Expression of gastrin-releasing peptide by excitatory interneurons in the mouse superficial dorsal horn

Background: Gastrin-releasing peptide (GRP) and its receptor have been shown to play an important role in the sensation of itch. However, although GRP immunoreactivity has been detected in the spinal dorsal horn, there is debate about whether this originates from primary afferents or local excitatory interneurons. We therefore examined the relation of GRP immunoreactivity to that seen with antibodies that label primary afferent or excitatory interneuron terminals. We tested the specificity of the GRP antibody by preincubating with peptides with which it could potentially cross-react. We also examined tissue from a mouse line in which enhanced green fluorescent protein (EGFP) is expressed under control of the GRP promoter.<p></p> Results: GRP immunoreactivity was seen in both primary afferent and non-primary glutamatergic axon terminals in the superficial dorsal horn. However, immunostaining was blocked by pre-incubation of the antibody with substance P, which is present at high levels in many nociceptive primary afferents. EGFP+ cells in the GRP-EGFP mouse did not express Pax2, and their axons contained the vesicular glutamate transporter 2 (VGLUT2), indicating that they are excitatory interneurons. In most cases, their axons were also GRP-immunoreactive. Multiple-labelling immunocytochemical studies indicated that these cells did not express either of the preprotachykinin peptides, and that they generally lacked protein kinase Cγ, which is expressed by a subset of the excitatory interneurons in this region.<p></p> Conclusions: These results show that GRP is expressed by a distinct population of excitatory interneurons in laminae I-II that are likely to be involved in the itch pathway. They also suggest that the GRP immunoreactivity seen in primary afferents in previous studies may have resulted from cross-reaction of the GRP antibody with substance P or the closely related peptide neurokinin A

Spinal neurons that contain gastrin-releasing peptide seldom express Fos or phosphorylate extracellular signal-regulated kinases in response to intradermal chloroquine

Background: Gastrin-releasing peptide (GRP) is thought to play a role in the itch evoked by intradermal injection of chloroquine. Although some early studies suggested that GRP was expressed in pruriceptive primary afferents, it is now thought that GRP in the spinal cord is derived mainly from a population of excitatory interneurons in lamina II, and it has been suggested that these are involved in the itch pathway. To test this hypothesis, we used the transcription factor Fos and phosphorylation of extracellular signal-regulated kinases (ERK) to look for evidence that interneurons expressing GRP were activated following intradermal injection of chloroquine into the calf, in mice that express enhanced green fluorescent protein (EGFP) in these cells. Results: Injection of chloroquine resulted in numerous Fos- or phospho-ERK (pERK) positive cells in the somatotopically appropriate part of the superficial dorsal horn. The proportion of all neurons in this region that showed Fos or pERK was 18% and 21%, respectively. However, among the GRP–EGFP, only 7% were Fos-positive and 3% were pERK-positive. As such, GRP–EGFP cells were significantly less likely than other neurons to express Fos or to phosphorylate ERK. Conclusions: Both expression of Fos and phosphorylation of ERK can be used to identify dorsal horn neurons activated by chloroquine injection. However, these results do not support the hypothesis that interneurons expressing GRP are critical components in the itch pathway

The organisation of spinoparabrachial neurons in the mouse

The anterolateral tract (ALT), which originates from neurons in lamina I and the deep dorsal horn, represents a major ascending output through which nociceptive information is transmitted to brain areas involved in pain perception. Although there is detailed quantitative information concerning the ALT in the rat, much less is known about this system in the mouse, which is increasingly being used for studies of spinal pain mechanisms because of the availability of genetically modified lines. The aim of this study was therefore to determine the extent to which information about the ALT in the rat can be extrapolated to the mouse. Our results suggest that as in the rat, most lamina I ALT projection neurons in the lumbar enlargement can be retrogradely labelled from the lateral parabrachial area, that the great majority of these cells (~90%) express the neurokinin 1 receptor (NK1r), and that these are larger than other NK1r-expressing neurons in this lamina. This means that many lamina I spinoparabrachial cells can be identified in NK1r-immunostained sections from animals that have not received retrograde tracer injections. However, we also observed certain species differences, in particular we found that many spinoparabrachial cells in lamina III-IV lack the NK1r, meaning that they cannot be identified based solely on expression of this receptor. We also provide evidence that the vast majority of spinoparabrachial cells are glutamatergic, and that some express substance P. These findings will be important for studies designed to unravel the complex neuronal circuitry that underlies spinal pain processing

Substance P-expressing excitatory interneurons in the mouse superficial dorsal horn provide a propriospinal input to the lateral spinal nucleus

