Peripheral oxygen-sensing cells directly modulate the output of an identified respiratory central pattern generating neuron

Breathing is an essential homeostatic behavior regulated by central neuronal networks, often called central pattern generators (CPGs). Despite ongoing advances in our understanding of the neural control of breathing, the basic mechanisms by which peripheral input modulates the activities of the central respiratory CPG remain elusive. This lack of fundamental knowledge vis-à-vis the role of peripheral influences in the control of the respiratory CPG is due in large part to the complexity of mammalian respiratory control centres. We have therefore developed a simpler invertebrate model to study the basic cellular and synaptic mechanisms by which a peripheral chemosensory input affects the central respiratory CPG. Here we report on the identification and characterization of peripheral chemoreceptor cells (PCRCs) that relay hypoxia-sensitive chemosensory information to the known respiratory CPG neuron right pedal dorsal 1 in the mollusk Lymnaea stagnalis. Selective perfusion of these PCRCs with hypoxic saline triggered bursting activity in these neurons and when isolated in cell culture these cells also demonstrated hypoxic sensitivity that resulted in membrane depolarization and spiking activity. When cocultured with right pedal dorsal 1, the PCRCs developed synapses that exhibited a form of short-term synaptic plasticity in response to hypoxia. Finally, osphradial denervation in intact animals significantly perturbed respiratory activity compared with their sham counterparts. This study provides evidence for direct synaptic connectivity between a peripheral regulatory element and a central respiratory CPG neuron, revealing a potential locus for hypoxia-induced synaptic plasticity underlying breathing behavior.     

Axonal regeneration of an identified Helisoma neuron depends on the site of axotomy.

Axotomy of molluscan neurons usually results in axonal regeneration. In this study, we tested the axotomy response of an identified neuron of the pond snail Helisoma trivolvis (buccal neuron 4, B4). This neuron has two primary axonal branches, the ipsilateral axon and the contralateral axon, each innervating one of the paired salivary glands. The ipsilateral axon projects via the ipsilateral esophageal nerve trunk whereas the contralateral axon crosses both buccal ganglia and projects via the contralateral esophageal nerve trunk. We tested various procedures of axotomy: injury to one axon or both axons, close to the ganglion or more distal. Surprisingly, we found that proximal axotomy of the ipsilateral axon was not usually followed by axonal regeneration. By contrast, all other procedures of axotomy (e.g., distal ipsilateral, or proximal bilateral) resulted in robust axonal regeneration and target reinnervation. Thus, in this preparation, axotomy may or may not result in axonal regeneration, depending on the site(s) of axotomy. To the best of our knowledge, such a differential result has not yet been found in any other preparation. We conclude that axotomy is not always a sufficient condition for axonal regeneration of molluscan neurons. We hypothesize that a damaged axonal stump may be a necessary condition for the initiation of regeneration. An alternative hypothesis is that neurite outgrowth is inhibited in normal mature neurons by a target-derived factor. This hypothetical factor would be transported retrogradely, but not anterogradely, along axons.     

An identified central pattern-generating neuron co-ordinates sensory-motor components of respiratory behavior in Lymnaea

Defining the attributes of individual central pattern-generating (CPG) neurons underlying various rhythmic behaviors are fundamental to our understanding of how the brain controls motor programs, such as respiration and locomotion. To this end, we have explored a simple invertebrate preparation in which the neuronal basis of respiratory rhythmogenesis can be investigated from the whole animal to a single cell level. An identified dopaminergic neuron, termed right pedal dorsal 1 (RPeD1), is a component of the CPG network which controls hypoxia-driven, aerial respiration in the fresh water snail Lymnaea stagnalis. Using intact, semi-intact and isolated brain preparations, we have discovered that in addition to its role as a respiratory CPG neuron, RPeD1 co-ordinates sensory-motor input from the pneumostome (the respiratory orifice) at the water/air interface to initiate respiratory rhythm generation. An additional, novel role of RPeD1 was also found. Specifically, direct intracellular stimulation of RPeD1 induced pneumostome openings, in the absence of motor neuronal activity. To determine further the role of RPeD1 in the respiratory behavior of intact animals, either its axon was severed or the soma selectively killed. Many components of the respiratory behavior in the intact animals were found to be perturbed following RPeD1 axotomy or 'somatomy' (soma removed). Taken together, the data presented provide a direct demonstration that RPeD1 is a multifunctional CPG neuron, which also serves many additional roles in the control of breathing behavior, ranging from co-ordination of mechanosensory input to the motor control of the respiratory orifice.     

