Developmental expression of CagMdkb during gibel carp embryogenesis

Midkine (Mdk) genes have been revealed to have different expression patterns in vertebrates and therefore, additional studies on Mdk expression patterns are required in more species. In this study, CagMdkb has been cloned and characterized from a SMART cDNA library of 10-somite stage embryos of Carassius auratus gibelio. Its full length cDNA is 1091 bp and encodes a sequence of 147 amino acids, which shows 97.3% identity to zebrafish Mdkb on the amino acid level. RT-PCR analysis reveals that CagMdkb is first transcribed in gastrula embryos and maintains a relatively stable expression level during subsequent embryogenesis. Western blot analysis reveals a 19 kDa maternal CagMdkb protein band and the zygotic CagMdkb protein is expressed from gastrula stage. At around 10 somite stage, the 19 kDa CagMdkb is processed to another protein band of about 17 kDa, which might be the secreted form with the 21-residue signal peptide removed. With immunofluorescence analysis, maternal CagMdkb protein was found to be localized in each blastamere cell of early embryos. The zygotic CagMdkb positive fluorescence signal was detected from a pair of large neurons at 18-somite stage. At the later stages, CagMdkb protein was also extended to numerous small neurons in the forebrain, midbrain and hindbrain, as well as to nerve fibers in the spinal cord. Co-localization with 3A10 antibody revealed CagMdkb immunoreactivity on developing Mauthner neurons, a member of reticulospinal neurons. In addition, ectopic expression of CagMdkb in early embryos of gibel carp and zebrafish suppressed head formation and CagMdkb function was found to depend on secretory activity. All these findings indicate that CagMdkb plays an important role in neural development during gibel carp embryogenesis and there is functional conservation of Mdkb in fish head formation.

Motor systems: reaching out and grasping the molecular tools.

Two recent studies provide important insights into the organization of premotor circuitries, showing that control of highly-specific skilled forelimb movements, such as reaching and grasping, requires activation of specific subpopulations of neurons in the brainstem and spinal cord.

Twitching in sensorimotor development from sleeping rats to robots.

It is still not known how the 'rudimentary' movements of fetuses and infants are transformed into the coordinated, flexible and adaptive movements of adults. In addressing this important issue, we consider a behavior that has been perennially viewed as a functionless by-product of a dreaming brain: the jerky limb movements called myoclonic twitches. Recent work has identified the neural mechanisms that produce twitching as well as those that convey sensory feedback from twitching limbs to the spinal cord and brain. In turn, these mechanistic insights have helped inspire new ideas about the functional roles that twitching might play in the self-organization of spinal and supraspinal sensorimotor circuits. Striking support for these ideas is coming from the field of developmental robotics: when twitches are mimicked in robot models of the musculoskeletal system, the basic neural circuitry undergoes self-organization. Mutually inspired biological and synthetic approaches promise not only to produce better robots, but also to solve fundamental problems concerning the developmental origins of sensorimotor maps in the spinal cord and brain.

Fisiopatologia da Dor

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

As neurotoxinas de clostridium sp.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Neural Control of Interlimb Coordination and Gait Timing in Bipeds and Quadrupeds

1) A large body of behavioral data conceming animal and human gaits and gait transitions is simulated as emergent properties of a central pattern generator (CPG) model. The CPG model incorporates neurons obeying Hodgkin-Huxley type dynamics that interact via an on-center off-surround anatomy whose excitatory signals operate on a faster time scale than their inhibitory signals. A descending cornmand or arousal signal called a GO signal activates the gaits and controL their transitions. The GO signal and the CPG model are compared with neural data from globus pallidus and spinal cord, among other brain structures. 2) Data from human bimanual finger coordination tasks are simulated in which anti-phase oscillations at low frequencies spontaneously switch to in-phase oscillations at high frequencies, in-phase oscillations can be performed both at low and high frequencies, phase fluctuations occur at the anti-phase in-phase transition, and a "seagull effect" of larger errors occurs at intermediate phases. When driven by environmental patterns with intermediate phase relationships, the model's output exhibits a tendency to slip toward purely in-phase and anti-phase relationships as observed in humans subjects. 3) Quadruped vertebrate gaits, including the amble, the walk, all three pairwise gaits (trot, pace, and gallop) and the pronk are simulated. Rapid gait transitions are simulated in the order--walk, trot, pace, and gallop--that occurs in the cat, along with the observed increase in oscillation frequency. 4) Precise control of quadruped gait switching is achieved in the model by using GO-dependent modulation of the model's inhibitory interactions. This generates a different functional connectivity in a single CPG at different arousal levels. Such task-specific modulation of functional connectivity in neural pattern generators has been experimentally reported in invertebrates. Phase-dependent modulation of reflex gain has been observed in cats. A role for state-dependent modulation is herein predicted to occur in vertebrates for precise control of phase transitions from one gait to another. 5) The primary human gaits (the walk and the run) and elephant gaits (the amble and the walk) are sirnulated. Although these two gaits are qualitatively different, they both have the same limb order and may exhibit oscillation frequencies that overlap. The CPG model simulates the walk and the run by generating oscillations which exhibit the same phase relationships. but qualitatively different waveform shapes, at different GO signal levels. The fraction of each cycle that activity is above threshold quantitatively distinguishes the two gaits, much as the duty cycles of the feet are longer in the walk than in the run. 6) A key model properly concerns the ability of a single model CPG, that obeys a fixed set of opponent processing equations to generate both in-phase and anti-phase oscillations at different arousal levels. Phase transitions from either in-phase to anti-phase oscillations, or from anti-phase to in-phase oscillations, can occur in different parameter ranges, as the GO signal increases.

