Sympathectomy-induced immune changes are not abrogated by the glucocorticoid receptor blocker RU-486.

Removal of sympathetic noradrenergic input to the immune system by injection of 6-hydroxydopamine (6-OHDA) triggers increases in antigen-specific in vitro splenocyte proliferation and cytokine production in BALB/cJ and C57B1/6J mice. This examines the possible role of glucocorticoids in these previously reported changes. In both strains, chemical sympathectomy triggers an elevation of glucocorticoid levels immediately following injection of 6-OHDA, returning to normal within one to two days. In the BALB/cJ strain, glucocorticoid elevation is seen only after the initial 6-OHDA injection; levels in chronically denervated animals are not different from controls. In the C57B1/6J strain, the increase is seen even with chronically denervated animals. Prior implantation of mice with pellets containing the glucocorticoid receptor antagonist RU-486 does not abrogate denervation-induced increases in cytokine production or proliferation in either strain. In addition to the previously reported increased interleukin (IL)-2 and IL-4 production, there is an increase in IFN-gamma production in the C57B1/6J strain following either acute or chronic denervation. The persistence of denervation-induced changes even when the effect of corticosterone is blocked with RU-486 or diminished with chronic denervation indicates that the changes are driven mainly by a glucocorticoid-independent mechanism.

Properties of presympathetic neurones in the rostral ventrolateral medulla in the rat: an intracellular study "in vivo'.

1. Intracellular recordings were made in pentobarbitone-anaesthetized rats from sixty-eight neurones located in the rostral ventrolateral medulla (RVLM), which responded with inhibition (latency, 33.6 +/- 9.3 ms) after stimulation of the aortic depressor nerve with short bursts of pulses. This inhibition was due to chloride- and voltage-dependent IPSPs. 2. Seventeen neurones could be excited antidromically after stimulation in the T2 spinal segment (conduction velocity 1.9-8.5 m.s-1) and were classified as RVLM presympathetic vasomotor neurones. 3. "Spontaneously' active neurones (n = 29) displayed a largely irregular pattern of firing, with no clear relationship between the level of the membrane potential and cycles of phrenic nerve activity at end-tidal CO2 < 5.0%. Cardiac cycle-related shifts of the membrane potential were not considered indicative of baroreceptor input as they could be due to movement artifacts. 4. All neurones displayed large synaptic activity (EPSPs and IPSPs, peak-to-peak amplitude > 5.0 mV). The depolarizing IPSPs observed during injection of chloride and/or negative current consisted of a phasic and a tonic component. 5. The on-going activity of these neurones resulted from synaptic inputs, with individual action potentials usually preceded by identifiable fast EPSPs. 6. No evidence was found for the presence of gradual depolarizations (autodepolarizations) between individual action potentials, and therefore under these experimental conditions the activity of RVLM presympathetic neurones did not depend on intrinsic pacemaker properties. 7. These results are consistent with the "network' hypothesis for the generation of sympathetic vasomotor tone.

Pre-sympathetic neurones in the rostral ventrolateral medulla of the rat: electrophysiology, morphology and relationship to adjacent neuronal groups.

The activity of preganglionic sympathetic neurones largely depends on synaptic excitation from antecedent reticulospinal neurones located in the rostral ventrolateral medulla (RVLM). Our study, conducted in anaesthetized rats, showed that all RVLM pre-sympathetic neurones display a substantial synaptic noise and their action potentials are usually preceded by fast EPSPs. No evidence was found for presence of gradual depolarizations (autodepolarizations) between individual spikes. Therefore our results are consistent with the "network" hypothesis for the generation of sympathetic vasomotor tone. Axons of some pre-sympathetic neurones intracellularly labelled with Neurobiotin or Lucifer Yellow had collaterals arborizing in several medullary regions. Thus these neurones have synaptic inputs not only to preganglionic sympathetic neurones, but also to other, yet unidentified cells in the brainstem. Finally, our results show that anatomically adjacent RVLM pre-sympathetic and Bötzinger respiratory neurones from two functionally distinct neuronal subpopulations, and that some pre-sympathetic neurones have an adrenergic phenotype.

