Ultrastructural changes of receptor endings in prolonged action of local anesthetics

Ultrastructural changes of the terminal plates of the bushy receptors in the frog urinary bladder have been studied after two hours' exposition in 0.05% novocaine solution and one hour's exposition in 0.05% dicaine and trimecaine solution. During these periods a steady block of the receptor impulse activity develops. The local anesthetics essentially change ultramicroscopic structure of the terminals. The reaction to the anesthetics investigated has both some features in common and certain peculiarities. At each effect three types of changes can be determined, characterized with various degree of rearrangement in neurilemma, neuroplasm and organelles. Each type of the changes is supposed to reflect a certain phase of the plate reactive response. Specificities of the reaction to novocaine are minimal changes of mitochondria, accumulation of glycogen granules, deformity and decreasing amount of vesicles. Under dicaine effect mitochondria do not change, amount of vesicles increases, their form does not change; under trimecaine effect mitochondria undergo most noticeable alterations. The changes of the terminal plates observed are interpreted as adaptive. The effect of the local anesthetics on the receptors is not limited with the blockade of the sodium canals of the afferent fibers, in parallel, biochemical processes, occurring in cytosol of the terminals also change; their morphological manifestations are the ultrastructural changes observed.     

Structuro-functional changes in bushy interoceptors during protracted anoxia

The bushy receptors of the frog urinary bladder respond to the effect of 60-minute-long anoxia with a complex combination of morphological. tinctorial and electrophysiological reactions. A biphase change in dynamics of bioelectrical activity takes place (an initial increase of frequency with its successive decrease), prolongation of the vital staining periods with methylene blue, change in the character of staining and decoloration of the receptory plates: increase in homogeneity and in intensity of staining, discontinuance of granuloformation and weakening of processes of the plates mobility. The ultrastructural changes are mainly concerned with mitochondria, where either crists reduce and osmiophility decreases, or increases. A part of mitochondria does not change. Certain heterogeneity in reactive changes of the receptors is noted. This is considered as manifestation of functional heterogeneity of the receptory units and their structural elements.     

Morphological Characterization of Bushy Cells and Their Inputs in the Laboratory Mouse (Mus musculus) Anteroventral Cochlear Nucleus

Spherical and globular bushy cells of the AVCN receive huge auditory nerve endings specialized for high fidelity neural transmission in response to acoustic events. Recent studies in mice and other rodent species suggest that the distinction between bushy cell subtypes is not always straightforward. We conducted a systematic investigation of mouse bushy cells along the rostral-caudal axis in an effort to understand the morphological variation that gives rise to reported response properties in mice. We combined quantitative light and electron microscopy to investigate variations in cell morphology, immunostaining, and the distribution of primary and non-primary synaptic inputs along the rostral-caudal axis. Overall, large regional differences in bushy cell characteristics were not found; however, rostral bushy cells received a different complement of axosomatic input compared to caudal bushy cells. The percentage of primary auditory nerve terminals was larger in caudal AVCN, whereas non-primary excitatory and inhibitory inputs were more common in rostral AVCN. Other ultrastructural characteristics of primary auditory nerve inputs were similar across the rostral and caudal AVCN. Cross sectional area, postsynaptic density length and curvature, and mitochondrial volume fraction were similar for axosomatic auditory nerve terminals, although rostral auditory nerve terminals contained a greater concentration of synaptic vesicles near the postsynaptic densities. These data demonstrate regional differences in synaptic organization of inputs to mouse bushy cells rather than the morphological characteristic of the cells themselves.     

Sensory endings in the intramural ganglia of the stomach. Light optical and electron microscopic study

Puppies and young dogs at the age of 3 days up to 2 months were used in the investigation. In total stomach preparations neuronal structures were stained with methylene blue after A. S. Dogel. Routine electron microscopic studies were carried out. Within the structure of intramuscular vegetative plexus of the stomach a sensory plexus was demonstrated, its terminals forming receptive bushy structures in all the tissues. Especially numerous the sensory endings were in neuronal cords and ganglia. The sensory endings innervated all the elements in ganglia. Ultrastructure of receptor terminals situated in the intramural ganglia of the stomach corresponded to that of terminals in tissue mechanoreceptors described for skin, auriculum, carotid sinus and trachea. Peculiar multilayer lamillar structures formed by cytoplasmic axonal processes of lemmocytes around lance-shaped, arranged in rosary fashion receptor varicosities were described. Contact of receptor terminals with the neuronal cell body was demonstrated. Functional nature of the tissue receptors described was discussed.     

