Alterations in the duodenum myenteric neurons of Wistar rats after ingesting of 2,4 dichlorophenoxyacetic acid

The 2,4 dichlorophenoxyacetic acid (2,4-D) is a systemic herbicide whose effects in animal organic systems have been examined in previous studies, being the neurotoxicity considered the predominant effect. However, the studies that detect the 2,4-D neurotoxicity have merely focused in the central nervous system, and therefore, little is known about the effect of this herbicide in the enteric nervous system. This study aimed to verifying the 2,4-D effects on the myenteric neurons in duodenum of Wistar rats. Ten 60-day-old male Wistar rats (Rattus norvegicus) were divided in two groups: control group (C) that did not receive 2,4-D and experimental group (E) that received 5.0 mg of 2,4-D/kg for 15 days. At the end of experimental period, the animal were euthanized, the duodenum was collected and processed for NADPH-diaphorase histochemical analysis in order to expose the nitrergic myenteric neurons (NADPH-dp). In the light microscopy analysis, the whole-mount preparation obtained from duodenum of each animal were image-captured in 120 and 40 fields, for quantitative and morphometric analyses of myenteric neurons, respectively. The neuronal density was not affected when comparing the two groups, but an increase (p > 0.05) of 8.5% was observed in the cell body area of neurons in the E group. In conclusion, the ingestion of 2,4-D at a dosage of 5.0 mg/kg body weight for 15 days does not change the neuronal density, but promotes the hypertrophy of NADPH-dp myenteric neurons in duodenum of the rats of this study.

LESION OF THE COMMISSURAL NUCLEUS OF THE SOLITARY TRACT/A2 NORADRENERGIC NEURONS FACILITATES THE ACTIVATION OF ANGIOTENSINERGIC MECHANISMS IN RESPONSE TO HEMORRHAGE

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Phox2b-expressing retrotrapezoid neurons and the integration of central and peripheral chemosensory control of breathing in conscious rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

IB4(+) and TRPV1(+) sensory neurons mediate pain but not proliferation in a mouse model of squamous cell carcinoma

Background: Cancer pain severely limits function and significantly reduces quality of life. Subtypes of sensory neurons involved in cancer pain and proliferation are not clear.Methods: We produced a cancer model by inoculating human oral squamous cell carcinoma (SCC) cells into the hind paw of athymic mice. We quantified mechanical and thermal nociception using the paw withdrawal assays. Neurotoxins isolectin B4-saporin (IB4-SAP), or capsaicin was injected intrathecally to selectively ablate IB4(+) neurons or TRPV1(+) neurons, respectively. JNJ-17203212, a TRPV1 antagonist, was also injected intrathecally. TRPV1 protein expression in the spinal cord was quantified with western blot. Paw volume was measured by a plethysmometer and was used as an index for tumor size. Ki-67 immunostaining in mouse paw sections was performed to evaluate cancer proliferation in situ.Results: We showed that mice with SCC exhibited both mechanical and thermal hypersensitivity. Selective ablation of IB4(+) neurons by IB4-SAP decreased mechanical allodynia in mice with SCC. Selective ablation of TRPV1(+) neurons by intrathecal capsaicin injection, or TRPV1 antagonism by JNJ-17203212 in the IB4-SAP treated mice completely reversed SCC-induced thermal hyperalgesia, without affecting mechanical allodynia. Furthermore, TRPV1 protein expression was increased in the spinal cord of SCC mice compared to normal mice. Neither removal of IB4(+) or TRPV1(+) neurons affected SCC proliferation.Conclusions: We show in a mouse model that IB4(+) neurons play an important role in cancer-induced mechanical allodynia, while TRPV1 mediates cancer-induced thermal hyperalgesia. Characterization of the sensory fiber subtypes responsible for cancer pain could lead to the development of targeted therapeutics.

Activity of neurons in the preoptic area and their participation in reproductive senescence: preliminary findings

Alterations in the hypothalamic-pituitary-gonadal axis in females determine the transition from regular to irregular reproductive cycles, with loss of fertility. Stimulation of noradrenergic neurons of the anteroventral periventricular neurons (AVPV) is essential for regular reproductive cycles. Therefore, we examined the activity of neurons of the AVPV and measure the noradrenaline (NE) of acyclic rats, in constant estrus, and compared it with that of cyclic rats in estrus. Female cyclic (4-5months) and acyclic (17-18months) rats were euthanized at 10, 14, and 18h in estrus. Brains were processed for immunoreactivity to antigens related to Fos (FRA) in AVPV, and the NE was determined by HPLC-ED. Plasma concentrations of LH, FSH, E2 and P4 were determined. In the acyclic animals, plasma LH was higher but the FSH was lower. There was decreasing P4 at different times, while the E2 was constant and lower in acyclic rats. FRA-ir expression in AVPV neurons of acyclic rats as well as turnover of NE was higher when compared with cyclic group. The preliminary findings showed increased activity in AVPV neurons in aging contribute to changes in the temporal pattern of neuroendocrine signaling, compromising the accuracy of inhibitory and stimulatory effects, causing irregularity in the estrous cycle and determining reproductive senescence.