Anatomical regeneration and behavioral recovery following crush injury of the trigeminal root in lamprey

In normal larval lamprey, bilateral application of horseradish peroxidase (HRP) to the dorsal part of the anterior oral hood labeled subpopulations of trigeminal components on both sides of the brain; peripherally projecting motoneurons, medullary dorsal cells (sensory), and spinal dorsal cells (sensory), as well as centrally projecting afferents in the trigeminal descending tracts. Following unilateral crush injury of the right trigeminal root, HRP labeling of sensory and motor trigeminal components on the right side gradually increased with increasing recovery time, between 2 weeks and 12 weeks postcrush (PC). Axons of trigeminal motoneurons appeared to exhibit robust regeneration, whereas restoration of projections in the descending trigeminal tract ipsilateral to the injury was incomplete. Control experiments indicated that motor and sensory axons from the intact side of the oral hood did not sprout across the midline to the denervated side. Several results suggested that regenerated trigeminal sensory fibers made synapses with brain neurons that have direct or indirect inputs to reticulospinal (RS) neurons. Following a unilateral crush injury of the right trigeminal root, escape behavior in response to stimulation of the right side of the oral hood gradually returned to normal. Muscle recordings at various recovery times confirmed that anatomical regeneration of trigeminal sensory axons was functional. In addition, at 8 or 12 weeks PC, brief stimulation of the oral hood ipsilateral or contralateral to the crush injury elicited synaptic responses in RS neurons on either side of the brain, similar to that in normal animals. In the lamprey, compensatory mechanisms probably allow recovery of behavioral function despite incomplete regeneration of trigeminal sensory axons within the central nervous system. J. Comp. Neurol. 396:322–337, 1998. © 1998 Wiley-Liss, Inc.     

Early cytoskeletal changes following injury of giant spinal axons in the lamprey

The spinal cord of the larval sea lamprey contains identified giant axons that readily regenerate following spinal transection. In this study, we used serial light and electron microscopy to analyze the early ultrastructural consequences of axotomy in the proximal stumps of these axons near the lesion site. Axotomy results in two types of striking ultrastructural changes: (1) changes associated with the degeneration of axoplasm and subsequent retraction of the cut axon from the lesion and (2) changes associated with the early stages of axonal regeneration. Degenerative changes include the disruption of mitochondria to form large vacuoles, the collapse of neurofilaments into closely packed masses (condensed filamentous cores; CFCs), and the appearance of amorphous electron-dense bodies (dense granular masses; DGMs). Events associated with regeneration include the disappearance of vacuoles, DGMs, and CFCs and the appearance of small, sprout-like projections from the axon stump. Thus, we show that degenerative and regenerative events can be clearly separated from one another in identified axons, unlike the situation in the central nervous systems of amniote vertebrates such as mammals. © 1995 Wiley-Liss, Inc.     

Coexistence of dopamine and serotonin in an identified neuron of the lobster nervous system

The combination of several analytical methods, i.e. chemical analysis (high performance liquid chromatography), biochemical analysis (radioimmunoassay) and immunohistochemistry, has shown that a single neuron can contain two ‘classical’ neurotransmitters.     

Distribution of membrane glycoproteins among the organelles of a single identified neuron of aplysia. I. association of a [H]glycoprotein with vesicles

[³H]-acetylgalactosamine injected into the cell body of R2, the giant cholinergic neuron in the abdominal ganglion, is rapidly incorporated into membrane glycoprotein and glycolipid. Incorporation, which is localized to the injected cells, occurs at a constant rate for approximately 15 h. By that time, 83% of the labeled macromolecules are associated with membranes. Quantitative electron microscopic radioautography of the cell body shows that labeling of membranous organelles is selective: the Golgi apparatus, endoplasmic reticulum, and lucent and compound vesicles are labeled, while the nucleus, end-stage lysosomes, and mitochondria are not. SDS-polyacrylamide gel electrophoresis of the total membranes from more than 40 R2s examined individually resolves reproducibly 5 major labeled glycoprotein components. In order to determine which of these are associated with vesicles, we isolated a labeled vesicle fraction from R2 using a combination of differential centrifugation and filtration on a column of glass beads with 200 nm pores. This fraction was consistently enriched in [³H]glycoproteins I (180,000 Daltons) and V (90,000 Daltons) relative to those fractions containing larger organelles. These experiments suggest that different organelles contain characteristic membrane components.     

