Multiple Adrenergic Receptor Subtypes Controlling Cyclic AMP Formation: Comparison of Brain Slices and Primary Neuronal and Glial Cultures

The adrenergic receptor subtypes involved in cyclic AMP responses to norepinephrine (NE) were compared between slices of rat cerebral cortex and primary neuronal and glial cultures from rat brain. In neuronal cultures, NE and the beta-adrenergic receptor agonist isoproterenol (ISO) caused similar increases in cyclic AMP, which were not altered by the alpha-adrenergic receptor antagonist phentolamine. In glial cultures, NE caused a much smaller cyclic AMP response than did ISO, and this difference was reversed by alpha-adrenergic receptor antagonists (phentolamine greater than yohimbine greater than prazosin). alpha 2-Adrenergic receptor agonists partially inhibited the ISO response in glial cultures to a level similar to that observed with NE alone (clonidine = UK 14,304 greater than NE greater than 6-fluoro-NE greater than epinephrine). In slices from cerebral cortex, NE caused a much larger increase in cyclic AMP than did ISO, and this difference was reversed by alpha-adrenergic receptor antagonists with a different order of potency (prazosin greater than phentolamine greater than yohimbine). alpha 1-Adrenergic receptor agonists potentiated the response to ISO to a level similar to that observed with NE alone (epinephrine = NE greater than phenylephrine greater than 6-fluoro-NE greater than methoxamine). In all three tissue preparations, large responses to both alpha 1-receptor activation (increases in inositol phosphate accumulation) and alpha 2-receptor activation (decreases in forskolin-stimulated cyclic AMP accumulation) were observed. These data indicate that all of the major adrenergic receptor subtypes (beta, alpha 1, alpha 2) are present in each tissue preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

结核分枝杆菌感染小鼠的脾脏和肺脏组织荷菌量与病理变化

目的分析结核急性感染小鼠脾脏和肺脏的组织荷菌量以及病理的动态变化。方法以3×105CFU结核分枝杆菌标准株H37Rv尾静脉途径感染雌性C57BL/6J小鼠,测定感染后1 d、1周、2周、3周、4周、6周和8周肺脏及脾脏组织的荷菌量,脾脏和肺脏组织经HE染色分析病理变化。结果感染动物的脾脏荷菌量在前3周逐步提高,在4～8周脾脏荷菌量维持稳定,肺脏组织在2～8周组织荷菌量逐步升高。病理分析显示动物感染3周后肺脏和脾脏出现肉芽肿,在6～8周病理损伤加重。结论小鼠经尾静脉感染高剂量结核分枝杆菌在8周前表现为急性感染状态,适用于抗结核药物的体内药效学评价。     

Microtubule Deacetylases,SirT2 and HDAC6, in the Nervous System

Examination of the cytoskeleton has demonstrated the pivotal role of regulatory proteins governing cytoskeletal dynamics. Most work has focused on cell cycle and cell migration regarding cancer. However, these studies have yielded tremendous insight for development, particularly in the nervous system where all major cell types remodel their shape, generate unsurpassed quantities of membranes and extend cellular processes to communicate, and regulate the activities of other cells. Herein, we analyze two microtubule regulatory alpha-tubulin deacetylases, histone deacetylase-6 (HDAC6) and SirT2. HDAC6 is expressed by most neurons but is abundant in cerebellar Purkinje cells. In contrast, SirT2 is targeted to myelin sheaths. Expression of these proteins by post-mitotic cells indicates novel functions, such as process outgrowth and membrane remodeling. In oligodendrocytes, targeting of SirT2 to paranodes coincides with the presence of the microtubule-destabilizing protein stathmin-1 during early myelinogenesis and suggests the existence of a microtubule regulatory network that modulates cytoskeletal dynamics. Special issue dedicated to Dr. Anthony Campagnoni.     

Immunocytochemistry of GABA in the antennal lobes of the sphinx moth Manduca sexta

Summary The ganglionated plexus of the trachea of mice was studied quantitatively with a histochemical method that stains electively the ganglion nerve cells in whole-mount preparations. The plexus lies exclusively over the muscular part of the trachea, dorsal to the muscle itself, and it varies considerably in pattern and extent between individual animals. In young adult mice the plexus contains on average 235 neurons, occurring singly or gathered in packed ganglia. The ganglion neurons are relatively small, the profile area of three quarters of them measuring between 150 and 275 m² with an average of 251 m². In ageing mice the average number of ganglion neurons is the same as in young animals; however, cell sizes are markedly increased, the average being 341 m². Among the ultrastructural features of the ganglia, is a capsule (perineurium) of very regular structure, the presence of collagen, capillaries and myelinated axons inside the ganglia, and the presence of only few and short dendrites, some of which are abutted by synapsing nerve endings.     

