Parafacial neurons in the human brainstem express specific markers for neurons of the retrotrapezoid nucleus.

The retrotrapezoid nucleus (RTN) is a hub for respiratory chemoregulation in the mammal brainstem that integrates chemosensory information from peripheral sites and central relays. Chemosensitive neurons of the RTN express specific genetic and molecular determinants, which have been used to identify RTN precise location within the brainstem of rodents and nonhuman primates. Based on a comparative approach, we hypothesized that among mammals, neurons exhibiting the same specific molecular and genetic signature would have the same function. The co-expression of preprogalanin (PPGAL) and SLC17A6 (VGluT2) mRNAs with duplex in situ hybridization has been studied in formalin fixed paraffin-embedded postmortem human brainstems. Two specimens were processed and analyzed in line with RTN descriptions in adult rats and macaques. Double-labeled PPGAL+/SLC17A6+ neurons were only identified in the parafacial region of the brainstem. These neurons were found surrounding the nucleus of the facial nerve, located ventrally to the nucleus VII on caudal sections, and slightly more dorsally on rostral sections. The expression of neuromedin B (NMB) mRNA as a single marker of chemosensitive RTN neurons has not been confirmed in humans. The location of the RTN in human adults is provided. This should help to develop investigation tools combining anatomic high-resolution imaging and respiratory functional investigations to explore the pathogenic role of the RTN in congenital or acquired neurodegenerative diseases.

Tridimensional mapping of Phox2b expressing neurons in the brainstem of adult Macaca fascicularis and identification of the retrotrapezoid nucleus.

Chemosensitivity is a key mechanism for the regulation of breathing in vertebrates. The retrotrapezoid nucleus is a crucial hub for respiratory chemoreception within the brainstem. It integrates chemosensory information that are both peripheral from the carotid bodies (via the nucleus of the solitary tract) and central through the direct sensing of extracellular protons. To date, the location of a genetically defined RTN has only been ascertained in rodents. We first demonstrated that Phox2b, a key determinant for the development of the visceral nervous system and branchiomotor nuclei in the brainstem including the RTN, had a similar distribution in the brainstem of adult macaques compared to adult rats. Second, based on previous description of a specific molecular signature for the RTN in rats, and on an innovative technique for duplex in situ hybridization, we identified parafacial neurons which coexpressed Phox2b and ppGal mRNAs. They were located ventrally to the nucleus of the facial nerve and extended from the caudal part of the nucleus of the superior olive to the rostral tip of the inferior olive. Using the previously described blockface technique, deformations were corrected to allow the proper alignment and stacking of digitized sections, hence providing for the first time a 3D reconstruction of the macaque brainstem, Phox2b distribution and the primate retrotrapezoid nucleus. This description should help bridging the gap between rodents and humans for the description of key respiratory structures in the brainstem.

Efficacy and Safety Profile of Tricyclo-DNA Antisense Oligonucleotides in Duchenne Muscular Dystrophy Mouse Model.

Antisense oligonucleotides (AONs) hold promise for therapeutic splice-switching correction in many genetic diseases. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake and sufficient therapeutic efficacy is still difficult to achieve. A novel class of AONs made of tricyclo-DNA (tcDNA) is considered very promising for the treatment of Duchenne muscular dystrophy (DMD), a neuromuscular disease typically caused by frameshifting deletions or nonsense mutations in the gene-encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, and respiratory failure in addition to cognitive impairment. Herein, we report the efficacy and toxicology profile of a 13-mer tcDNA in mdx mice. We show that systemic delivery of 13-mer tcDNA allows restoration of dystrophin in skeletal muscles and to a lower extent in the brain, leading to muscle function improvement and correction of behavioral features linked to the emotional/cognitive deficiency. More importantly, tcDNA treatment was generally limited to minimal glomerular changes and few cell necroses in proximal tubules, with only slight variation in serum and urinary kidney toxicity biomarker levels. These results demonstrate an encouraging safety profile for tcDNA, albeit typical of phosphorothiate AONs, and confirm its therapeutic potential for the systemic treatment of DMD patients.

Human spinal ejaculation generator.

OBJECTIVE: A spinal ejaculation generator (SEG) has been identified in the rat with lumbar galaninergic interneurons playing a pivotal role (Science 2002;297:1566-1569). The aim was to evidence a SEG in humans. METHODS: Spatial distribution of galaninergic neurons was studied in postmortem spinal cord segments of 6 men and compared with that of 6 women for evidencing sexual dimorphism. Based on the identified segmental distribution of galaninergic neurons, the ability for penile vibratory stimulation (PVS) to elicit ejaculation when the concerned spinal segments were injured was studied in 384 patients with clinically complete spinal cord injury (SCI) and consequent anejaculation. Such patients represent a unique model to investigate the role of defined spinal segments in the control of ejaculation. RESULTS: Galaninergic neurons were mostly located between L2 and L5 segments in medial lamina VII, with a maximal density within L4. Three-dimensional 3D reconstruction showed that these neurons were grouped into single columns bilaterally to the central canal. In addition, galaninergic neuron density was found higher in L3 and L4 segments in men as compared to women supporting sexual dimorphism. In the patients' cohort, injury of L3-L5 segments was the sole independent predictor for failure of PVS to induce ejaculation. Although evidence from clinical observations was indirect, there is close correspondence to neuroanatomical data. INTERPRETATION: Organization and sexual dimorphism of human spinal galaninergic neurons were similar to the rat's SEG. Neurohistological data, together with clinical results, corroborate the existence of an SEG in humans in L3-L5 segments. Such a generator could be targeted to treat neurogenic and non-neurogenic ejaculatory disorders. ANN NEUROL 2017;81:35-45.