Input-output relationships of identified buccal neurones involved in feeding control in Aplysia

A group of about 28 neurones located in the lateral portion of the caudal face of Aplysia buccal ganglion and projecting into the cerebro-buccal connective were identified by retrograde cobalt staining, and designated as L neurones. It was found that the L neurones did not establish synaptic relations with the known buccal neurones, which are mainly involved in the production of the consummatory phase of feeding, nor with several cerebral neurones tested, including the well-known serotonin giant cell. Neither did they show responses to stimulation of the nerves directed to the buccal mass. On the other hand, the L neurones showed depolarizing responses, with the possible addition of a weak, slower hyperpolarizing phase, to stimulation of the ipsi- and contralateral oesophageal nerves, which innervate the portion of the gut posterior to the buccal mass. These findings, together with several properties of the oesophageal nerve input, suggest that one function of the L cells is to transmit information about gut regions posterior to the buccal mass towards the cerebral ganglia, and that they may mediate the inhibitory influence which in Aplysia is known to be exerted upon feeding by the presence of bulk in the anterior gut. The L neurones showed synaptic responses - consisting mainly or exclusively of depolarizations - to stimulation of the cerebro-buccal connectives. Besides this, large, tonic EPSPs, which often occurred in the 'spontaneous' activity of the L neurones, were found to be generated by spikes that travelled in the cerebro-buccal connective towards the buccal ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transplantation and restoration of functional synapses between an identified neuron and its targets in the intact brain of Lymnaea stagnalis

Most information available to date regarding the cellular and synaptic mechanisms of target cell selection and specific synapse formation has primarily come from in vitro cell culture studies. Whether fundamental mechanisms of synapse formation revealed through in vitro studies are similar to those occurring in vivo has not yet been determined. Taking advantage of the regenerative capabilities of adult molluscan neurons, we demonstrate that when transplanted into the host ganglia an identified neuron reestablishes its synaptic connections with appropriate targets in vivo. This synaptogenesis, however, was possible only if the targets were denervated from the host cell. Specifically, the giant dopamine neuron right pedal dorsal 1 (RPeD1) located in the pedal ganglia was isolated from a donor brain and transplanted into the visceral ganglia of the recipient brain. We discovered that within 2-4 days the transplanted RPeD1 exhibited extensive regeneration. However, simultaneous intracellular recordings failed to reveal synapses between the transplanted cell and its targets in the visceral ganglia, despite physical overlap between the neurites. To test whether the failure of a transplanted cell to innervate its target was due to the fact that the targets continued to receive input from the native RPeD1, the latter soma was surgically removed prior to the transplantation of RPeD1. Even after the removal of host soma, the transplanted RPeD1 failed to innervate the targets such as visceral dorsal 4 (VD4)-despite extensive regeneration by the transplanted cell. However, when RPeD1 axon was allowed to degenerate completely, the transplanted RPeD1 successfully innervated all of its targets and these synapses were similar to those seen between host RPeD1 and its targets. Taken together, our data demonstrate that the transplanted cells will innervate their potential targets only if the targets were denervated from the host cell. These data also lend support to the idea that, irrespective of their physical location in the brain, the displaced neurons are able to regenerate, recognize their targets, and establish specific synapses in the nervous system.     

Response to axotomy of an identified leech neuron, in vivo and in culture

Membrane properties of identified leech neurons with non-spiking somata were studied after axotomy. Two distinct procedures were used: the section of ganglionic roots in vivo and the isolation of cell somata in culture. The results indicated that axotomized neurons progressively developed the excitability of somatic membrane, both in vivo and in culture.     

Two rates of fast axonal transport of [H]glycoprotein in an identified invertebrate neuron

Recently, the kinetics of fast axonal transport of a single type of organelle, the serotonergic storage vesicle, was described in an identified axon of Aplysia using a pulsing technique combined with intracellular injection of [³H]serotonin. Here we extend the single axon studies by analyzing the movement of pulses of [³H]glycoprotein, following injection into the giant Aplysia cell, R2, of the amino sugar [³H]N-acetylgalactosamine. This glycoprotein precursor has been shown to label several organelles in this neuron. [³H]Glycoprotein is found to move in the axon of R2 at 2 rates of fast transport, 174 and 105 mm per day at room temperature. We suggest that the 2 rates reflect movements of 2 different types of organelle.     

