatostatin/allatostatin receptor system, a highly specific, powerful inhibitory chemogenetic approach, has this advantage, because the ligand, being a peptide, is genetically encoded. We developed viral vector-based systems to express biologically active allatostatin in neurons in vivo and allatostatin receptors in subpopulations of postsynaptic neurons. We demonstrate that activity-dependent release of allatostatin induces inhibition of allatostatin receptor-expressing neurons. We validate the approach in the vagal viscerosensory system where inhibitory, rather than the usual excitatory, viscerosensory input leads to sustained decreases in baroreceptor reflex sensitivity and bodyweight.

Improving data integrity in regulated bioanalysis: proposal for a generic data transfer process for LC-MS from the European Bioanalysis Forum.

In this paper, the European Bioanalysis Forum reports back from the discussions with software developers, involved in regulated bioanalysis software solutions, on agreeing to data transfer specification in the bioanalytical labs' LC-MS workflows as part of today's Data Integrity (DI) challenges. The proposed specifications aim at identifying what consists of a minimum dataset, that is, which are the pre-identified fields to be included in DI proof bidirectional data transfer between LC-MS and information management systems. The proposal is an attempt from the European Bioanalysis Forum to facilitate new software solutions becoming available to increase compliance related to DI in today's LC-MS workflows. The proposal may also serve as a template and inspiration for new data transfer solutions in other workflows.

PreBötzinger complex neurons drive respiratory modulation of blood pressure and heart rate.

Heart rate and blood pressure oscillate in phase with respiratory activity. A component of these oscillations is generated centrally, with respiratory neurons entraining the activity of pre-sympathetic and parasympathetic cardiovascular neurons. Using a combination of optogenetic inhibition and excitation in vivo and in situ in rats, as well as neuronal tracing, we demonstrate that preBötzinger Complex (preBötC) neurons, which form the kernel for inspiratory rhythm generation, directly modulate cardiovascular activity. Specifically, inhibitory preBötC neurons modulate cardiac parasympathetic neuron activity whilst excitatory preBötC neurons modulate sympathetic vasomotor neuron activity, generating heart rate and blood pressure oscillations in phase with respiration. Our data reveal yet more functions entrained to the activity of the preBötC, with a role in generating cardiorespiratory oscillations. The findings have implications for cardiovascular pathologies, such as hypertension and heart failure, where respiratory entrainment of heart rate is diminished and respiratory entrainment of blood pressure exaggerated.

Organotypic Cocultures of Human Pluripotent Stem Cell Derived-Neurons with Mammalian Inner Ear Hair Cells and Cochlear Nucleus Slices.

Stem cells have been touted as a source of potential replacement neurons for inner ear degeneration for almost two decades now; yet to date, there are few studies describing the use of human pluripotent stem cells (hPSCs) for this purpose. If stem cell therapies are to be used clinically, it is critical to validate the usefulness of hPSC lines in vitro and in vivo. Here, we present the first quantitative evidence that differentiated hPSC-derived neurons that innervate both the inner ear hair cells and cochlear nucleus neurons in coculture, with significantly more new synaptic contacts formed on target cell types. Nascent contacts between stem cells and hair cells were immunopositive for both synapsin I and VGLUT1, closely resembling expression of these puncta in endogenous postnatal auditory neurons and control cocultures. When hPSCs were cocultured with cochlear nucleus brainstem slice, significantly greater numbers of VGLUT1 puncta were observed in comparison to slice alone. New VGLUT1 puncta in cocultures with cochlear nucleus slice were not significantly different in size, only in quantity. This experimentation describes new coculture models for assessing auditory regeneration using well-characterised hPSC-derived neurons and highlights useful methods to quantify the extent of innervation on different cell types in the inner ear and brainstem.

Extensive Inhibitory Gating of Viscerosensory Signals by a Sparse Network of Somatostatin Neurons.