Catecholaminergic neurons in the comissural region of the nucleus of the solitary tract modulate hyperosmolality-induced responses

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Regulation of ventral surface CO2/H+-sensitive neurons by purinergic signalling

Central chemoreception is the mechanism by which the brain regulates breathing in response to changes in tissue CO2/H+. Abrainstemregion called the retrotrapezoid nucleus (RTN) contains a population of CO2/H+-sensitive neurons that appears to function as an important chemoreceptor. Evidence also indicates that CO2-evoked ATP release from RTN astrocytes modulates activity of CO2/H+-sensitive neurons; however, the extent to which purinergic signalling contributes to chemoreception by RTN neurons is not clear and the mechanism(s) underlying CO2/H+-evoked ATP release is not fully elucidated. The goals of this study are to determine the extent to which ATP contributes to RTN chemoreception both in vivo and in vitro, andwhether purinergic drive to chemoreceptors relies on extracellularCa(2+) or gap junction hemichannels. We also examine the possible contribution of P2Y1 receptors expressed in theRTNto the purinergic drive to breathe. We showthat purinergic signalling contributes, in part, to the CO2/H+ sensitivity of RTN neurons. In vivo, phrenic nerve recordings of respiratory activity in adult rats show that bilateral injections of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS, a P2 receptor blocker) decreased the ventilatory response to CO2 by 30%. In vitro, loose-patch recordings from RTN neurons show that P2 receptor blockers decreased responsiveness to both 10% and 15% CO2 also by 30%. In the slice, the contribution of purinergic signalling to RTN chemoreception did not increase with temperature (22-35 degrees C) and was retained in low extracellular Ca2+ medium. Conversely, the gap junction blockers carbenoxolone and cobalt decreased neuronal CO2/H+ sensitivity by an amount similar to P2 receptor antagonists. Inhibition of the P2Y1 receptor in the RTN had no effect on CO2 responsivness in vitro or in vivo; thus, the identity of P2 receptors underlying the purinergic component of RTN chemoreception remains unknown. These results support the possibility that CO2/H+-evoked ATP release is mediated by a mechanism involving gap junction hemichannels.

Morphological Homogeneity of Neurons: Searching for Outlier Neuronal Cells

We report a morphology-based approach for the automatic identification of outlier neurons, as well as its application to the NeuroMorpho.org database, with more than 5,000 neurons. Each neuron in a given analysis is represented by a feature vector composed of 20 measurements, which are then projected into a two-dimensional space by applying principal component analysis. Bivariate kernel density estimation is then used to obtain the probability distribution for the group of cells, so that the cells with highest probabilities are understood as archetypes while those with the smallest probabilities are classified as outliers. The potential of the methodology is illustrated in several cases involving uniform cell types as well as cell types for specific animal species. The results provide insights regarding the distribution of cells, yielding single and multi-variate clusters, and they suggest that outlier cells tend to be more planar and tortuous. The proposed methodology can be used in several situations involving one or more categories of cells, as well as for detection of new categories and possible artifacts.

Phenotypic alterations of neuropeptide Y and calcitonin gene-related peptide-containing neurons innervating the rat temporomandibular joint during carrageenan-induced arthritis

The aim of this study was to identify immunoreactive neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) neurons in the autonomic and sensory ganglia, specifically neurons that innervate the rat temporomandibular joint (TMJ). A possible variation between the percentages of these neurons in acute and chronic phases of carrageenan-induced arthritis was examined. Retrograde neuronal tracing was combined with indirect immunofluorescence to identify NPY-immuno-reactive (NPY-IR) and CGRP-immunoreactive (CGRP-IR) neurons that send nerve fibers to the normal and arthritic temporomandibular joint. In normal joints, NPY-IR neurons constitute 78 +/- 3%, 77 +/- 6% and 10 +/- 4% of double-labeled nucleated neuronal profile originated from the superior cervical, stellate and otic ganglia, respectively. These percentages in the sympathetic ganglia were significantly decreased in acute (58 +/- 2% for superior cervical ganglion and 58 +/- 8% for stellate ganglion) and chronic (60 +/- 2% for superior cervical ganglion and 59 +/- 15% for stellate ganglion) phases of arthritis, while in the otic ganglion these percentages were significantly increased to 19 +/- 5% and 13 +/- 3%, respectively. In the trigeminal ganglion, CGRP-IR neurons innervating the joint significantly increased from 31 +/- 3% in normal animals to 54 +/- 2% and 49 +/- 3% in the acute and chronic phases of arthritis, respectively. It can be concluded that NPY neurons that send nerve fibers to the rat temporomandibular joint are located mainly in the superior cervical, stellate and otic ganglia. Acute and chronic phases of carrageenan-induced arthritis lead to an increase in the percentage of NPY-IR parasympathetic and CGRP-IR sensory neurons and to a decrease in the percentage of NPY-IR sympathetic neurons related to TMJ innervation.

Exercise training normalizes an increased neuronal excitability of NTS-projecting neurons of the hypothalamic paraventricular nucleus in hypertensive rats

Stern JE, Sonner PM, Son SJ, Silva FC, Jackson K, Michelini LC. Exercise training normalizes an increased neuronal excitability of NTS-projecting neurons of the hypothalamic paraventricular nucleus in hypertensive rats. J Neurophysiol 107: 2912-2921, 2012. First published February 22, 2012; doi:10.1152/jn.00884.2011.-Elevated sympathetic outflow and altered autonomic reflexes, including impaired baroreflex function, are common findings observed in hypertensive disorders. Although a growing body of evidence supports a contribution of preautonomic neurons in the hypothalamic paraventricular nucleus (PVN) to altered autonomic control during hypertension, the precise underlying mechanisms remain unknown. Here, we aimed to determine whether the intrinsic excitability and repetitive firing properties of preautonomic PVN neurons that innervate the nucleus tractus solitarii (PVN-NTS neurons) were altered in spontaneously hypertensive rats (SHR). Moreover, given that exercise training is known to improve and/or correct autonomic deficits in hypertensive conditions, we evaluated whether exercise is an efficient behavioral approach to correct altered neuronal excitability in hypertensive rats. Patch-clamp recordings were obtained from retrogradely labeled PVN-NTS neurons in hypothalamic slices obtained from sedentary (S) and trained (T) Wistar-Kyoto (WKY) and SHR rats. Our results indicate an increased excitability of PVN-NTS neurons in SHR-S rats, reflected by an enhanced input-output function in response to depolarizing stimuli, a hyperpolarizing shift in Na+ spike threshold, and smaller hyperpolarizing afterpotentials. Importantly, we found exercise training in SHR rats to restore all these parameters back to those levels observed in WKY-S rats. In several cases, exercise evoked opposing effects in WKY-S rats compared with SHR-S rats, suggesting that exercise effects on PVN-NTS neurons are state dependent. Taken together, our results suggest that elevated preautonomic PVN-NTS neuronal excitability may contribute to altered autonomic control in SHR rats and that exercise training efficiently corrects these abnormalities.

NADPH- Diaphorase positive cardiac neurons in the atria of mice. A morphoquantitative study

Abstract Background The present study was conducted to determine the location, the morphology and distribution of NADPH-diaphorase positive neurons in the cardiac nerve plexus of the atria of mice (ASn). This plexus lies over the muscular layer of the atria, dorsal to the muscle itself, in the connective tissue of the subepicardium. NADPH- diaphorase staining was performed on whole-mount preparations of the atria mice. For descriptive purposes, all data are presented as means ± SEM. Results The majority of the NADPH-diaphorase positive neurons were observed in the ganglia of the plexus. A few single neurons were also observed. The number of NADPH-d positive neurons was 57 ± 4 (ranging from 39 to 79 neurons). The ganglion neurons were located in 3 distinct groups: (1) in the region situated cranial to the pulmonary veins, (2) caudally to the pulmonary veins, and (3) in the atrial groove. The largest group of neurons was located cranially to the pulmonary veins (66.7%). Three morphological types of NADPH-diaphorase neurons could be distinguished on the basis of their shape: unipolar cells, bipolar cells and cells with three processes (multipolar cells). The unipolar neurons predominated (78.9%), whereas the multipolar were encountered less frequently (5,3%). The sizes (area of maximal cell profile) of the neurons ranged from about 90 μm2to about 220 μm2. Morphometrically, the three types of neurons were similar and there were no significant differences in their sizes. The total number of cardiac neurons (obtained by staining the neurons with NADH-diaphorase method) was 530 ± 23. Therefore, the NADPH-diaphorase positive neurons of the heart represent 10% of the number of cardiac neurons stained by NADH. Conclusion The obtained data have shown that the NADPH-d positive neurons in the cardiac plexus of the atria of mice are morphologically different, and therefore, it is possible that the function of the neurons may also be different.