An investigation into mechanisms of visceral pain in rodents

Visceral pain is a debilitating symptom of irritable bowel syndrome (IBS), a disorder affecting up to 30% of adults. A better understanding of the mechanisms underlying visceral hypersensitivity may facilitate development of more targeted therapies, improving the quality of life of these individuals. The studies performed in this thesis were designed to investigate important factors of visceral pain, including early-life manipulations, genetic predisposition and sex hormones. Maternal separation (MS) consistently reproduces visceral hypersensitivity and altered anxiety-like behaviours in rats, symptoms associated with IBS. It has been found that 5-HT2B receptor antagonism blocks visceral pain but no difference in relative 5-HT2B receptor mRNA expression was found in hippocampus, amygdala and colon. The neuronal activation patterns of prefrontal cortex and amygdala of MS rats were then investigated. MS animals are characterised by differential activation of the prefrontal cortex (anterior cingulate cortex (ACC), infralibic cortex, prelimbic cortex) as well as the central nucleus of the amygdala (CeA). Genetic factors also contribute to pain syndromes such as IBS. We utilised the Wistar Kyoto (WKY) rat, a stress-sensitive strain, as an animal model of brain-gut axis dysfunction. WKY rats have a lower expression of the glutamate transporter EAAT2 and mGlu4 receptor in the ACC. Another early-life factor that can increase susceptibility to functional gastrointestinal symptoms later life is disruption of the gut microbiota, thus early-life antibiotic treatment was used to assess this effect. Antibiotic treatment induced visceral hypersensitivity in adulthood and may be related to observed reductions in spinal cord alpha-2A adrenoreceptor (adra2A) mRNA. Lastly, we investigated sex differences in visceral sensitivity. EAAT1 & 2 mRNA levels are lower in females, potentially increasing glutamatergic concentration at the symaptic level. Moreover, NR1 and NR2B subunits mRNA of NMDA receptor were increased in caudal ACC of females. These findings may account for sex differences in visceral sensitivity.

Differential mechanisms of morphine antinociceptive tolerance revealed in (beta)arrestin-2 knock-out mice.

Morphine induces antinociception by activating mu opioid receptors (muORs) in spinal and supraspinal regions of the CNS. (Beta)arrestin-2 (beta)arr2), a G-protein-coupled receptor-regulating protein, regulates the muOR in vivo. We have shown previously that mice lacking (beta)arr2 experience enhanced morphine-induced analgesia and do not become tolerant to morphine as determined in the hot-plate test, a paradigm that primarily assesses supraspinal pain responsiveness. To determine the general applicability of the (beta)arr2-muOR interaction in other neuronal systems, we have, in the present study, tested (beta)arr2 knock-out ((beta)arr2-KO) mice using the warm water tail-immersion paradigm, which primarily assesses spinal reflexes to painful thermal stimuli. In this test, the (beta)arr2-KO mice have greater basal nociceptive thresholds and markedly enhanced sensitivity to morphine. Interestingly, however, after a delayed onset, they do ultimately develop morphine tolerance, although to a lesser degree than the wild-type (WT) controls. In the (beta)arr2-KO but not WT mice, morphine tolerance can be completely reversed with a low dose of the classical protein kinase C (PKC) inhibitor chelerythrine. These findings provide in vivo evidence that the muOR is differentially regulated in diverse regions of the CNS. Furthermore, although (beta)arr2 appears to be the most prominent and proximal determinant of muOR desensitization and morphine tolerance, in the absence of this mechanism, the contributions of a PKC-dependent regulatory system become readily apparent.