SET translocation is associated with increase in caspase cleaved amyloid precursor protein in CA1 of Alzheimer and Down syndrome patients.

Caspase cleaved amyloid precursor protein (APPcc) and SET are increased and mislocalized in the neuronal cytoplasm in Alzheimer Disease (AD) brains. Translocated SET to the cytoplasm can induce tau hyperphosphorylation. To elucidate the putative relationships between mislocalized APPcc and SET, we studied their level and distribution in the hippocampus of 5 controls, 3 Down syndrome and 10 Alzheimer patients. In Down syndrome and Alzheimer patients, APPcc and SET levels were increased in CA1 and the frequency of both localizations in the neuronal cytoplasm was high in CA1, and low in CA4. As the increase of APPcc is already present at early stages of AD, we overexpressed APPcc in CA1 and the dentate gyrus neurons of adult mice with a lentiviral construct. APPcc overexpression in CA1 and not in the dentate gyrus induced endogenous SET translocation and tau hyperphosphorylation. These data suggest that increase in APPcc in CA1 neurons could be an early event leading to the translocation of SET and the progression of AD through tau hyperphosphorylation.

Citrulline diet supplementation improves specific age-related raft changes in wild-type rodent hippocampus.

The levels of molecules crucial for signal transduction processing change in the brain with aging. Lipid rafts are membrane microdomains involved in cell signaling. We describe here substantial biophysical and biochemical changes occurring within the rafts in hippocampus neurons from aging wild-type rats and mice. Using continuous sucrose density gradients, we observed light-, medium-, and heavy raft subpopulations in young adult rodent hippocampus neurons containing very low levels of amyloid precursor protein (APP) and almost no caveolin-1 (CAV-1). By contrast, old rodents had a homogeneous age-specific high-density caveolar raft subpopulation containing significantly more cholesterol (CHOL), CAV-1, and APP. C99-APP-Cter fragment detection demonstrates that the first step of amyloidogenic APP processing takes place in this caveolar structure during physiological aging of the rat brain. In this age-specific caveolar raft subpopulation, levels of the C99-APP-Cter fragment are exponentially correlated with those of APP, suggesting that high APP concentrations may be associated with a risk of large increases in beta-amyloid peptide levels. Citrulline (an intermediate amino acid of the urea cycle) supplementation in the diet of aged rats for 3 months reduced these age-related hippocampus raft changes, resulting in raft patterns tightly close to those in young animals: CHOL, CAV-1, and APP concentrations were significantly lower and the C99-APP-Cter fragment was less abundant in the heavy raft subpopulation than in controls. Thus, we report substantial changes in raft structures during the aging of rodent hippocampus and describe new and promising areas of investigation concerning the possible protective effect of citrulline on brain function during aging.

Effect of dapoxetine on ejaculatory performance and related brain neuronal activity in rapid ejaculator rats.

INTRODUCTION: A brain network specifically activated when ejaculation occurs has been described in rats. Increasing serotonin (5-hydroxytryptamine [5-HT]) tone impairs ejaculation and chronic 5-HT selective serotonin reuptake inhibitors (SSRIs) are known to inhibit ejaculation. However, efficacy of acute treatment with SSRI varies from one compound to another. The SSRI dapoxetine has been reported to delay ejaculation when given on demand to men with premature ejaculation (PE), although the mechanism of action is unclear. Effects of acute SSRIs on activity of the brain ejaculation circuit in relation with ejaculation have never been examined. AIM: To test the effects of acute administration of the short half-life SSRI dapoxetine on ejaculatory performance and activity in brain ejaculation circuit in rapid ejaculator rats taken as PE model. METHODS: Standard copulatory test was used to attribute one sexual category (sluggish, middle, or rapid) to male rats on the basis of their ejaculatory performance. Parameters of sexual, including ejaculatory, behavior, and Fos level of expression in discrete brain areas were assessed in the three sexual categories and in rapid category following acute oral treatment with dapoxetine. MAIN OUTCOME MEASURES: Ejaculation frequency (EF) and latency (EL) were measured as primary end points of ejaculatory behavior. Density of Fos-immunopositive cells in specific brain areas of brain stem, hypothalamus, and thalamus was determined as marker of neuronal activity. RESULTS: EL and Fos level of expression in hypothalamic and thalamic structures were found related. Dapoxetine acute oral administration (300 mg/kg) to rapid ejaculator rats resulted in (i) diminution of ejaculatory performance (lengthened EL and decreased EF); and (ii) modulation of Fos level of expression in hypothalamic and thalamic nuclei of the brain ejaculatory circuit. CONCLUSION: Acute treatment with dapoxetine, which reduced ejaculatory performance in rapid ejaculator rats, was also accompanied with changes in neuronal activity in components of the brain ejaculatory network.