Modality of the bush-like receptors

In an isolated preparation of the Rana temporaria urinary bladder after a simultaneous morphological and physiological investigation a, structural-functional differentiation of free bushy receptors has been demonstrated. According to arborization character and to the appearance of deferent fibers, the receptors are divided into two types. The first type receptors have a simple structure, a long deferent poorly branching myelin fiber, terminating in a diffuse bush near blood vessels. The second type receptors are of a more complex structure. Their myelin fibers, when leaving the fasciculus, are 4-8 times shorter than the first type receptors, undergo dichotomic and trichotomic divisions several times, and in their distal part they form two initial myelin segments. Their receptory apparatus has a tree-like bush-shaped form and consists of several compact bushes. The impulse activity of the receptors is also characterized by two types of action potentials, differing in their amplitude. When responding to a mechanical stimulation, the high voltage impulse frequency changes, when sodium chloride concentration is increased, the low voltage impulse frequency changes. There is a certain localization of the zones in the preparation from which it is possible to obtain predominantly either low voltage or high voltage responses. The response to the low voltage reaction proves to be obtained from the first type receptors, and that to the high voltage reaction--from the second type receptors. Thus, in the frog, that is on a low phylogenetic stage, differentiation of the free bushy sensitive terminal into mechano- and chemoreceptors is already outlined.     

Morphophysiological characteristics of bushy interoceptors

The survey of the existing morphological and physiological classifications of the bushy interoceptors is presented. Dependence of adaptive properties of the receptors on their structure is demonstrated. The bushy receptors can be both quickly- and slowly-adapting: Their adaptive properties evidently depend on viscous-elastic peculiarities of the surrounding tissues. The data on a possible connection between impulse activity of the receptors and the level of their oxidative metabolism are reported. Similarity and differences of the morpho-functional characteristics of the bushy receptors in various animal organs and tissues are established.     

Dopamine D1 and D2 Receptor Immunoreactivities in the Arcuate-Median Eminence Complex and their Link to the Tubero-Infundibular Dopamine Neurons

Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tuberoinfundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially modulate the activity and/or Dopamine synthesis of substantial numbers of tubero-infundibular dopamine neurons at the somatic and terminal level. The immunohistochemical work also gives support to the view that dopamine D1 receptors and/or dopamine D2 receptors in the lateral palisade zone by mediating dopamine volume transmission may contribute to the inhibition of luteinizing hormone releasing hormone release from nerve terminals in this region.Key words: Dopamine D1 and D2 receptors, tubero-infundibular dopamine neurons, dopamine receptor colocalization, arcuate-median eminence complex, volume transmission, luteinizing hormone releasing hormone     

Structural and functional reactive changes in the bushy interceptor of the frog urinary bladder upon adequate stimulation]

Morphological and functional changes in the bushy receptors of the frog urinary bladder were studied after a definite cystectasia (bladder wall distention). Morphological and electrophysiological parameters were registered synchronously. Under the effect of distention the total rate of afferent impulse activity was demonstrated to increase with a simultaneous increase of the average size of the receptor end-plates and with changing (growing round) in their form. Increment of the average size of the end-plates overtakes that of the distance between two points chosen on the urinary bladder wall; certain shifts are also observed in time parameters for staining the receptors with methylene blue. A possibility is suggested on interconnections of morphological and electrophysiological shifts.     

Neurochemical pattern of the complex innervation of neuroepithelial bodies in mouse lungs