Adrenergic Receptors and Fat Cells: Differential Recruitment by Physiological Amines and Homologous Regulation

The control of fat cell lipolysis by the catecholamines involves at least four different adrenoceptor subtypes; three β (β1-, β2-, and β3-ARs) and one α2-adrenoceptor(α2-AR). The physiological importance of the β- and α2A-ARs varies according to the species, the sex, the age, the anatomical location of fat deposits and the degree of obesity in humans and animals. The physiological amines operate through differential recruitment of these sites on the basis of their relative affinities. This point has been assessed by in vitro studies and has partly been confirmed in in vivo experiments using selected a/β-AR antagonists and in situ microdialysis. The affinity of the β3-AR for catecholamines is less than that of the classical β1- and β2-ARs in the various species investigated. Conversely, it is the α2-AR which exhibit the highest affinity for the physiological amines in all fat cells. The relative order of affinity of the various fat cell ARs for the physiological amines defined in binding studies and in vitro ass ays is α2 > β1 > β2 > β3 for norepinephrine and α2 >β2 > β1> β3 for epinephrine. When considering differential β-AR recruitment by catecholamines, it is the β1-AR which is always activated at the lowest norepinephrine levels, whatever the species, while the activation of the β3-AR requires higher norepinephrine levels. In addition to the differential recruitment, differential regulation by hormones could also occur for each fat cell AR subtype. The α2-and β3-ARs are less prone to desensitization and down-regulation by comparison with the β1- and β2-AR.     

Modulation by GABA of neuroplasticity in the central and peripheral nervous system

Apart from being a prominent (inhibitory) neurotransmitter that is widely distributed in the central and peripheral nervous system, -aminobutyric acid (GABA) has turned out to exert trophic actions. In this manner GABA may modulate the neuroplastic capacity of neurons and neuron-like cells under various conditions in situ and in vitro. In the superior cervical ganglion (SCG) of adult rat, GABA induces the formation of free postsynaptic-like densities on the dendrites of principal neurons and enables implanted foreign (cholinergic) nerves to establish functional synaptic contacts, even while preexisting connections of the preganglionic axons persist. Apart from postsynaptic effects, GABA inhibits acetylcholine release from preganglionic nerve terminals and changes, at least transiently, the neurochemical markers of cholinergic innervation (acetylcholinesterase and nicotinic receptors). In murine neuroblastoma cells in vitro, GABA induces electron microscopic changes, which are similar in principle to those seen in the SCG. Both neuroplastic effects of GABA, in situ and in vitro, could be mimicked by sodium bromide, a hyperpolarizing agent. In addition, evidence is available that GABA via A- and/or B-receptors may exert direct trophic actions. The regulation of both types of trophic actions (direct, receptor-mediated vs. indirect, bioelectric activity dependent) is discussed.Special issue dedicated to Dr. Claude Baxter.     

Cholinergic innervation of the mouse superior cervical ganglion: light-and electron-microscopic immunocytochemistry for choline acetyltransferase

Summary The cholinergic innervation of the mouse superior cervical ganglion was investigated by means of immunocytochemistry using a well-characterized monoclonal antibody against choline acetyltransferase (ChAT). Immunopositive nerve fibers entered the superior cervical ganglion from the cervical sympathetic trunk. Light-microscopically, these fibers appeared to be heterogeneously distributed among the principal ganglion cells. The rostral part of the ganglion contained more ChAT-positive fibers then the middle or the caudal one. The axons branched several times before forming numerous varicosities. Most of the ChAT-stained fibers and varicosities aggregated in glomerula-like neuropil structures that were surrounded by principal ganglion cell bodies, whereas others were isolated or formed little bundles among principal neurons. None of the neurons or other cell types in the ganglion exhibited ChAT-positivity. ChAT-immunoreactive fibers disappeared from the ganglion 5 or 13 days after transection of the cervical sympathetic trunk. At the ultrastructural level, most axon terminals and synapses showed ChAT-immunoreactivity. An ultrastructural analysis indicated that immunostained synapses occurred directly on the surface of neuronal soma (1.8%) and dendritic shafts (17.6%). Synapses were often seen on soma spines (18.4%) and on dendritic spines (62.2%). All immunoreactive synapses were of the asymmetric type. The results provide immunocytochemical evidence for a heterogeneous cholinergic innervation of the ganglion and the principal neurons.     