Integration and modulation of primary afferent sensory information begins at the first terminating sites within the CNS, where central inhibitory circuits play an integral role. Viscerosensory information is conveyed to the nucleus of the solitary tract (NTS) where it initiates neuroendocrine, behavioral, and autonomic reflex responses that ensure optimal internal organ function. This excitatory input is modulated by diverse, local inhibitory interneurons, whose functions are not clearly understood. Here we show that, in male rats, 65% of somatostatin-expressing (SST) NTS neurons also express GAD67, supporting their likely role as inhibitory interneurons. Using whole-cell recordings of NTS neurons, from horizontal brainstem slices of male and female SST-yellow fluorescent protein (YFP) and SST-channelrhodopsin 2 (ChR2)-YFP mice, we quantified the impact of SST-NTS neurons on viscerosensory processing. Light-evoked excitatory photocurrents were reliably obtained from SST-ChR2-YFP neurons (n = 16) and the stimulation-response characteristics determined. Most SST neurons (57%) received direct input from solitary tract (ST) afferents, indicating that they form part of a feedforward circuit. All recorded SST-negative NTS neurons (n = 72) received SST-ChR2 input. ChR2-evoked PSCs were largely inhibitory and, in contrast to previous reports, were mediated by both GABA and glycine. When timed to coincide, the ChR2-activated SST input suppressed ST-evoked action potentials at second-order NTS neurons, demonstrating strong modulation of primary viscerosensory input. These data indicate that the SST inhibitory network innervates broadly within the NTS, with the potential to gate viscerosensory input to powerfully alter autonomic reflex function and other behaviors.SIGNIFICANCE STATEMENT Sensory afferent input is modulated according to state. For example the baroreflex is altered during a stress response or exercise, but the basic mechanisms underpinning this sensory modulation are not fully understood in any sensory system. Here we demonstrate that the neuronal processing of viscerosensory information begins with synaptic gating at the first central synapse with second-order neurons in the NTS. These data reveal that the somatostatin subclass of inhibitory interneurons are driven by visceral sensory input to play a major role in gating viscerosensory signals, placing them within a feedforward circuit within the NTS.

Data integrity in regulated bioanalysis: a summary from the European Bioanalysis Forum Workshop in collaboration with the MHRA.

In this conference report, we summarize the main findings and messages from a workshop on 'Data Integrity'. The workshop was held at the 11th European Bioanalysis Forum Open (EBF) Symposium in Barcelona (21-23 November 2018), in collaboration with the Medicines and Health products Regulatory Agency to provide insight and understanding of regulatory data integrity expectations. The workshop highlighted the importance of engaging with software developers to address the gap between industry's data integrity needs and current system software capabilities. Delegates were also made aware of the importance of implementing additional procedural controls to mitigate the risk associated with using systems that do not fully meet data integrity requirements.

Characterization of the relaxin family peptide receptor 3 system in the mouse bed nucleus of the stria terminalis.

The bed nucleus of the stria terminalis (BNST) is a critical node involved in stress and reward-related behaviors. Relaxin family peptide receptor 3 (RXFP3) signaling in the BNST has been implicated in stress-induced alcohol seeking behavior. However, the neurochemical phenotype and connectivity of BNST RXFP3-expressing (RXFP3+) cells have yet to be elucidated. We interrogated the molecular signature and electrophysiological properties of BNST RXFP3+ neurons using a RXFP3-Cre reporter mouse line. BNST RXFP3+ cells are circumscribed to the dorsal BNST (dBNST) and are neurochemically heterogeneous, comprising a mix of inhibitory and excitatory neurons. Immunohistochemistry revealed that ~48% of BNST RXFP3+ neurons are GABAergic, and a quarter of these co-express the calcium-binding protein, calbindin. A subset of BNST RXFP3+ cells (~41%) co-express CaMKIIα, suggesting this subpopulation of BNST RXFP3+ neurons are excitatory. Corroborating this, RNAscope® revealed that ~35% of BNST RXFP3+ cells express vVGluT2 mRNA, indicating a subpopulation of RXFP3+ neurons are glutamatergic. RXFP3+ neurons show direct hyperpolarization to bath application of a selective RXFP3 agonist, RXFP3-A2, while around 50% of cells were depolarised by exogenous corticotrophin releasing factor. In behaviorally naive mice the majority of RXFP3+ neurons were Type II cells exhibiting Ih and T type calcium mediated currents. However, chronic swim stress caused persistent plasticity, decreasing the proportion of neurons that express these channels. These studies are the first to characterize the BNST RXFP3 system in mouse and lay the foundation for future functional studies appraising the role of the murine BNST RXFP3 system in more complex behaviors.

Pharmacogenetic stimulation of neuronal activity increases myelination in an axon-specific manner.

Mounting evidence suggests that neuronal activity influences myelination, potentially allowing for experience-driven modulation of neural circuitry. The degree to which neuronal activity is capable of regulating myelination at the individual axon level is unclear. Here we demonstrate that stimulation of somatosensory axons in the mouse brain increases proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) within the underlying white matter. Stimulated axons display an increased probability of being myelinated compared to neighboring non-stimulated axons, in addition to being ensheathed with thicker myelin. Conversely, attenuating neuronal firing reduces axonal myelination in a selective activity-dependent manner. Our findings reveal that the process of selecting axons for myelination is strongly influenced by the relative activity of individual axons within a population. These observed cellular changes are consistent with the emerging concept that adaptive myelination is a key mechanism for the fine-tuning of neuronal circuitry in the mammalian CNS.