As best characterized for rats, it is clear that pulmonary neuroepithelial bodies (NEBs) are contacted by a plethora of nerve fiber populations, suggesting that they represent an extensive group of multifunctional intraepithelial airway receptors. Because of the importance of genetically modified mice for functional studies, and the current lack of data, the main aim of the present study was to achieve a detailed analysis of the origin and neurochemical properties of nerve terminals associated with NEBs in mouse lungs. Antibodies against known selective markers for sensory and motor nerve terminals in rat lungs were used on lungs from control and vagotomized mice of two different strains, i.e., Swiss and C57-Bl6. NEB cells were visualized by antibodies against either the general neuroendocrine marker protein gene-product 9.5 (PGP9.5) or calcitonin gene-related peptide (CGRP). Thorough immunohistochemical examination of NEB cells showed that some of these NEB cells also exhibit calbindin D-28 k (CB) and vesicular acetylcholine transporter (VAChT) immunoreactivity (IR). Mouse pulmonary NEBs were found to receive intraepithelial nerve terminals of at least two different populations of myelinated vagal afferents: (1) Immunoreactive (ir) for vesicular glutamate transporters (VGLUTs) and CB; (2) expressing P2X₂ and P2X₃ ATP receptors. CGRP IR was seen in varicose vagal nerve fibers and in delicate non-vagal fibers, both in close proximity to NEBs. VAChT immunostaining showed very weak IR in the NEB-related intraepithelial vagal sensory nerve terminals. nNOS- or VIP-ir nerve terminals could be observed at the base of pulmonary NEBs. While a single NEB can be contacted by multiple nerve fiber populations, it was clear that none of the so far characterized nerve fiber populations contacts all pulmonary NEBs. The present study revealed that mouse lungs harbor several populations of nerve terminals that may selectively contact NEBs. Although at present the physiological significance of the innervation pattern of NEBs remains enigmatic, it is likely that NEBs are receptor–effector end-organs that may host complex and/or multiple functional properties in normal airways. The neurochemical information on the innervation of NEBs in mouse lungs gathered in the present study will be essential for the interpretation of upcoming functional data and for the study of transgenic mice.     

Vesicular glutamate transporter 2 is expressed in different nerve fibre populations that selectively contact pulmonary neuroepithelial bodies

Pulmonary neuroepithelial body (NEB) receptors in rats receive at least four different nerve fibre populations. In addition to a spinal sensory innervation that contacts NEBs at their basal side, extensive vagal nodose sensory terminals and separate nitrergic and cholinergic nerve endings protrude between NEB cells. In the present study, antibodies against the vesicular glutamate transporter 2 (VGLUT2), a transmembrane protein responsible for loading glutamate into synaptic vesicles, were used to investigate whether some of the nerve terminals contacting NEBs in rat lungs might use glutamate as a neurotransmitter. VGLUT2 immunoreactivity (IR) was detected in extensive intraepithelial arborising nerve terminals that appeared to contact most of the NEBs. Multiple immunostaining showed VGLUT2 IR in the vagal nodose and spinal sensory nerve terminals contacting NEBs, and in another, most likely sensory, intraepithelial nerve fibre population, the origin and further characteristics of which remain to be elucidated. At least part of the VGLUT2-immunoreactive nerve fibres that contact NEBs were shown to be myelinated. The expression of VGLUT2 indicates that glutamate is stored and released as a neurotransmitter in terminals of several pulmonary (sensory) nerve fibre populations that selectively relate to the complex NEB receptors. The present study strongly suggests an involvement of glutamatergic mechanisms in the peripheral transduction of sensory stimuli from the lungs, via the release of glutamate from nerve terminals, thereby modulating the activity of NEB receptor cells or the excitability of afferent nerves.     

Multireceptor activation of the pulmonary chemoreflex

Schertel et al. (J. Appl. Physiol. 61: 1237-1240, 1984) reported that pulmonary C fibers initiate the prompt apnea followed by rapid shallow breathing evoked by pulmonary arterial injections of capsaicin. However, doubt has remained as to whether these changes in breathing pattern are induced exclusively by direct stimulation of pulmonary C fibers or whether secondary stimulation of slowly adapting pulmonary stretch receptors by capsaicin-induced reflex bronchoconstriction also contributes to the response. To determine the contribution of this secondary mechanism to changes in breathing pattern, we evoked the pulmonary chemoreflex in spontaneously breathing dogs before and after blockade of muscarinic receptors with atropine. Right atrial injections of capsaicin before the administration of atropine induced a classical pulmonary chemoreflex, i.e., apnea, hypotension, and bradycardia followed by rapid shallow breathing and bronchoconstriction. After atropine, all components of the pulmonary chemoreflex induced by right atrial injections of capsaicin remained intact except bronchoconstriction. However, the absolute magnitude of the change in each component of the reflex except apnea was significantly attenuated. We conclude that the classic pulmonary chemoreflex is a complex phenomenon initiated primarily by stimulation of pulmonary C fibers but significantly influenced by secondary stimulation of slowly adapting pulmonary stretch receptors.     

实验性肺纤维化形成过程中肺组织血管生成的动态变化

目的观察大鼠肺纤维化形成过程中肺组织血管生成的动态变化及其与肺纤维化形成的关系.方法免疫组织化学方法结合透射电镜观察博莱霉素组和对照组肺组织血管生成的变化,同时测定羟脯氨酸含量评估肺纤维化程度.结果博莱霉素组大鼠早期有血管计数一过性的明显升高,到第八天达到高峰,血管新生的区域与肺纤维化区域相吻合;随着胶原纤维的沉积,血管逐渐减少最后近于消失.电镜显示肺损伤后早期毛细血管内皮细胞表现出细胞核增大,常染色质增多,细胞器丰富等增生旺盛表现.结论肺纤维化早期血管生成显著,血管生成在肺纤维化发生发展中可能起重要作用.     

Autotomy of sensory nerve endings in the area of microvessels]

The phenomenon of autotomy of peripheral receptor terminals, in the area of the intestinal microvessel bed has been analysed. This phenomenon is accompanied with a real separation of the terminal patches or large fragments of receptors with their successive degeneration. The effect of the terminals autotomy is succeeded in reproducing and analysing in dynamics in tissue culture. The computer analysis of the image demonstrates that autotomy is observed not only in developing structures during early period of ontogenesis, but in well developed, as regards all morphological criteria, tissue receptors of mature animals. The investigations have been performed by means of silver nitrate impregnation after Bielschowsky--Gros, as well as using vital microscopy. An idea has been formed that autotomy is a part of the cyclic process of autotomy--regeneration of the terminals. This process is connected with retractile cytopoiesis, apocrine secretion. A high concentration of proteolytic enzymes in the amputated degrading fragment of the receptor can serve as a trophic factor, activating the local metabolic process in the area of microvessels.     

蛤蚧豆状核的结构及其与顶盖前端的纤维联系

运用Nissl法和辣根过氧化物酶(horseradish peroxidase,HRP)追踪标记技术,研究蛤蚧(Gekko gecko)豆状核的结构及其与顶盖前端的纤维联系。Nissl染色显示,蛤蚧豆状核细胞大小没有明显差别,由背内侧细胞密集部和腹外侧细胞稀疏部组成。将HRP注射于顶盖前端,结果豆状核背内侧部和腹外侧部分别接受同侧顶盖前端脑室内、外侧纤维的传入,核内标记有浓密的神经丛和大量纤维末梢,并在该核腹外侧部及其邻近区域发现少量大胞体标记细胞。推测豆状核腹外侧部的大胞体细胞及其邻近区域的大胞体细胞可能具有相同的功能,且该核可能形成离顶盖通路和副视系统相联系的交通要道。     

Ultrastructural changes in the gracile nucleus of alloxan-induced diabetic rats

The present study reports ultrastructural changes in the gracile nucleus of male Wistar rats after alloxan-induced diabetes. During the acute phase (3-7 days) degenerating electron-dense dendrites and axon terminals were dispersed in the neuropil. Degenerating dendrites were characterized by an electron-dense cytoplasm, swollen mitochondria, dilated endoplasmic reticulum and randomized ribosomes. Degenerating axon terminals were characterized by an electron-dense cytoplasm and clustering of small spherical agranular vesicles. Degenerating axon terminals may form the central element or part of a synaptic glomerulus. Macrophages were present in the neuropil and in the process of engulfing neuronal elements. During the medium phase (1-6 months), most of the degenerating dendrites and axon terminals had been engulfed or removed by macrophages. During the late phase (9-12 months), a second wave of degeneration occurred in the gracile nucleus, similar to the acute phase.     

Mediator mechanisms involved in TRPV1 and P2X receptor-mediated, ROS-evoked bradypneic reflex in anesthetized rats.

Inhalation of H2O2 is known to evoke bradypnea followed by tachypnea, which are reflexes resulting from stimulation by reactive oxygen species of vagal lung capsaicin-sensitive and myelinated afferents, respectively. This study investigated the pharmacological receptors and chemical mediators involved in triggering these responses. The ventilatory responses to 0.2% aerosolized H2O2 were studied before and after various pharmacological pretreatments in anesthetized rats. The initial bradypneic response was reduced by a transient receptor potential vanilloid 1 (TRPV1) receptor antagonist [capsazepine; change (Delta) = -53%] or a P2X purinoceptor antagonist [iso-pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (PPADS); Delta = -47%] and was further reduced by capsazepine and iso-PPADS in combination (Delta = -78%). The initial bradypneic response was reduced by a cyclooxygenase inhibitor (indomethacin; Delta = -48%), ATP scavengers (apyrase and adenosine deaminase in combination; Delta = -50%), or capsazepine and indomethacin in combination (Delta = -47%), was further reduced by iso-PPADS and indomethacin in combination (Delta = -75%) or capsazepine and ATP scavengers in combination (Delta = -83%), but was not affected by a lipoxygenase inhibitor (nordihydroguaiaretic acid) or by any of the various vehicles. No pretreatment influenced delayed tachypnea. We concluded that 1) the initial bradypneic response to H2O2 results from activation of both TRPV1 and P2X receptors, possibly located at terminals of vagal lung capsaicin-sensitive afferent fibers; 2) the functioning of the TRPV1 and P2X receptors in triggering the initial bradypnea is, in part, mediated through the actions of cyclooxygenase metabolites and ATP, respectively; and 3) these mechanisms do not contribute to the H2O2-evoked delayed tachypnea.     

