CRF release from a unique subpopulation of accumbal neurons constrains action-outcome acquisition in reward learning.

Background: The nucleus accumbens (NAc) mediates reward learning and motivation. Despite an abundance of neuropeptides, peptidergic neurotransmission from the NAc has not been integrated into current models of reward learning. The existence of a sparse population of neurons containing corticotropin releasing factor (CRF) has been previously documented. Here we provide a comprehensive analysis of their identity and functional role in shaping reward learning. Methods: To do this, we took a multidisciplinary approach that included florescent in situ hybridization (N mice ≥ 3), tract tracing (N mice = 5), ex vivo electrophysiology (N cells ≥ 30), in vivo calcium imaging with fiber photometry (N mice ≥ 4) and use of viral strategies in transgenic lines to selectively delete CRF peptide from NAc neurons (N mice ≥ 4). Behaviors used were instrumental learning, sucrose preference and spontaneous exploration in an open field. Results: Here we show that the vast majority of NAc CRF-containing (NAc CRF ) neurons are spiny projection neurons (SPNs) comprised of dopamine D1-, D2- or D1/D2-containing SPNs that primarily project and connect to the ventral pallidum and to a lesser extent the ventral midbrain. As a population, they display mature and immature SPN firing properties. We demonstrate that NAc CRF neurons track reward outcomes during operant reward learning and that CRF release from these neurons acts to constrain initial acquisition of action-outcome learning, and at the same time facilitates flexibility in the face of changing contingencies. Conclusion: We conclude that CRF release from this sparse population of SPNs is critical for reward learning under normal conditions.

Cholinergic interneurons in the nucleus accumbens are a site of cellular convergence for corticotropin release factor and estrogen regulation.

Cholinergic interneurons (ChIs) act as master regulators of striatal output, finely tuning neurotransmission to control motivated behaviors. ChIs are a cellular target of many peptide and hormonal neuromodulators, including corticotropin releasing factor, opioids, insulin and leptin, which can influence an animal's behavior by signaling stress, pleasure, pain and nutritional status. However, little is known about how sex hormones via estrogen receptors influence the function of these other neuromodulators. Here, we performed in situ hybridization on mouse striatal tissue to characterize the effect of sex and sex hormones on choline acetyltransferase ( Chat ), estrogen receptor alpha ( Esr1 ), and corticotropin releasing factor type 1 receptor ( Crhr1 ) expression. Although we did not detect sex differences in ChAT protein levels in the striatum, we found that female mice have more Chat mRNA-expressing neurons than males. At the population level, we observed a sexually dimorphic distribution of Esr1 - and Crhr1 -expressing ChIs in the ventral striatum that demonstrates an antagonistic correlational relationship, which is abolished by ovariectomy. Only in the NAc did we find a significant population of ChIs that co-express Crhr1 and Esr1 . At the cellular level, Crhr1 and Esr1 transcript levels were negatively correlated only during estrus, indicating that changes in sex hormones levels can modulate the interaction between Crhr1 and Esr1 mRNA levels. Together, these data provide evidence for the unique expression and interaction of Esr1 and Crhr1 in ventral striatal ChIs, warranting further investigation into how these transcriptomic patterns might underlie important functions for ChIs at the intersection of stress and reproductive behaviors.

Neonatal screening for spinal muscular atrophy: A pilot study in Brazil.

Spinal muscular atrophy (SMA) is considered one of the most common autosomal recessive disorders, with an estimated incidence of 1 in 10,000 live births. Testing for SMA has been recommended for inclusion in neonatal screening (NBS) panels since there are several therapies available and there is evidence of greater efficacy when introduced in the pre/early symptomatic phases. In Brazil, the National Neonatal Screening Program tests for six diseases, with a new law issued in 2021 stating that it should incorporate more diseases, including SMA. In the present study, dried blood spot (DBS) samples collected by the Reference Services of Neonatal Screening of RS and SP, to perform the conventional test were also screened for SMA, using real-time PCR, with SALSA MC002 technique. A total of 40,000 samples were analyzed, enabling the identification of four positive cases of SMA, that were confirmed by MLPA. Considering our sampling, Brazil seems to have an incidence comparable to the described in other regions. This work demonstrated that the use of the MC002 technique in samples routinely collected for the conventional NBS program is suitable to screen for SMA in our conditions and can be included in the expansion of the neonatal screening programs.