PAT1 induces cell death signal and SET mislocalization into the cytoplasm by increasing APP/APLP2 at the cell surface.

The cleavage of amyloid precursor protein (APP) by caspases unmasks a domain extending from membrane to caspase cleavage site. This domain induces apoptosis in vitro and in vivo when overexpressed in neurons through the help of an internalization vector. In this model, we previously showed that SET rapidly binds to the internalized domain and is involved in downstream deleterious effects. Under these conditions SET mislocalizes from the nucleus to the cytoplasm, as in Alzheimer's disease (AD). In this report using the same model, we show that PAT1 attaches to the internalized domain earlier than SET and that this binding causes an increase in the levels of APP and APLP2 at the cell surface. Down regulation experiments of PAT1 and of APP and APLP2 show that the increase of the levels of APP and APLP2 at the cell surface triggers the cell death signal and SET mislocalization into the cytoplasm. In the context of AD these data suggest that mislocalization of SET into the cytoplasm may occur downstream of first cell death signal events involving PAT1 protein.

Neuroanatomical distribution of the melanocortin-4 receptors in male and female rodent brain.

The melanocortin-4 receptor (MC4-R) plays a critical role in several physiological functions, from food intake, energy homeostasis, neuroendocrine and cardiovascular function, to sexual responses. The brain regions and the central neuronal pathways mediating the different actions of MC4-R remain largely unknown. We aimed to use immunocytochemistry using a specific antibody against rat MC4-R, to establish the detailed neuroanatomical distribution of MC4-R in brain slices of male and estrous female rats. We demonstrated that MC4-R-positive neurons were widely distributed in several brain regions including the cortex, thalamus, hypothalamus, and brainstem. In both male and female brains, MC4-R-positive cells were especially abundant in the hypothalamus, including the paraventricular hypothalamic nucleus, lateral septal nucleus, arcuate nucleus, supraoptic nucleus, medial preoptic area and lateral hypothalamic area. A moderate number of MC4-R-positive neurons were found in the piriform cortex, bed nucleus of the stria terminalis, medial and basolateral nuclei of amygdala, periaqueductal gray, red nucleus and raphe nucleus. A dimorphic sexual difference in the number of MC4-R-positive neurons was observed in some brain regions. In the medial preoptic area and arcuate nucleus, MC4-R-positive neurons were significantly more abundant in female than in males, whereas in the lateral hypothalamus the opposite proportion was observed. This is the first time the neuroanatomical distribution, and sex differences, of brain MC4-R localisation have been described. The distribution of MC4-R is consistent with the proposed roles of MC4-R-positive neurons and provides further information about the circuitry controlling food intake, energy balance and sexual responses in both males and females.

Neuroanatomical evidence for a role of central melanocortin-4 receptors and oxytocin in the efferent control of the rodent clitoris and vagina.

INTRODUCTION: The clitoris and the vagina are the main peripheral anatomical structures involved in physiological changes related to sexual arousal and orgasm. Their efferent control and, more particularly, the neurochemical phenotype of these descending neuronal pathways remain largely uncharacterized. AIM: To examine if brain neurons involved in the efferent control of the clitoris and the vagina possess melanocortin-4 receptor (MC4-R) and/or contain oxytocin (OT). METHODS: Neurons involved in the efferent control of the vagina and clitoris were identified following visualization of pseudorabies virus (PRV) retrograde tracing. PRV was injected into the vagina and clitoris in adult rats in estrous. On the fifth day postinjection, animals were humanely sacrificed, and brains were removed and sectioned, and processed for PRV visualization. The neurochemical phenotype of PRV-positive neurons was identified using double or triple immunocytochemical labeling against PRV, MC4-R, and OT. Double and triple labeling were quantified using confocal laser scanning microscopy. MAIN OUTCOME MEASURE: Neuroanatomical brain distribution, number and percentage of double-labeled PRV/MC4-R and PRV-/OT-positive neurons, and triple PRV-/MC4-R-/OT-labeled neurons. RESULTS: The majority of PRV immunopositive neurons which also expressed immunoreactivity for MC4-R were located in the paraventricular and arcuate nuclei of the hypothalamus. The majority of PRV positive neurons which were immunoreactive (IR) for OT were located in the paraventricular nucleus (PVN), medial preoptic area (MPOA), and lateral hypothalamus. PRV positive neurons were more likely to be IR for MC4-R than for OT. Scattered triple-labeled PRV/MC4-R/OT neurons were detected in the MPOA and the PVN. CONCLUSION: These data strongly suggest that MC4-R and, to a less extent, OT are involved in the efferent neuronal control of the clitoris and vagina, and consequently facilitate our understanding of how the melanocortinergic pathway regulates female sexual function.