Alterations in cytokine and antibody production following chemical sympathectomy in two strains of mice.

It is becoming clear that immune responses are subject to modulation by the sympathetic nervous system. We examined the effect of chemical sympathectomy (to ablate peripheral sympathetic nerve fibers) on cytokine and Ab production in two strains of mice that are known to differ in their response to a variety of pathogens and in the dominant types of cytokines produced. C57Bl/6J mice produce a strong cell-mediated response, characterized by production of IL-2 and IFN-gamma, whereas BALB/cJ have a dominant humoral response, with production of IL-4 and IL-10. Animals were denervated by injection with 6-hydroxydopamine and immunized with keyhole limpet hemocyanin, and spleens were removed at various times after immunization. Denervation significantly increased the keyhole-limpet-hemocyanin-stimulated in vitro proliferation and IL-2 and IL-4 production by splenocytes from both strains. The increases were prevented by pretreatment with desipramine, which blocks the uptake of 6-hydroxydopamine into the nerve fibers and subsequent nerve fiber destruction. Serum titers of IgM, IgG, IgG1, and IgG2a were also enhanced in the C57Bl/6J strain; BALB/cJ mice had a small increase in IgG1 only. These results suggest that one function of splenic innervation and transmitter release may be to modulate T helper cytokines, thereby partially regulating immune effector function. Our evidence is consistent with a model of immune regulation in which removal of sympathetic nervous system input enhances at least some parameters of immune responses.

A comparative study of pre-sympathetic and Bötzinger neurons in the rostral ventrolateral medulla (RVLM) of the rat.

The aim of this study was to investigate the degree of functional and anatomical overlap between two major neuronal subpopulations in the rostral ventrolateral medulla: pre-sympathetic (sympathoexcitatory) neurons, and expiratory neurons of the Bötzinger complex. Extracellular recordings were made with dye-filled microelectrodes in pentobarbital anesthetized, paralyzed and artificially ventilated adult Wistar rats. Tests applied included stimulation of baroreceptor afferents, activation of peripheral chemoreceptors and lung stretch receptors, changes in central respiratory drive with hyper- or hypoventilation, nociceptive stimulation, and antidromic stimulation from the T2 segment of the spinal cord or medulla oblongata at obex level. The two groups of neurons showed different patterns of spontaneous activity and generally different responses to these stimuli. The recording positions showed some overlap, but the majority of Bötzinger neurons were dorsolateral to pre-sympathetic neurons. There was a large overlap between the location of pre-sympathetic neurons and the lateral part of the C1 adrenergic group, but only a small overlap between these adrenergic neurons and Bötzinger neurons. These results indicate that the anatomically adjacent pre-sympathetic and Bötzinger expiratory neurons form two functionally distinct neuronal subpopulations.

Barosensitive neurons in the rostral ventrolateral medulla of the rat in vivo: morphological properties and relationship to C1 adrenergic neurons.

The aim of this study, conducted in anaesthetized rats, was to examine the morphology of barosensitive neurons in the rostral ventrolateral medulla and their immunoreactivity for a catecholamine synthesizing enzyme, tyrosine hydroxylase. Thirty neurons displaying inhibitory postsynaptic potentials following stimulation of the aortic depressor nerve were intracellularly labelled with Lucifer Yellow or Neurobiotin. Some of these neurons could be excited antidromically from the second thoracic segment of the spinal cord, with conduction velocities of spinal axons ranging from 1.9 to 7.2 m/s. The filled somas were found immediately caudal to the facial nucleus and ventral or ventromedial to compact formation of the nucleus ambiguus. Some dendrites reached the ventral medullary surface. Axons usually projected dorsomedially and then made a sharp rostral and/or caudal turn. The caudally projecting axon could, in some cases, be followed to the first cervical segment of the spinal cord. Seven cells issued fine axon collaterals on the ipsilateral side. These were identified mainly in two areas: in the rostral ventrolateral medulla (or immediately dorsomedial to that region), and within the dorsal vagal complex. Seven of 27 examined cells (26%) were tyrosine hydroxylase-immunoreactive and were classified as C1 adrenergic neurons. No clear relationship was found between the presence or absence of adrenergic phenotype and the morphology of filled cells. However, the amplitude of aortic nerve-evoked inhibitory postsynaptic potentials was significantly larger in tyrosine hydroxylase-positive neurons. Possible reasons for the low percentage of barosensitive cells with tyrosine hydroxylase immunoreactivity found in this study, in comparison with previously published estimates, are discussed. This is the first study describing the morphology of neurons in this part of the medulla identified as barosensitive in vivo, and directly demonstrating adrenergic phenotype in a subset of these neurons.