The role of caveolin-1 in pulmonary matrix remodeling and mechanical properties

Caveolin-1 (cav1) is a 22-kDa membrane protein essential to the formation of small invaginations in the plasma membrane, called caveolae. The cav1 gene is expressed primarily in adherent cells such as endothelial and smooth muscle cells and fibroblasts. Caveolae contain a variety of signaling receptors, and cav1 notably downregulates transforming growth factor (TGF)-beta signal transduction. In pulmonary pathologies such as interstitial fibrosis or emphysema, altered mechanical properties of the lungs are often associated with abnormal ECM deposition. In this study, we examined the physiological functions and the deposition of ECM in cav1(-/-) mice at various ages (1-12 mo). Cav1(-/-) mice lack caveolae and by 3 mo of age have significant reduced lung compliance and increased elastance and airway resistance. Pulmonary extravasation of fluid, as part of the cav1(-/-) mouse phenotype, probably contributed to the alteration of compliance, which was compounded by a progressive increase in deposition of collagen fibrils in airways and parenchyma. We also found that the increased elastance was caused by abundant elastic fiber deposition primarily around airways in cav1(-/-) mice at least 3 mo old. These observed changes in the ECM composition probably also contribute to the increased airway resistance. The higher deposition of collagen and elastic fibers was associated with increased tropoelastin and col1alpha2 and col3alpha1 gene expression in lung tissues, which correlated tightly with increased TGF-beta/Smad signal transduction. Our study illustrates that perturbation of cav1 function may contribute to several pulmonary pathologies as the result of the important role played by cav1, as part of the TGF-beta signaling pathway, in the regulation of the pulmonary ECM.     

Pharmacological evidence for the existence of presynaptic alpha-like receptors in Mytilus muscle

The effects of alpha-adrenoceptor agonists and antagonists on contractions of the ABRM of M. edulis were examined. Naphazoline (10(-11)-10(-7)M) as well as octopamine potentiated both contractions in response to ACh and to repetitive electrical stimulation with brief pulses, while clonidine (10(-12)-10(-6)M), imidazole (10(-4)-10(-3)M) and tolazoline (10(-6)-10(-4)M) potentiated only the contraction in response to repetitive electrical stimulation. Potentiating actions of octopamine and naphazoline on ACh contraction were blocked by tolazoline but were little affected by clonidine and imidazole. Potentiating after-effect of repetitive electrical stimulation on subsequent ACh-contraction was not affected by clonidine and imidazole but was blocked by tolazoline. These results suggest that in the ABRM there are probably at least two classes of alpha-like receptors for octopamine; one may be on the muscle fibres and the other may be on the excitatory nerve terminals. Activation of the latter class of receptors may result in enhancement of ACh release from the terminals.     

Potential mechanisms of effects of endogenous neuromodulators on the interdependent activity of neurons in various nuclei of the basal ganglia

A possible mechanism of influence of neuromodulators on interdependent activity of neurons in the diverse basal ganglia nuclei is suggested. According to modulation rules, an activation of postsynaptic Gs- or Gq/11-(Gi/0-) protein coupled receptors promotes induction of long-term potentiation (depression) of excitatory inputs to different neurons and augmentation (lowering) of their activity; an activation of presynaptic Gs- or Gq/11-(Gi/0-) protein coupled receptors promotes a rise (decrease) of release of GABA and co-peptides from striatal terminals and glutamate release from subthalamic terminals in the globus pallidus and output nuclei. It follows from the modulation rules that, since identical receptors are present on striatal neuron and their axon terminals, effects of neuromodulator action in diverse basal ganglia nuclei can be summarized. Neuromodulators released from striato-nigral and striato-pallidal fibers could promote interdependent activity of neurons in "direct" and "indirect" pathways through the basal ganglia due to convergence of these fibers on cholinergic interneurons and pallido-striatal cells.