The superficial dorsal horn (laminae I and II) of the spinal cord contains numerous excitatory and inhibitory interneurons, and recent studies have shown that each of these groups can be divided into several neurochemically distinct populations. Although it has long been known that some neurons in this region have intersegmental (propriospinal) axonal projections, there have been conflicting reports concerning the number of propriospinal cells and the extent of their axons. In addition, little is known about the neurochemical phenotype of propriospinal neurons or about the termination pattern of their axons. In the present study we show, using retrograde tracing, that around a third of lamina I–II neurons in the lumbar enlargement project at least five segments cranially. Substance P-expressing excitatory neurons are over-represented among these cells, accounting for one-third of the propriospinal neurons. In contrast, inhibitory interneurons and excitatory PKCγ neurons are both under-represented among the retrogradely labelled cells. By combining viral vector-mediated Cre-dependent anterograde tracing with immunocytochemistry, we provide evidence that the lateral spinal nucleus (LSN), rather than the superficial dorsal horn, is the main target for axons belonging to propriospinal substance P-expressing neurons. These findings help to resolve the discrepancies between earlier studies and have implications for the role of the LSN in pain mechanisms

Immunostaining for Homer reveals the majority of excitatory synapses in laminae I-III of the mouse spinal dorsal horn

The spinal dorsal horn processes somatosensory information before conveying it to the brain. The neuronal organization of the dorsal horn is still poorly understood, although recent studies have defined several distinct populations among the interneurons, which account for most of its constituent neurons. All primary afferents, and the great majority of neurons in laminae I–III are glutamatergic, and a major factor limiting our understanding of the synaptic circuitry has been the difficulty in identifying glutamatergic synapses with light microscopy. Although there are numerous potential targets for antibodies, these are difficult to visualize with immunocytochemistry, because of protein cross-linking following tissue fixation. Although this can be overcome by antigen retrieval methods, these lead to difficulty in detecting other antigens. The aim of this study was to test whether the postsynaptic protein Homer can be used to reveal glutamatergic synapses in the dorsal horn. Immunostaining for Homer gave punctate labeling when viewed by confocal microscopy, and this was restricted to synapses at the ultrastructural level. We found that Homer puncta were colocalized with the AMPA receptor GluR2 subunit, but not with the inhibitory synapse-associated protein gephyrin. We also examined several populations of glutamatergic axons and found that the great majority of boutons were in contact with at least one Homer punctum. These results suggest that Homer antibodies can be used to reveal the great majority of glutamatergic synapses without antigen retrieval. This will be of considerable value in tracing synaptic circuits, and also in investigating plasticity of glutamatergic synapses in pain states

A quantitative study of neurochemically-defined populations of inhibitory interneurons in the superficial dorsal horn of the mouse spinal cord

Around a quarter of neurons in laminae I-II of the dorsal horn are inhibitory interneurons. These play an important role in modulating somatosensory information, including that perceived as pain or itch. Previous studies in rat identified four largely non-overlapping neurochemical populations among these cells, defined by expression of galanin, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) or parvalbumin. The galanin cells were subsequently shown to coexpress dynorphin. Several recent studies have used genetically-modified mice to investigate the function of different interneuron populations, and it is therefore important to determine whether the same pattern applies in mouse, and to estimate the relative sizes of these populations. We show that the neurochemical organisation of inhibitory interneurons in mouse superficial dorsal horn is similar to that in the rat, although a larger proportion of these neurons (33%) express NPY. Between them, these four populations account for ∼75% of inhibitory cells in laminae I-II. Since ∼25% of inhibitory interneurons in this region belong to a novel calretinin-expressing type, our results suggest that virtually all inhibitory interneurons in superficial dorsal horn can be assigned to one of these five neurochemical populations. Although our main focus was inhibitory neurons, we also identified a population of excitatory dynorphin-expressing cells in laminae I-II that are largely restricted to the medial part of the mid-lumbar dorsal horn, corresponding to glabrous skin territory. These findings are important for interpretation of studies using molecular-genetic techniques to manipulate the functions of interneuron populations to investigate their roles in somatosensory processing

Scientific Rationale for the Use of Alpha-Adrenergic Agonists and Glucocorticoids in the Therapy of Pediatric Stridor