Criteria for intracellular identification of pre-sympathetic neurons in the rostral ventrolateral medulla in the rat.

Previous studies revealed that a relatively small group of reticulospinal neurons located in the rostral ventrolateral medulla (RVLM) plays a key role in the generation of resting vasomotor tone and in reflex control of arterial blood pressure. These medullary pre-sympathetic neurons have been extensively studied with extracellular microelectrodes, but so far few attempts have been made to examine their intracellular properties in vivo. This report, based on intracellular recordings from 8 RVLM pre-sympathetic neurons in anaesthetised rats, sets out criteria for intracellular identification of such neurons. We propose that two features are sufficient to classify RVLM neurons as pre-sympathetic during intracellular recording: inhibitory response to stimulation of the aortic depressor nerve with short bursts of pulses applied at low frequency; and antidromic stimulation from the upper thoracic segments. Cardiac oscillations in the membrane potential or responses during large changes in blood pressure can be due to movement artefact, and are therefore not reliable as a means of demonstrating baroreceptor input. Further intracellular studies of these neurons will undoubtedly result in further progress in understanding their function.

Morphological study of long axonal projections of ventral medullary inspiratory neurons in the rat.

The aim of this study was to examine medullary and spinal axonal projections of inspiratory bulbospinal neurons of the rostral ventral respiratory group (VRG) in the rat. A direct visualization of long (9.8-33 mm) axonal branches, including those projecting to the contralateral side of the medulla oblongata and the spinal cord, was possible due to intracellular labeling with neurobiotin and long survival times (up to 22 h) after injections. Seven of the nine labeled neurons had bilateral descending axons, which were located in discrete regions of the spinal white matter; ipsilateral axons in the lateral and dorsolateral funiculus, contralateral in the ventral and ventromedial funiculus. The collaterals issued by these axons at the mid-cervical level formed close appositions with dendrites of phrenic motoneurons, which had also been labeled with neurobiotin. None of these collaterals crossed the midline. The significance of this finding is discussed in relation to the crossed-phrenic phenomenon. Additional spinal collaterals were identified in the C1 and T1 segments. Within the medulla, collaterals with multiple varicosities were identified in the lateral tegmental field and in the dorsomedial medulla (in the hypoglossal nucleus and in the nucleus of the solitary tract). These results demonstrate that inspiratory VRG neurons in the rat have some features which have not been previously described in the cat, including frequent bilateral spinal projection and projection to the nucleus of the solitary tract. In addition, this study shows that intracellular labeling with neurobiotin offers an effective way of tracing long axonal projections, supplementing results previously obtainable only with antidromic mapping, and providing morphological details which could not be observed in previous studies using labeling with horseradish peroxidase.

An electrophysiological and morphological study of esophageal motoneurons in rats.

Previous anatomic studies have shown that motoneurons supplying the striated musculature of the esophagus form a tightly grouped cluster in the rostral portion of the nucleus ambiguus, known as the compact formation. This study, conducted in anesthetized rats, presents the first in vivo intracellular and extracellular recordings from this group of motoneurons, which were identified by antidromic stimulation directly from the esophagus (latency 7-68 ms). The motoneurons were silent at rest, and those impaled intracellularly (n = 44) showed no respiratory modulation of their membrane potential. Intracellular labelling with Lucifer Yellow (n = 3) or Neurobiotin (n = 15) revealed multipolar somas with longitudinally oriented dendritic trees mainly confined to the compact formation. No axon collaterals were found. When swallowing-like activity was induced by muscarine applied to the dorsal medullary surface, the motoneurons displayed bursting activity, with the majority of bursts occurring during expiration. These results show that antidromic stimulation of esophageal motoneurons with an electrode inserted into the esophagus provides a simple way of identifying these motoneurons. In the absence of pharmacological stimulation, these motoneurons receive no respiratory-modulated synaptic input, in contrast to adjacent motoneurons in the semicompact formation (supplying the upper airways), which are known to display respiratory activity. However, some synchronization of respiratory and swallowing-like activity was observed after pharmacological activation of the swallowing pattern generator in the dorsal medulla.