TRH: Cardiovascular and sympathetic modulation in brain nuclei of the rat

The cardiovascular and sympathetic effects of TRH in discrete cardiovascular-related brain nuclei were studied. Microinjections of TRH were made into the nucleus preopticus medialis (POM) of conscious rats and the nucleus tractus solitarius (NTS) of pentobarbitone-anesthetized, artificially respired rats. POM injections (1 μl, 0.8–80 nM) elicited dose dependent pressor and tachycardic responses which were accompanied by increased levels of norepinephrine (NE) and epinephrine (EPI) in the plasma. These pressor/tachycardic effects of TRH were also elicited in adrenal demedullated (ADM-x) rats, but completely abolished in ADM-x rats pretreated with bretylium (30 mg/kg, IA). NTS injections (0.1 μl, 30 and 150 nM) had a short depressor effect on blood pressure (BP) and a delayed increase in heart rate (HR). From these findings we suggest that the POM, a central nucleus in the AV3V region, may be an important forebrain site for autonomic regulation by TRH, mediated through the sympathetic nervous system.     

Thermal sensitivity of heart rate and insensitivity of blood pressure in the Antarctic nototheniid fish <Emphasis Type="Italic">Pagothenia borchgrevinki</Emphasis>

The Antarctic notothenioids are among the most stenothermal of fishes, well adapted to their stable, cold and icy environment. The current study set out to investigate the thermal sensitivity/insensitivity of heart rate and ventral aortic blood pressure of the Antarctic nototheniid fish Pagothenia borchgrevinki over a range of temperatures. The heart rate increased rapidly from –1 to 6°C (Q₁₀=2.0–3.3), but was relatively insensitive to temperature above the ~6°C lethal limit of the species (Q₁₀=1.2). The increase in heart rate from –1 to 6°C was the result of a 45% increase in excitatory adrenergic tone, masking a 37% increase in inhibitory cholinergic tone. Ventral aortic pressure was regulated well above the lethal limit, up to at least 10°C. With the return of the fish to environmental temperatures, the heart rate rapidly decreased back to control levels, while ventral aortic pressure increased and remained elevated for over an hour following a 6°C exposure.     

Adrenergic innervation of the dorsal vagal motor nucleus: possible involvement in inhibitory control of gastric acid and pancreatic insulin secretion

Summary Morphological and physiological approaches were used to investigate the possible role of an adrenergic innervation of the dorsal vagal complex in the control of basal gastric acid and pancreatic insulin secretion in the rat. The use of retrograde-tracing methods with injections of True Blue or of wheat-germ agglutinin into the stomach or pancreas first confirmed that most vagal preganglionic neurons innervating these two viscera are localized in the dorsal motor nucleus of the vagus, a number of them connected to both viscera. Light- and electron-microscopic investigation of the organization of adrenergic neuronal structures immunoreactive to phenylethanolamine-N-methyltransferase within this medullary nucleus further revealed: (i) that adrenergic axons establish profuse synaptic connections of the symmetrical type with perikarya and dendrites of this nucleus, and (ii) that several of these adrenergic fibers are connected with retrogradely labeled neurons innervating the stomach and/or pancreas. Lastly, measurements of basal gastric acid output and plasma insulin clearly indicated that both visceral secretions are rapidly and conspicuously decreased by local infusion of 2 nM adrenaline within the dorsal vagal complex. Taken together, these data strongly suggest that the adrenergic innervation of the dorsal medulla oblongata is involved in direct synaptic inhibition of the parasympathetic preganglionic neurons of the vagus that control secretion of gastric acid and pancreatic insulin.     

Differential effects of hydrocortisone on sympathetic and hemodynamic responses to sympathoexcitatory manoeuvres in men

Aim of the present study was to investigate the influence of hydrocortisone on muscle sympathetic nerve activity (MSNA) and hemodynamic parameters during different sympathoexcitatory manoeuvres in humans. The study focuses on the interaction of the hypothalamo-pituitary-adrenal system and the sympathetic nervous system. Hydrocortisone 100 mg or placebo was administered intravenously to eight young healthy subjects in a double-blind crossover design. After 6 h, blood pressure, heart rate and MSNA from the peroneal nerve were recorded at rest, during an arithmetic stress task, an apnea and a cold pressor test. Hydrocortisone treatment increased serum cortisol levels to the upper physiological range and suppressed basal levels of adrenocorticotropin. During mental stress, MSNA, heart rate and blood pressure levels were elevated independently of hydrocortisone pre-treatment. However, hydrocortisone induced a sustained increase in basal heart rate throughout the whole experiment. A stronger increase in diastolic blood pressure was observed during apnea and cold pressor test in the hydrocortisone experiments. MSNA or plasma catecholamines at rest or during the manoeuvres were not affected by hydrocortisone. The observed hydrocortisone effects may be due to an increased responsiveness of adrenergic receptors towards catecholamines or a central modulation of the baroreflex involving parasympathetic mechanisms. Further studies are needed to confirm that the increase in MSNA during mental stress does not depend on a concomitant activation of the hypothalamo-pituitary-adrenal system.     

Regulation of Dipeptidyl Aminopeptidase I and Angiotensin Converting Enzyme Activities in Cultured Murine Brain Cells by Cortisol and Thyroid Hormone

Cultures of dissociated brain cells from 15-day-old fetal mice were grown in the presence and absence of 20 or 50 nM triiodothyronine (T3), 30 or 300 nM cortisol, and 30 nM cortisol plus 50 nM T3 added to chemically defined media or in media supplemented with 15% serum from control and hypothyroid calves. The specific activities of five lysosomal enzymes--N-acetyl galactosaminidase, beta-glucuronidase, beta-galactosidase, cathepsin B, and dipeptidyl aminopeptidase I (DAP-I)--were higher in cells grown in calf serum than in cells grown in defined media. Of these enzymes, only DAP-I was elevated in activity when the cells were grown in hypothyroid calf serum instead of control calf serum. Elevation of DAP-I activity was reversed by addition of 20 nM T3 to hypothyroid calf serum. The enzymatic properties of DAP-I were similar whether the cells were grown in control or hypothyroid calf serum and were similar to those reported for human fibroblasts and the purified enzyme. When the cells were grown in defined media, cortisol decreased the activities of all lysosomal enzymes, with 300 nM cortisol being more effective than 30 nM cortisol. Addition of 50 nM T3 to 30 nM cortisol decreased DAP-I activity more than 30 nM cortisol alone, but 50 nM T3 alone in defined media did not alter DAP-I levels. The reduction of DAP-I activity in these cells by T3 required cortisol, unidentified components in serum, or both.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of chronic psychogenic stress exposure on enkephalin neuronal activity and expression in the rat hypothalamic paraventricular nucleus

This study tested the hypothesis that the activation pattern of enkephalinergic (ENKergic) neurons within the paraventricular nucleus of the hypothalamus (PVH) in response to psychogenic stress is identical whether in response to repeated exposure to the same stress (homotypic; immobilization) or to a novel stress (heterotypic; air jet puff). Rats were assigned to either acute or chronic immobilization stress paradigms (90 min/day for 1 or 10 days, respectively). The chronic group was then subjected to an additional 90-min session of either heterotypic or homotypic stress. A single 90-min stress session (immobilization or air jet) increased PVH-ENK heteronuclear (hn) RNA expression. In chronically stressed rats, exposure to an additional stress session (whether homotypic or heterotypic) continued to stimulate ENK hnRNA expression. Acute immobilization caused a marked increase in the numbers of Fos-immunoreactive and Fos-ENK double-labeled cells in the dorsal and ventral medial parvicellular, and lateral parvicellular subdivisions of the PVH. Chronic immobilization caused an attenuated Fos response ( approximately 66%) to subsequent immobilization. In contrast, chronic immobilization did not impair ENKergic neuron activation within the PVH following homotypic or heterotypic stress. These results indicate that within the PVH, chronic psychogenic stress markedly attenuates the Fos response, whereas ENKergic neurons resist habituation, principally within the ventral neuroendocrine portion of the nucleus. This suggests an increase in ENK effect during chronic stress exposure. Homotypic (immobilization) and heterotypic (air jet) psychogenic stressors produce similar responses, including Fos, ENK-Fos, and ENK hnRNA, within each subdivision of the PVH, suggesting similar processing for painless neurogenic stimuli.     

Projections and chemistry of Dogiel type II neurons in the mouse colon

The physiological properties, shapes, projections and neurochemistries of Dogiel type II neurons have been thoroughly investigated in the guinea-pig intestine in which these neurons have been identified as intrinsic primary afferent neurons. Dogiel type II neurons in the myenteric ganglia of mice have similar physiological properties to those in guinea-pigs but whether other features of the neurons are similar is unknown. We have used intracellular dye-filling, retrograde tracing, immunohistochemistry and nerve lesions to determine salient features of Dogiel type II neurons of the mouse colon. Dye-filling showed that the neurons provide profuse terminal networks in the myenteric ganglia and also have axons that project towards the mucosa. Retrograde tracing and lesion studies showed that these axons provide direct innervation to the mucosa. High proportions of the neurons had immunoreactivity for calretinin, calbindin, choline acetyltransferase, the purine P2X₂ receptor and calcitonin gene-related peptide (CGRP). CGRP was the most selective marker of the neurons. Following surgery to remove an area of myenteric plexus, the CGRP-immunoreactive nerve fibres in the mucosa degenerated. Thus, Dogiel type II neurons in mice have similar shapes and projections but some differences in chemistry from those in guinea-pigs. The close similarities between the two species in the shapes, projections and electrophysiology of these neurons suggest that they serve the same functions in both species.These studies were funded by the National Health and Medical Research Council (Australia)     

Regulation of neuroinflammation: the role of CXCL10 in lymphocyte infiltration during autoimmune encephalomyelitis

The movement of lymphocytes from the microvasculature into the central nervous system (CNS) parenchyma is an essential step in the pathogenesis of a variety of infectious and autoimmune neuroinflammatory diseases. The lymphocyte chemoattractant CXCL10 and its receptor, CXCR3, are expressed by the CNS and by CNS infiltrating lymphocytes, respectively, only in patients with ongoing CNS inflammation, suggesting an important role for these molecules in the pathogenic process. Numerous studies utilizing animal models and transgenic approaches have indeed supported a role for CXCL10 in the intraparenchymal trafficking of lymphocytes during acute CNS inflammation; however, other studies suggest that its expression is not required for the development of autoimmune forms of CNS inflammation and, in fact, that interference with CXCL10 signaling could lead to increased neuroinflammation. This review will consider the data from these studies and attempt to reconcile them through comparisons of both the neuroinflammatory models and the effects of CXCL10 in the CNS versus lymphoid tissues. Finally, it will define directions for future analyses of CXCL10 and CXCR3 in CNS inflammation so that their potential therapeutic utility can be more completely determined.     

Twenty-Four-Hour Variation in the Occurrence and Survival of Potentially Lethal Ventricular Tachyarrhythmia

The temporal pattern in the occurrence and primary outcome of resuscitation of out-of-hospital ventricular fibrillation/tachycardia was studied. The onset time of 117 cases of tachyarrhythmia exhibited a double-peaking 24-h variation with a first prominent peak in the midmorning and a second less prominent one during the late afternoon and early evening hours. Survival after resuscitation also was time dependent, showing a quite different temporal pattern. Survival was lowest during the daytime and greatest (four to five times greater) during the night. The findings are consistent with the hypothesis that out-of-hospital ventricular fibrillation/tachycardia may be dependent on autonomic regulation.     

Nerve fibers in the gut and pancreas of the rat displaying neuropeptide-Y immunoreactivity

Summary Immunoreactive neuropeptide Y (NPY) was demonstrated in neuronal elements in the gut and pancreas of the rat. Immunoreactive endocrine cells could not be detected. The occurrence of NPY containing nerve-cell bodies in the submucosal and myenteric ganglia indicates an intrinsic origin of the NPY fibers. However, an additional extrinsic supply of NPY fibers is suggested by the finding that abdominal sympathectomy caused the disappearance of some NPY fibers, notably those around blood vessels. The distribution of NPY fibers in all layers of the gut wall suggests multiple functions of NPY, including a role in the regulation of intramural neuronal activities, smooth muscle tone, and local blood flow.     

Distribution of peptide-immunoreactive nerves in the foetal and newborn guinea-pig caecum

Summary The distribution of vasoactive intestinal polypeptide-, substance P-, [met]enkephalin- and somatostatin-like immunoreactive nerves was studied in the caecum from foetal guinea-pigs of 6–9 weeks gestation (i.e., approximately 1–4 weeks before birth) and 4–5-day-old guinea-pigs. Peptide-immunoreactive nerves were first detected in the myenteric and submucous plexuses and circular muscle layer at 6 weeks of gestation and in the mucosa at 7 weeks of gestation. The density of fibres in these layers increased during prenatal development until, by 9 weeks of gestation, their distribution resembled that seen in the postnatal animals. This distribution was similar to that described previously in adult animals. A different pattern of development was observed in the caecal taenia coli muscle. Peptide-immunoreactive fibres were not detected until 8 weeks of gestation in this tissue layer, and were then only sparsely distributed. A dramatic increase in the number of labelled fibres, however, occurred between 8 and 9 weeks of gestation. Further, vasoactive intestinal polypeptide- and substance P-immunoreactive fibres were more numerous in the taeniae coli of 9-week-old embryos than in those of postnatal animals. Thus, the guinea-pig enteric nervous system, which in many respects is well-developed at the time of birth, may still be undergoing developmental changes at this time.