Adaptive stereotactic body radiation therapy planning for lung cancer.

PURPOSE: To investigate the dosimetric effects of adaptive planning on lung stereotactic body radiation therapy (SBRT). METHODS AND MATERIALS: Forty of 66 consecutive lung SBRT patients were selected for a retrospective adaptive planning study. CBCT images acquired at each fraction were used for treatment planning. Adaptive plans were created using the same planning parameters as the original CT-based plan, with the goal to achieve comparable comformality index (CI). For each patient, 2 cumulative plans, nonadaptive plan (PNON) and adaptive plan (PADP), were generated and compared for the following organs-at-risks (OARs): cord, esophagus, chest wall, and the lungs. Dosimetric comparison was performed between PNON and PADP for all 40 patients. Correlations were evaluated between changes in dosimetric metrics induced by adaptive planning and potential impacting factors, including tumor-to-OAR distances (dT-OAR), initial internal target volume (ITV1), ITV change (ΔITV), and effective ITV diameter change (ΔdITV). RESULTS: 34 (85%) patients showed ITV decrease and 6 (15%) patients showed ITV increase throughout the course of lung SBRT. Percentage ITV change ranged from -59.6% to 13.0%, with a mean (±SD) of -21.0% (±21.4%). On average of all patients, PADP resulted in significantly (P=0 to .045) lower values for all dosimetric metrics. ΔdITV/dT-OAR was found to correlate with changes in dose to 5 cc (ΔD5cc) of esophagus (r=0.61) and dose to 30 cc (ΔD30cc) of chest wall (r=0.81). Stronger correlations between ΔdITV/dT-OAR and ΔD30cc of chest wall were discovered for peripheral (r=0.81) and central (r=0.84) tumors, respectively. CONCLUSIONS: Dosimetric effects of adaptive lung SBRT planning depend upon target volume changes and tumor-to-OAR distances. Adaptive lung SBRT can potentially reduce dose to adjacent OARs if patients present large tumor volume shrinkage during the treatment.

Chronic opioid therapy and opioid tolerance: a new hypothesis.

Opioids are efficacious and cost-effective analgesics, but tolerance limits their effectiveness. This paper does not present any new clinical or experimental data but demonstrates that there exist ascending sensory pathways that contain few opioid receptors. These pathways are located by brain PET scans and spinal cord autoradiography. These nonopioid ascending pathways include portions of the ventral spinal thalamic tract originating in Rexed layers VI-VIII, thalamocortical fibers that project to the primary somatosensory cortex (S1), and possibly a midline dorsal column visceral pathway. One hypothesis is that opioid tolerance and opioid-induced hyperalgesia may be caused by homeostatic upregulation during opioid exposure of nonopioid-dependent ascending pain pathways. Upregulation of sensory pathways is not a new concept and has been demonstrated in individuals impaired with deafness or blindness. A second hypothesis is that adjuvant nonopioid therapies may inhibit ascending nonopioid-dependent pathways and support the clinical observations that monotherapy with opioids usually fails. The uniqueness of opioid tolerance compared to tolerance associated with other central nervous system medications and lack of tolerance from excess hormone production is discussed. Experimental work that could prove or disprove the concepts as well as flaws in the concepts is discussed.

Absence of aquaporin-4 expression in lesions of neuromyelitis optica but increased expression in multiple sclerosis lesions and normal-appearing white matter.

Aquaporin-4 (AQP4) has recently been implicated in the pathogenesis of neuromyelitis optica(NMO) where it has been identifed as the first defined autoantigen pertinent to an infammatory demyelinating disorder of the human CNS. Furthermore, a recent case report has shown a lack of AQP4 expression in the spinal cord lesions of NMO. However, the pattern of AQP4 expression in multiple sclerosis (MS) tissues has not been well-defned. In the present investigation we have confirmed a lack of expression of AQP4 in optic and spinal cord lesions in NMO which contrasted sharply with the increased levels of AQP4 expression seen in MS lesions. Furthermore a detailed immunohistochemical and semi-quantitative analysis is used to describe the expression pattern of AQP4 on well-characterized tissue microarray samples of MS and control white matter. Anatomically AQP4 was more highly expressed in all categories of MS tissue compared to normal control tissues with the most abundant expression in active lesions. Within active lesions AQP4 expression was significantly correlated with expression of the pro-infammatory cytokine osteopontin. At the cellular level dual-labelling immunofluoresence demonstrated that increased expression of AQP4 was most pronounced at the astrocytic endfeet but was also associated with the cell bodies of astrocytes in the tissue parenchyma. The finding of increased AQP4 expression in MS lesions in contrast to the lack of expression in NMO lesions may suggest different mechanisms of initiation and progression between the two disease states.