Specific second messengers activate the caudal photoreceptor of crayfish.

The caudal photoreceptor (CPR) found in the last abdominal ganglion of crayfish is a well-known example of a non-retinal photosensitive element. In addition to light sensitivity, this cell has been assigned a command role for a walking behavior. The molecular mechanism of transduction in this cell has not been previously studied. The involvement of an intermediate messenger substance is suggested by its long latency to response, its prolonged afterdischarge, and by the requirement for an amplification process for the efficient transduction of light. We tested the effect of some putative second messengers by pressure injecting them into the CPR and noting the physiological response. Here we report that intracellular injection of inositol 1,4,5-trisphosphate (IP3), calcium, and the guanosine nucleotide GTP mimics the light response, while cAMP, IP1 and IP2 have no effect on the firing rate. The key intermediate in transduction in vertebrate photoreceptors, cGMP, was ineffective in this system. This work adds to the growing body of evidence that IP3 plays a role in invertebrate phototransduction.

Upper cervical inspiratory neurons in the rat: an electrophysiological and morphological study.

Upper cervical inspiratory neurons from a distinct neuronal column located near the lateral edge of the intermediate grey matter in the rostral spinal segments. Previous studies conducted in cats have demonstrated synaptic inputs to these neurons from several respiratory-related regions of the medulla, and long descending axonal projections mainly towards the motoneurons supplying the intercostal muscles. The aim of this study was to examine the electrophysiological and morphological properties of this propriospinal system in the rat. Extracellular recordings were made from 127 cervical inspiratory units, mainly in the C1 and C2 segments. Eighty-two percent could be antidromically activated from the C7/C8 border. No evidence of monosynaptic connection was obtained by cross-correlating the activity of some of these units with the discharge of the phrenic nerve. Intracellular recordings were made from seven neurons, three of which were labelled with biotinamide (neurobiotin). Long "survival times" after intracellular injections (up to 23 h) resulted in staining of axons for long distances, at least to the C5 segment. Each of the three labelled axons issued only one short collateral which arborized in the region of the phrenic nucleus. These results demonstrate that upper cervical inspiratory neurons in the rat have features similar to those previously described in the cat, including only a limited projection to the phrenic nucleus. In addition, this study provides the first morphological identification of these neurons.

Membrane fractions display different lipid and enzyme content in three cell types in 16-cell stage embryos of sea urchins.

Three cell types were isolated from dissociated 16-cell sea urchin embryos. Four membrane density fractions from discontinuous gradients have different proportions of lipids, surfacer markers and enzymes for the three cell types. Assays of lipid content, CH/PLIPID and SPH/PC ratios, acyl chain length, level of unsaturation by proton NMR and assays of enzyme activity revealed variation at the same density between the three cell types and among different densities from one cell type. There were also differences between whole embryos and dissociated embryo cells. There was no typical membrane domain at a particular density common to the cell types. Cell surface characteristics and polarity of adult cells rely on which lipid domains and enzymes are present, their association with cytoskeleton and how they are localized. At the 16-cell stage these characteristics are still very dynamic as revealed by cytochemical localization of Na+/K(+)-ATPase which varied with cell type and suggests endocytosis at set times in the division cycle. Polarity has not been permanently set for Na+/K(+)-ATPase yet. Membrane enzyme and lipid distributions unique to the three cell types seen in this study suggest parcelling out or insertion of new membrane domains occurs during early sea urchin cleavage. Perturbation of membrane density distribution and lipid content occurs after treatment of embryos with animalizing and vegetalizing teratogens which alter development.