Purpose. The most common pharmacological therapies used in the treatment of stridor in children are glucocorticosteroids (GC) and alpha-adrenergic (αAR) agonists. Despite the long-standing reported efficacy of these medications, there is a paucity of data relating to their actual mechanisms of action in the upper airway. Summary. There is compelling scientific evidence supporting the use of αAR-agonists and GCs in pediatric stridor. αAR signaling and GCs regulate the vasomotor tone in the upper airway mucosa. The latter translates into better airflow dynamics, as delineated by human and nonhuman upper airway physiological models. In turn, clinical trials have demonstrated that GCs and the nonselective αAR agonist, epinephrine, improve respiratory distress scores and reduce the need for further medical care in children with stridor. Future research is needed to investigate the role of selective αAR agonists and the potential synergism of GCs and αAR-signaling in the treatment of upper airway obstruction and stridor

Novel Mutation of Interferon-γ Receptor 1 Gene Presenting as Early Life Mycobacterial Bronchial Disease

Mendelian susceptibility to mycobacterial diseases (MSMD) are a spectrum of inherited disorders characterized by localized or disseminated infections caused by atypical mycobacteria. Interferon-γ receptor 1 (IFNGR1) deficiency was the first identified genetic disorder recognized as MSMD. Mutations in the genes encoding IFNGR1 can be recessive or dominant and cause complete or partial receptor deficiency. We present the case of a 2½-year-old boy with a history of recurrent wheezing, diagnosed with endobronchial mycobacterial infection. Immunological workup revealed a homozygous nonsense mutation in the IFNGR1 gene, a novel mutation predicted in silico to cause complete IFNGR1 deficiency. This case demonstrates that (a) Interferon-γ receptor deficiency can present resembling common disorders of the lung; (b) mycobacterial infections should be suspected when parenchymal lung disease, hilar lymphadenopathy, and endobronchial disease are present; and (c) high index of suspicion for immunodeficiency should be maintained in patients with disseminated nontubercular mycobacterial infection

Preprotachykinin A (PPTA) is expressed by a distinct population of excitatory neurons in the mouse superficial spinal dorsal horn including cells that respond to noxious and pruritic stimuli

The superficial dorsal horn, which is the main target for nociceptive and pruritoceptive primary afferents, contains a high density of excitatory interneurons. Our understanding of their roles in somatosensory processing has been restricted by the difficulty of distinguishing functional populations among these cells. We recently defined three non-overlapping populations among the excitatory neurons, based on the expression of neurotensin, neurokinin B (NKB) and gastrin-releasing peptide (GRP). Here we identify and characterise another population: neurons that express the tachykinin peptide substance P. We show with immunocytochemistry that its precursor protein (preprotachykinin A, PPTA) can be detected in ~14% of lamina I-II neurons, and these are concentrated in the outer part of lamina II. Over 80% of the PPTA-positive cells lack the transcription factor Pax2 (which determines an inhibitory phenotype), and these account for ~15% of the excitatory neurons in this region. They are different from the neurotensin, NKB or GRP neurons, although many of them contain somatostatin, which is widely expressed among superficial dorsal horn excitatory interneurons. We show that many of these cells respond to noxious thermal and mechanical stimuli, and to intradermal injection of pruritogens. Finally, we demonstrate that these cells can also be identified in a knock-in Cre mouse line (Tac1Cre), although our findings suggest that there is an additional population of neurons that transiently express PPTA. This population of substance P-expressing excitatory neurons is likely to play an important role in transmission of signals that are perceived as pain and itch

Stakeholder´s analysis in e-learning software process development

The success of an e-learning project depends largely on the quality of applications and tools used to support the learning process. Meanwhile, the quality of software depends largely on proper stakeholders? identification in the requirements stage during the development process. Although some studies exist which have identified key stakeholders in e-learning domain, most of them do not perform a deep analysis of the stakeholders and their contexts, necessary to consider educational and cultural issues in this type of environment. The aim of this paper is to present the application of a method for the identification and analysis of stakeholders, which is adapted to e-learning domain. This method was used in a case study considering university environments.Fil: Romero, Lucila J.. Universidad Nacional del Litoral. Facultad de Ingeniería y Ciencias Hídricas; ArgentinaFil: Ballejos, Luciana Cristina. Universidad Tecnológica Nacional. Facultad Regional Santa Fe. Centro de Investigación y Desarrollo de Ingeniería en Sistemas de Información; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gutierrez, Maria de Los Milagros. Universidad Tecnológica Nacional. Facultad Regional Santa Fe. Centro de Investigación y Desarrollo de Ingeniería en Sistemas de Información; ArgentinaFil: Caliusco, Maria Laura. Universidad Tecnológica Nacional. Facultad Regional Santa Fe. Centro de Investigación y Desarrollo de Ingeniería en Sistemas de Información; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin