RESUMO
BACKGROUND: Electrical stimulation on select areas of the external auricular dermatome influences the autonomic nervous system. It has been postulated that activation of the Auricular Branch of the Vagus Nerve (ABVN) mediates such autonomic changes. However, the underlying neural pathways mediating these effects are unknown and, further, our understanding of the anatomical distribution of the ABVN in the auricle has now been questioned. OBJECTIVE: To investigate the effects of electrical stimulation of the tragus on autonomic outputs in the rat and probe the underlying neural pathways. METHODS: Central neuronal projections from nerves innervating the external auricle were investigated by injections of the transganglionic tracer cholera toxin B chain (CTB) into the right tragus of Wistar rats. Physiological recordings of heart rate, perfusion pressure, respiratory rate and sympathetic nerve activity were made in an anaesthetic free Working Heart Brainstem Preparation (WHBP) of the rat and changes in response to electrical stimulation of the tragus analysed. RESULTS: Neuronal tracing from the tragus revealed that the densest CTB labelling was within laminae III-IV of the dorsal horn of the upper cervical spinal cord, ipsilateral to the injection sites. In the medulla oblongata, CTB labelled afferents were observed in the paratrigeminal nucleus, spinal trigeminal tract and cuneate nucleus. Surprisingly, only sparse labelling was observed in the vagal afferent termination site, the nucleus tractus solitarius. Recordings made from rats at night time revealed more robust sympathetic activity in comparison to day time rats, thus subsequent experiments were conducted in rats at night time. Electrical stimulation was delivered across the tragus for 5â¯min. Direct recording from the sympathetic chain revealed a central sympathoinhibition by up to 36% following tragus stimulation. Sympathoinhibition remained following sectioning of the cervical vagus nerve ipsilateral to the stimulation site, but was attenuated by sectioning of the upper cervical afferent nerve roots. CONCLUSIONS: Inhibition of the sympathetic nervous system activity upon electrical stimulation of the tragus in the rat is mediated at least in part through sensory afferent projections to the upper cervical spinal cord. This challenges the notion that tragal stimulation is mediated by the auricular branch of the vagus nerve and suggests that alternative mechanisms may be involved.
Assuntos
Vértebras Cervicais , Frequência Cardíaca/fisiologia , Células Receptoras Sensoriais/fisiologia , Medula Espinal/fisiologia , Estimulação Elétrica Nervosa Transcutânea/métodos , Nervo Vago/fisiologia , Vias Aferentes/fisiologia , Animais , Tronco Encefálico/fisiologia , Masculino , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Núcleo Solitário/fisiologia , Estimulação do Nervo Vago/métodosRESUMO
Sensory information arising from the upper neck is important in the reflex control of posture and eye position. It has also been linked to the autonomic control of the cardiovascular and respiratory systems. Whiplash associated disorders (WAD) and cervical dystonia, which involve disturbance to the neck region, can often present with abnormalities to the oromotor, respiratory and cardiovascular systems. We investigated the potential neural pathways underlying such symptoms. Simulating neck afferent activity by electrical stimulation of the second cervical nerve in a working heart brainstem preparation (WHBP) altered the pattern of central respiratory drive and increased perfusion pressure. Tracing central targets of these sensory afferents revealed projections to the intermedius nucleus of the medulla (InM). These anterogradely labelled afferents co-localised with parvalbumin and vesicular glutamate transporter 1 indicating that they are proprioceptive. Anterograde tracing from the InM identified projections to brain regions involved in respiratory, cardiovascular, postural and oro-facial behaviours--the neighbouring hypoglossal nucleus, facial and motor trigeminal nuclei, parabrachial nuclei, rostral and caudal ventrolateral medulla and nucleus ambiguus. In brain slices, electrical stimulation of afferent fibre tracts lateral to the cuneate nucleus monosynaptically excited InM neurones. Direct stimulation of the InM in the WHBP mimicked the response of second cervical nerve stimulation. These results provide evidence of pathways linking upper cervical sensory afferents with CNS areas involved in autonomic and oromotor control, via the InM. Disruption of these neuronal pathways could, therefore, explain the dysphagic and cardiorespiratory abnormalities which may accompany cervical dystonia and WAD.
Assuntos
Fenômenos Fisiológicos Cardiovasculares , Bulbo/fisiologia , Bulbo/ultraestrutura , Músculos do Pescoço/inervação , Músculos do Pescoço/fisiologia , Respiração , Vias Aferentes/fisiologia , Animais , Tronco Encefálico/fisiologia , Tronco Encefálico/ultraestrutura , Sistema Cardiovascular/inervação , Estimulação Elétrica , Nervo Hipoglosso/fisiologia , Masculino , Camundongos , Músculos do Pescoço/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Neurônios/ultraestrutura , Nervo Frênico/fisiologia , Ratos Wistar , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
Connexin (Cx) proteins localized to neuronal and glial syncytia provide the ultrastructural components for intercellular communication via gap junctions. In this study, a Cx45 reporter mouse model in which the Cx45 coding sequence is substituted for enhanced green fluorescent protein (eGFP) was used to characterize Cx45 expressing neurones within adult mouse spinal cord. eGFP-immunoreactive (eGFP-IR) cells were localized at all rostro-caudal levels to laminae I-III of the dorsal horn (DH), areas associated with nociception. The neuronal rather than glial phenotype of these cells in DH was confirmed by co-localisation of eGFP-IR with the neuronal marker NeuN. Further immunohistochemical studies revealed that eGFP-IR interneurones co-express the calcium-binding protein calbindin, and to a lesser extent calretinin. In contrast, eGFP-IR profiles did not co-localize with either parvalbumin or GAD-67, both of which are linked to inhibitory interneurones. Staining with the primary afferent markers isolectin-B4 (IB4) and calcitonin gene-related peptide revealed that eGFP-IR somata within laminae I-III receive close appositions from the former, presumed non-peptidergic nociceptive afferents of peripheral origin. The presence of 5-HT terminals in close apposition to eGFP-IR interneuronal somata suggests modulation via descending pathways. These data demonstrate a highly localized expression of Cx45 in a population of interneurones within the mouse superficial dorsal horn. The implications of these data in the context of the putative role of Cx45 and gap junctions in spinal somatosensory processing and pain are discussed.
Assuntos
Conexinas/genética , Conexinas/metabolismo , Regulação da Expressão Gênica/genética , Células do Corno Posterior/metabolismo , Medula Espinal/citologia , Animais , Colina O-Acetiltransferase/metabolismo , Glutamato Descarboxilase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Receptores Opioides mu , Serotonina/metabolismoRESUMO
A rapid method for fixation and embedding of plant materials, especially pteridophytes, is suggested. Addition of tannic acid following osmication improved the visualization of membranes. Staining en bloc with uranyl acetate between osmication and tannic acid is suggested for tissues infected with fungi and bacteria.