T cell biology in neuromuscular disorders: a focus on Duchenne Muscular Dystrophy and Amyotrophic Lateral Sclerosis.

Growing evidence demonstrates a continuous interaction between the immune system, the nerve and the muscle in neuromuscular disorders of different pathogenetic origins, such as Duchenne Muscular Dystrophy (DMD) and Amyotrophic Lateral Sclerosis (ALS), the focus of this review. Herein we highlight the complexity of the cellular and molecular interactions involving the immune system in neuromuscular disorders, as exemplified by DMD and ALS. We describe the distinct types of cell-mediated interactions, such as cytokine/chemokine production as well as cell-matrix and cell-cell interactions between T lymphocytes and other immune cells, which target cells of the muscular or nervous tissues. Most of these interactions occur independently of exogenous pathogens, through ligand-receptor binding and subsequent signal transduction cascades, at distinct levels of specificity. Although this issue reveals the complexity of the system, it can also be envisioned as a window of opportunity to design therapeutic strategies (including synthetic moieties, cell and gene therapy, as well as immunotherapy) by acting upon one or more targets. In this respect, we discuss ongoing clinical trials using VLA-4 inhibition in DMD, and in ALS, with a focus on regulatory T cells, both revealing promising results.

Corticotropin releasing factor alters the functional diversity of accumbal cholinergic interneurons.

Cholinergic interneurons (ChIs) provide the main source of acetylcholine in the striatum and have emerged as a critical modulator of behavioral flexibility, motivation, and associative learning. In the dorsal striatum, ChIs display heterogeneous firing patterns. Here, we investigated the spontaneous firing patterns of ChIs in the nucleus accumbens (NAc) shell, a region of the ventral striatum. Using male and female mice, we performed cell-attached patch clamp electrophysiology recordings from ChIs. We identified four distinct ChI firing signatures: regular single-spiking, irregular single-spiking, rhythmic bursting followed by pauses or low activity, and a mixed-mode pattern composed of bursting activity and regular single spiking. ChIs from females had lower firing rates compared to males and had both a higher proportion of mixed-mode firing patterns and a lower proportion of regular single-spiking neurons compared to males. We further observed that across the estrous cycle, the estrus phase was characterized by higher proportions of mixed-mode, rhythmic bursting, and irregular ChI firing patterns compared to other phases. ChI firing mode was not driven by glutamatergic synaptic transmission in the slice preparation. Using pooled data from males and females, we examined the how the stress-associated neuropeptide corticotropin releasing factor (CRF) impacts these firing patterns. ChI firing patterns showed differential sensitivity to CRF. Furthermore, CRF shifted the proportion of ChI firing patterns toward more regular spiking activity over bursting patterns. These findings highlight the heterogeneous nature of ChI firing patterns, which may have implications for accumbal-dependent motivated behaviors. New and Noteworthy: ChIs within the dorsal and ventral striatum can exert a huge influence on network output and motivated behaviors. However, the firing patterns and neuromodulation of ChIs within the ventral striatum,specifically the NAc shell, are understudied. Here we report that NAc shell ChIs have a heterogenous distribution of ChI firing patterns that are labile and can be modulated by the stress-linked neuropeptide CRF and by estrous cycle.

Spatial dysregulation of T follicular helper cells impairs vaccine responses in aging.

The magnitude and quality of the germinal center (GC) response decline with age, resulting in poor vaccine-induced immunity in older individuals. A functional GC requires the co-ordination of multiple cell types across time and space, in particular across its two functionally distinct compartments: the light and dark zones. In aged mice, there is CXCR4-mediated mislocalization of T follicular helper (TFH) cells to the dark zone and a compressed network of follicular dendritic cells (FDCs) in the light zone. Here we show that TFH cell localization is critical for the quality of the antibody response and for the expansion of the FDC network upon immunization. The smaller GC and compressed FDC network in aged mice were corrected by provision of TFH cells that colocalize with FDCs using CXCR5. This demonstrates that the age-dependent defects in the GC response are reversible and shows that TFH cells support stromal cell responses to vaccines.

WHIM Syndrome-linked CXCR4 mutations drive osteoporosis.

WHIM Syndrome is a rare immunodeficiency caused by gain-of-function CXCR4 mutations. Here we report a decrease in bone mineral density in 25% of WHIM patients and bone defects leading to osteoporosis in a WHIM mouse model. Imbalanced bone tissue is observed in mutant mice combining reduced osteoprogenitor cells and increased osteoclast numbers. Mechanistically, impaired CXCR4 desensitization disrupts cell cycle progression and osteogenic commitment of skeletal stromal/stem cells, while increasing their pro-osteoclastogenic capacities. Impaired osteogenic differentiation is evidenced in primary bone marrow stromal cells from WHIM patients. In mice, chronic treatment with the CXCR4 antagonist AMD3100 normalizes in vitro osteogenic fate of mutant skeletal stromal/stem cells and reverses in vivo the loss of skeletal cells, demonstrating that proper CXCR4 desensitization is required for the osteogenic specification of skeletal stromal/stem cells. Our study provides mechanistic insights into how CXCR4 signaling regulates the osteogenic fate of skeletal cells and the balance between bone formation and resorption.

Arterial stiffness indices, pulse wave velocity and central systolic blood pressure, are able to discriminate between obese and non-obese children.

The objectives of this study were to verify, first, if arterial stiffness indices can discriminate between obese and healthy children. Second, to evaluate arterial stiffness index predictors and hemodynamic parameters in obese children. Arterial stiffness indices evaluated were pulse wave velocity (PWV), central systolic blood pressure (SBPc), and central pulse pressure (PPc). A cross-sectional, descriptive, comparative study design was used. The sample consisted of 78 normal-weight children (8.1 ± 1.96 years) and 58 obese children (9.0 ± 1.87 years). PWV, PPc, and SBPc were significantly higher in the group of obese children than in the control group. The ROC curve analysis showed that maximum PWV and SBPc sensitivity and specificity in differentiating obese from non-obese children occurred at 4.09 m/s and 86.17 mmHg, respectively. PPc did not exhibit a discriminatory capacity between the two groups. Peripheral systolic blood pressure (SBPp), peripheral pulse pressure (PPp), and PPc (R2 = 0.98) were predictors of increased PWV. Augmentation pressure, PPp, and reflection coefficient (R2 = 0.873) were predictors of PPc. Age, augmentation index, total vascular resistance, cardiac index, and mean fat percentage (R2 = 0.801) were predictors of SBPc. CONCLUSION: This study shows for the first time that PWV > 4.09 m/s and SBPc > 86.17 mmHg are cut-off points associated with a higher risk of obesity. These results indicate that the simple, rapid, and noninvasive measurement of arterial stiffness adds prognostic information regarding cardiovascular risk, in addition to increased body mass index. WHAT IS KNOWN: • Overweight and obesity are strongly associated with comorbidities que contribute to the development of cardiovascular diseases. WHAT IS NEW: • This is the first study to show that PWV and SBPc can discriminate obese from non-obese children. These results show that, in addition to an increased BMI, a simple, rapid, and noninvasive measurement of arterial stiffness adds prognostic information on cardiovascular risk.

Chronic loss of muscarinic M5 receptor function manifests disparate impairments in exploratory behavior in male and female mice despite common dopamine regulation.

There are five cloned muscarinic acetylcholine receptors (M1-M5). Of these, the muscarinic type 5 receptor (M5) is the only one localized to dopamine neurons in the ventral tegmental area and substantia nigra. Unlike M1-M4, the M5 receptor has relatively restricted expression in the brain, making it an attractive therapeutic target. Here we performed an in-depth characterization of M5-dependent potentiation of dopamine transmission in the nucleus accumbens and accompanying exploratory behaviors in male and female mice. We show that M5 receptors potentiate dopamine transmission by acting directly on the terminals within the nucleus accumbens. Using the muscarinic agonist oxotremorine, we revealed a unique concentration-response curve and a sensitivity to repeated forced swim stress or restraint stress exposure. We found that constitutive deletion of M5 receptors reduced exploration of the center of an open field while at the same time impairing normal habituation only in male mice. In addition, M5 deletion reduced exploration of salient stimuli, especially under conditions of high novelty, yet had no effect on hedonia assayed using the sucrose preference test or on stress coping strategy assayed using the forced swim test. We conclude that M5 receptors are critical for both engaging with the environment and updating behavioral output in response to environment cues, specifically in male mice. A cardinal feature of mood and anxiety disorders is withdrawal from the environment. These data indicate that boosting M5 receptor activity may be a useful therapeutic target for ameliorating these symptoms of depression and anxiety.Significance Statement:The basic physiological and behavioral functions of the muscarinic M5 receptor remain understudied. Furthermore, its presence on dopamine neurons, relatively restricted expression in the brain, and recent crystallization make it an attractive target for therapeutic development. Yet, most preclinical studies of M5 receptor function have primarily focused on substance use disorders in male rodents. Here we characterized the role of M5 receptors in potentiating dopamine transmission in the nucleus accumbens, finding impaired functioning after stress exposure. Furthermore, we show that M5 receptors can modulate exploratory behavior in a sex-specific manner, without impacting hedonic behavior. These findings further illustrate the therapeutic potential of the M5 receptor, warranting further research in the context of treating mood disorders.

Ventral tegmental area GABAergic inhibition of cholinergic interneurons in the ventral nucleus accumbens shell promotes reward reinforcement.

The long-range GABAergic input from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) is relatively understudied, and therefore its role in reward processing has remained unknown. In the present study, we show, in both male and female mice, that long-range GABAergic projections from the VTA to the ventral NAc shell, but not to the dorsal NAc shell or NAc core, are engaged in reward and reinforcement behavior. We show that this GABAergic projection exclusively synapses on to cholinergic interneurons (CINs) in the ventral NAc shell, thereby serving a specialized function in modulating reinforced reward behavior through the inhibition of ventral NAc shell CINs. These findings highlight the diversity in the structural and functional topography of VTA GABAergic projections, and their neuromodulatory interactions across the dorsoventral gradient of the NAc shell. They also further our understanding of neuronal circuits that are directly implicated in neuropsychiatric conditions such as depression and addiction.

Culture, Expansion and Differentiation of Mouse Bone-Derived Mesenchymal Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) are multipotent adult cells that are present in several tissues including the bone marrow (BM), in which they can differentiate in a variety of cell types such as osteoblasts, chondrocytes and adipocytes. The isolation of MSCs has been carried out by many studies that aim to control their differentiation into cartilaginous and bone cells in vitro in order to use this technology in the repair of damaged tissues. Here we describe the minimum requirements and an efficient method for isolation, expansion of mouse bone-derived multipotent mesenchymal stromal cells and their differentiation into osteoblasts, responsible for the bone matrix synthesis and mineralization.

Hematopoietic Multipotent Progenitors and Plasma Cells: Neighbors or Roommates in the Mouse Bone Marrow Ecosystem?

The bone marrow is a complex ecosystem in which hematopoietic and non-hematopoietic cells reside. In this review, we discuss the bone marrow niches in mice that facilitate the survival, maintenance, and differentiation of cells of hematopoietic origin based on the recent literature. Our review places a special focus on the hematopoietic multipotent progenitors and on plasma cells, corresponding to the last stage of the B-cell lineage, that play a key role in the humoral memory response. We highlight the similarities between the microenvironments necessary for the establishment and the maintenance of these two immune cell subsets, and how the chemokine CXCL12/CXCR4 signaling axis contributes to these processes. Finally, we bring elements to address the following question: are multipotent progenitors and plasma cells neighbors or roommates within the bone marrow?

Insidious Transmission of a Stress-Related Neuroadaptation.

Stress is highly pervasive in humans, impacting motivated behaviors with an enormous toll on life quality. Many of the effects of stress are orchestrated by neuropeptides such as corticotropin-releasing factor (CRF). It has previously been shown that in stress-naïve male mice, CRF acts in the core of the nucleus accumbens (NAc) to produce appetitive effects and to increase dopamine release; yet in stress-exposed male mice, CRF loses its capacity to modulate NAc dopamine release and is aversive. In the current research, we tested whether this effect is comparable in females to males and whether the neuroadaptation is susceptible to social transmission. We found that, like in males, CRF increased dopamine release in stress-naïve but not stress-exposed female mice. Importantly, this persistent physiological change was not accompanied by overt behavioral changes that would be indicative of depression- or anxiety-like phenotype. Nonetheless, when these mice were housed for 7 days with stress-naïve conspecifics, the cage mates also exhibited a loss of dopamine potentiation by CRF. These data demonstrate the asymptomatic, yet pervasive transmission of stress-related neuroadaptations in the population.

Prefrontal Cortex-Driven Dopamine Signals in the Striatum Show Unique Spatial and Pharmacological Properties.

Dopamine (DA) signals in the striatum are critical for a variety of vital processes, including motivation, motor learning, and reinforcement learning. Striatal DA signals can be evoked by direct activation of inputs from midbrain DA neurons (DANs) as well as cortical and thalamic inputs to the striatum. In this study, we show that in vivo optogenetic stimulation of prelimbic (PrL) and infralimbic (IL) cortical afferents to the striatum triggers an increase in extracellular DA concentration, which coincides with elevation of striatal acetylcholine (ACh) levels. This increase is blocked by a nicotinic ACh receptor (nAChR) antagonist. Using single or dual optogenetic stimulation in brain slices from male and female mice, we compared the properties of these PrL/IL-evoked DA signals with those evoked by stimulation from midbrain DAN axonal projections. PrL/IL-evoked DA signals are undistinguishable from DAN evoked DA signals in their amplitudes and electrochemical properties. However, PrL/IL-evoked DA signals are spatially restricted and preferentially recorded in the dorsomedial striatum. PrL/IL-evoked DA signals also differ in their pharmacological properties, requiring activation of glutamate and nicotinic ACh receptors. Thus, both in vivo and in vitro results indicate that cortical evoked DA signals rely on recruitment of cholinergic interneurons, which renders DA signals less able to summate during trains of stimulation and more sensitive to both cholinergic drugs and temperature. In conclusion, cortical and midbrain inputs to the striatum evoke DA signals with unique spatial and pharmacological properties that likely shape their functional roles and behavioral relevance.SIGNIFICANCE STATEMENT Dopamine signals in the striatum play a critical role in basal ganglia function, such as reinforcement and motor learning. Different afferents to the striatum can trigger dopamine signals, but their release properties are not well understood. Further, these input-specific dopamine signals have only been studied in separate animals. Here we show that optogenetic stimulation of cortical glutamatergic afferents to the striatum triggers dopamine signals both in vivo and in vitro These afferents engage cholinergic interneurons, which drive dopamine release from dopamine neuron axons by activation of nicotinic acetylcholine receptors. We also show that cortically evoked dopamine signals have other unique properties, including spatial restriction and sensitivity to temperature changes than dopamine signals evoked by stimulation of midbrain dopamine neuron axons.

Respiratory muscle training in non-athletes and athletes with spinal cord injury: A systematic review of the effects on pulmonary function, respiratory muscle strength and endurance, and cardiorespiratory fitness based on the FITT principle of exercise prescription.