Differential effects of undernourishment on the differentiation and maturation of rat enteric neurons

The colocalization, number, and size of various classes of enteric neurons immunoreactive (IR) for the purinergic P2X2 and P2X7 receptors (P2X2R, P2X7R) were analyzed in the myenteric and submucosal plexuses of control, undernourished, and re-fed rats. Pregnant rats were exposed to undernourishment (protein-deprivation) or fed a control diet, and their offspring comprised the following experimental groups: rats exposed to a normal diet throughout gestation until postnatal day (P)42, rats protein-deprived throughout gestation and until P42, and rats protein-deprived throughout gestation until P21 and then given a normal diet until P42. Immunohistochemistry was performed on the myenteric and submucosal plexuses to evaluate immunoreactivity for P2X2R, P2X7R, nitric oxide synthase (NOS), choline acetyltransferase (ChAT), calbindin, and calretinin. Double-immunohistochemistry of the myenteric and submucosal plexuses demonstrated that 100% of NOS-IR, calbindin-IR, calretinin-IR, and ChAT-IR neurons in all groups also expressed P2X2R and P2X7R. Neuronal density increased in the myenteric and submucosal plexuses of undernourished rats compared with controls. The average size (profile area) of some types of neurons in the myenteric and submucosal plexuses was smaller in the undernourished than in the control animals. These changes appeared to be reversible, as animals initially undernourished but then fed a normal diet at P21 (re-feeding) were similar to controls. Thus, P2X2R and P2X7R are present in NOS-positive inhibitory neurons, calbindin- and calretinin-positive intrinsic primary afferent neurons, cholinergic secretomotor neurons, and vasomotor neurons in rats. Alterations in these neurons during undernourishment are reversible following re-feeding

Theoretical and implementation aspects in the mechanization of the metatheory of programming languages

Interactive theorem provers are tools designed for the certification of formal proofs developed by means of man-machine collaboration. Formal proofs obtained in this way cover a large variety of logical theories, ranging from the branches of mainstream mathematics, to the field of software verification. The border between these two worlds is marked by results in theoretical computer science and proofs related to the metatheory of programming languages. This last field, which is an obvious application of interactive theorem proving, poses nonetheless a serious challenge to the users of such tools, due both to the particularly structured way in which these proofs are constructed, and to difficulties related to the management of notions typical of programming languages like variable binding. This thesis is composed of two parts, discussing our experience in the development of the Matita interactive theorem prover and its use in the mechanization of the metatheory of programming languages. More specifically, part I covers: - the results of our effort in providing a better framework for the development of tactics for Matita, in order to make their implementation and debugging easier, also resulting in a much clearer code; - a discussion of the implementation of two tactics, providing infrastructure for the unification of constructor forms and the inversion of inductive predicates; we point out interactions between induction and inversion and provide an advancement over the state of the art. In the second part of the thesis, we focus on aspects related to the formalization of programming languages. We describe two works of ours: - a discussion of basic issues we encountered in our formalizations of part 1A of the Poplmark challenge, where we apply the extended inversion principles we implemented for Matita; - a formalization of an algebraic logical framework, posing more complex challenges, including multiple binding and a form of hereditary substitution; this work adopts, for the encoding of binding, an extension of Masahiko Sato's canonical locally named representation we designed during our visit to the Laboratory for Foundations of Computer Science at the University of Edinburgh, under the supervision of Randy Pollack.

The Social Principles of Jesus: A Critical Re-examination of Walter Rauschenbusch’s Social Gospel

It has been nine decades since Walter Rauschenbusch (1861-1918) published a slim volume entitled The Social Principles of Jesus. Though today less well known than his other works Christianity and the Social Crisis (1907) and Theology for the Social Gospel (1917), it is Social Principles that most adeptly summarizes the theological ethics of Rauschenbusch’s “social gospel.” Taking the form of a pedagogical treatise, Social Principles reads as both a finely tuned analysis of the modern relevance of the teachings of Jesus, and an impassioned plea on the part of its author for an end to the folly of interpreting Christianity solely in “individualistic” terms. It is Rauschenbusch’s expressed aim to resurrect the core teachings of Jesus, which are social and ethical, and apply these to a renewed, socially conscious liberal democracy, establishing a grand harmony between religion, ethics, and social evolution. How far this vision was from the burgeoning “fundamentalism” of his day (and ours) is more than evidenced by the critical reaction of many of his more conservative peers, but also indicates the continuing relevance of his work for theologians and others looking for alternative paths. The following exposition is supplemented with appreciative and critical comments.

Translational neuroprotection research in glaucoma: a review of definitions and principles

The maintenance of vision, through prevention and attenuation of neuronal injury in glaucoma, forms the basis of current clinical practice. Currently, the reduction of intraocular pressure is the only proven method to achieve these goals. Although this strategy enjoys considerable success, some patients progress to blindness; hence, additional management options are highly desirable. Several terms describing treatment modalities of neuronal diseases with potential applicability to glaucoma are used in the literature, including neuroprotection, neurorecovery, neurorescue and neuroregeneration. These phenomena have not been defined within a coherent framework. Here, we suggest a set of definitions, postulates and principles to form a foundation for the successful translation of novel glaucoma therapies from the laboratory to the clinic.