Intensity-modulated radiotherapy reduces lung irradiation in patients with carcinoma of the oesophagus.

The 2-year survival rate after conventional radiotherapy for carcinoma of the oesophagus is around 10–20% [8]. Concomitant chemoradiation schedules have produced survival figures of 25–30% at 5 years, and this is now considered standard treatment [1]. Conformal radiotherapy techniques offer the potential to deliver higher doses of radiation to oesophageal tumours [5], and this may improve local tumour control. However, concerns regarding late normal tissue damage to the lung parenchyma and spinal cord remain a concern. Intensitymodulated radiotherapy (IMRT) allows complex dose distributions to be produced, and can reduce the dose to radiosensitive organs close to the tumour [2]. The present study was designed to investigate the impact of beam intensity modulation on treatment planning for carcinoma of the oesophagus, by comparing a standard three-dimensional conformal radiotherapy (3DCRT) technique to an IMRT technique using the same number and orientation of treatment fields.

UROTENSIN II FROM THE RIVER LAMPREY (LAMPETRA-FLUVIATILIS), THE SEA LAMPREY (PETROMYZON-MARINUS), AND THE PADDLEFISH (POLYODON SPATHULA)

Urotensin II was isolated from extracts of the whole brain of the river lamprey (Lampetra fluviatilis) and the sea lamprey (Petromyzon marinus). The primary structure of the peptide from both species is the same (Asn-Asn-Phe-Ser-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val) and this amino acid sequence is identical to that of urotensin II from the dogfish and skate. Consistent with previous morphological studies indicating that the Agnatha lack a caudal neurosecretory system, urotensin II was not detected in an extract of P. marinus spinal cord. The data suggest that the urotensin II may have functioned in the earliest vertebrates as a neurotransmitter/neuromodulator in the central nervous system rather than as a neurohormone of the caudal neurosecretory system. Urotensin II was also isolated from an extract of the spinal cord of a chondrostean fish, the paddlefish (Polyodon spathula). The primary structure of the paddlefish urotensin II (Gly-Ser-Thr-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val) is the same as that of another chondrostean, the sturgeon (Acipenser ruthenus). The study provides further evidence for a widespread distribution of urotensin II in vertebrate species and suggests that the primary structure of the peptide is better conserved in these phylogenetically ancient fish than in teleosts. (C) 1995 Academic Press, Inc.

A PEPTIDE FROM THE CAUDAL NEUROSECRETORY-SYSTEM OF THE DOGFISH SCYLIORHINUS-CANICULA THAT IS STRUCTURALLY RELATED TO UROTENSIN-I

Using reversed-phase HPLC in combination with a radioimmunoassay for ovine corticotropin-releasing hormone (CRH), a peptide with CRH-like immunoreactivity was isolated in pure form from an extract of the caudal spinal cord region of the spotted dogfish, Scyliorhinus canicula. The primary structure of the peptide was established as Pro-Ala-Glu-Thr-Pro-Asn-Ser-Leu-Asp-Leu(10)-Thr-Phe-His-Leu-Leu-Arg-Glu-Met-Ile-Glu(20)-Ile-Ala-Lys-His-Glu-Asn-Gln-Gln-Met-Gln(30)-Ala-Asp-Ser-Asn-Arg-Arg-Ile-Met-Asp-Thr(40)-Ile . NH2. This amino acid sequence shows moderate structural similarity to Catostomus urotensin I (51%) and to human CRH (56%). The data provide, therefore, chemical evidence to support the conclusions of earlier immunohistochemical studies that the diffuse caudal neurosecretory system of elasmobranchs produces a peptide that is immunochemically related to teleost urotensin I peptides. However, the primary structure of urotensin I has been poorly conserved during evolution. (C) 1995 Academic Press, Inc.