BACKGROUND: Respiratory muscle training (RMT) has been recommended to mitigate impacts of spinal cord injuries (SCI), but the optimal dosage in terms of the frequency, intensity, time, and type (FITT principle) to promote health in SCI individuals remains unclear. OBJECTIVE: To discuss research related to the effects of RMT on pulmonary function, respiratory muscle strength and cardiorespiratory fitness in athletes and non-athletes with SCI, presenting the FITT principle. METHODS: We performed a systematic review. PubMed, Lilacs, Scopus, Web of Science, PEDro, SciELO and Cochrane databases were searched between 1989 and August 2018. Participants were athletes and non-athletes with SCI. RESULTS: 4,354 studies were found, of which only 17 met the eligibility criteria. Results indicated that RMT is associated with beneficial changes in pulmonary function and respiratory muscle strength and endurance among athletes and non-athletes, whereas no effect was reported for maximal oxygen uptake. It was not possible to establish an optimal RMT dose from the FITT principle, but combined inspiratory/expiratory muscle training seems to promote greater respiratory changes than isolated IMT or EMT. CONCLUSION: The use of RMT elicits benefits in ventilatory variables of athletes and non-athletes with SCI. However, it remains unclear which RMT type and protocol should be used to maximize benefits.

Zika virus infects human blood mononuclear cells.

BACKGROUND: Zika virus (ZIKV) infection gained public health concern after the 2015 outbreak in Brazil, when microcephaly rates increased in babies born from infected mothers. It was demonstrated that ZIKV causes a congenital Zika virus syndrome, including various alterations in the development of the central nervous system. Although the infection of cells from the nervous system has been well documented, less is known in respect of ZIKV ability to infect immune cells. Herein, we investigated if peripheral blood mononuclear cells (PBMCs), freshly-isolated from healthy donors, could be infected by ZIKV. METHODS: PBMCs from healthy donors were isolated and cultured in medium with ZIKV strain Rio-U1 (MOI = 0.1). Infection was analyzed by RT-qPCR and flow cytometry. RESULTS: We detected the ZIKV RNA in PBMCs from all donors by RT-qPCR analysis. The detection of viral antigens by flow cytometry revealed that PBMC from more than 50% the donors were infected by ZIKV, with CD3+CD4+ T cells, CD3-CD19+ B cells and CD3+CD8+ T cells being, respectively, the most frequently infected subpopulations, followed by CD14+ monocytes. Additionally, we observed high variability in PBMC infection rates among different donors, either by numbers or type infected cells. CONCLUSIONS: These findings raise the hypothesis that PBMCs can act as a reservoir of the virus, which may facilitate viral dissemination to different organs, including immune-privileged sites.

Striatal Cholinergic Interneurons Are a Novel Target of Corticotropin Releasing Factor.

Cholinergic interneurons (CINs) are critical regulators of striatal network activity and output. Changes in CIN activity are thought to encode salient changes in the environment and stimulus-response-outcome associations. Here we report that the stress-associated neuropeptide corticotropin releasing factor (CRF) produces a profound and reliable increase in the spontaneous firing of CINs in both dorsal striatum and nucleus accumbens (NAc) through activation of CRF type 1 receptors, production of cAMP and reduction in spike accommodation in male mice. The increase of CIN firing by CRF results in the activation muscarinic acetylcholine receptors type 5, which mediate potentiation of dopamine transmission in the striatum. This study provides critical mechanistic insight into how CRF modulates striatal activity and dopamine transmission in the NAc to likely account for CRF facilitation of appetitive behaviors.SIGNIFICANCE STATEMENT Although the presence of CRF receptors in the dorsal and ventral striatum has been acknowledged, the cellular identity and the functional consequences of receptor activation is unknown. Here we report that striatal cholinergic interneurons express CRF-R1 receptors and are acutely activated by the neuropeptide CRF that is released in response to salient environmental stimuli. Cholinergic interneurons make <1% of the cells in the striatum but are critical regulators of the striatal circuitry and its output. CRF's fast and potent activation of cholinergic interneurons could have far reaching behavioral implications across motivated behaviors controlled by the striatum.