A morphometric analysis of transmitter identified dendrites and nerve terminals

The present method is exemplified on coronal sections of the medulla oblongata containing phenylethanolamine-N-methyltransferase (PNMT) immunoreactive nerve cell bodies and their processes and on coronal sections of the pons containing the locus coeruleus, where PNMT immunoreactive nerve terminals have been demonstrated together with the dopamine-beta-hydroxylase immunoreactive nerve cell bodies. Morphometric analysis of the processes (both length and branches) and of the nerve terminals involve as a first step the division of the area under study into squares 100 microns wide, which are superimposed on a Cartesian plane. The uniformity of the density distribution of the nerve terminals and the processes (branches or length) can be analyzed by Lorenz curves, which in a quantitative way can measure the degree of unevenness and thus represent a measure of concentration. A concentration index can therefore be calculated. By the use of the densitometric approach it also becomes possible to study the density distribution of the nerve terminals with the highest antigen contents. The present method will make it possible to quantitate morphological changes occurring in processes and nerve terminals of transmitter-identified neurons.     

Reorganization of central terminals of myelinated primary afferents in the rat dorsal horn following peripheral axotomy

We have investigated the time course and extent to which peripheral nerve lesions cause a morphological reorganization of the central terminals of choleragenoid-horseradish peroxidase (B-HRP)-labelled primary afferent fibers in the mammalian dorsal horn. Choleragenoid horseradish peroxidase is retrogradely transported by myelinated (A) sensory axons to laminae I, III, IV and V of the normal dorsal horn of the spinal cord, leaving lamina II unlabelled. We previously showed that peripheral axotomy results in the sprouting of numerous B-HRP labelled large myelinated sensory axons into lamina II. We show here that this spread of B-HRP-labelled axons into lamina II is detectable at 1 week, maximal by 2 weeks and persists for over 6 months postlesion. By 9 months, however, B-HRP fibers no longer appear in lamina II. The sprouting into lamina II occurs whether regeneration is allowed (crush) or prevented (section with ligation), and does not reverse at times when peripheral fibers reinnervate the periphery. We also show that 15 times more synaptic terminals in lamina II are labelled by B-HRP 2 weeks after axotomy than in the normal. We interpret this as indicating that the sprouting fibers are making synaptic contacts with postsynaptic targets. This implies that A-fiber terminal reorganization is a prominent and long-lasting but not permanent feature of peripheral axotomy. We also provide evidence that this sprouting is the consequence of a combination of an atrophic loss of central synaptic terminals and the conditioning of the sensory neurons by peripheral axotomy. The sprouting of large sensory fibers into the spinal territory where postsynaptic targets usually receive only small afferent fiber input may bear on the intractable touch-evoked pain that can follow nerve injury. © 1995 Wiley-Liss, Inc.     

Specificity in mammalian peripheral nerve regeneration at the level of the nerve trunk

Previous studies indicate that distal stumps of transected rat peripheral nerves secrete 'tropic' factors which can attract/support axonal regeneration over distances of several mm in vivo. The present study was undertaken in order to determine if there is specificity of neurotropic interaction at the level of the nerve trunk. Proximal stumps of transected peroneal or tibial nerves were inserted into the single inlet end of Y-shaped Silastic implants and offered alternative 'lures' at the paired outlet ends (specifically, grafts of peroneal vs tibial distal stump tissue). Several weeks later, the overwhelming majority of preparations showed exclusive growth of nerve fibers in implant forks attached to 'native' (originally associated) nerve stumps. Inversion of the distal stump grafts (such that the proximal stump was facing an analogous native distal stump, but a different region of it) diminished the frequency and extent of native preference. Taken together, data suggest the possibility that there can be a specificity of nerve regeneration at the level of the nerve trunk.     

Resprouting and survival of guinea pig cochlear neurons in response to the administration of the neurotrophins brain-derived neurotrophic factor and neurotrophin-3

Degeneration of auditory neurons occurs after deafening and is associated with damage to the organ of Corti. The administration of neurotrophins can protect auditory neurons against degeneration if given shortly after deafening. However, it is not known whether the delayed administration of neurotrophins, when significant degeneration has already occurred, will provide similar protection. Furthermore, little is known about the effects of neurotrophins on the peripheral processes of the auditory neurons or whether these neurons can resprout. This study examined the morphological effects on auditory neurons following deafening and the administration of brain-derived neurotrophic factor and neurotrophin-3. Results showed that neurotrophins were effective in preventing death of auditory neurons if administered 5 days after deafening and were also effective in preventing the continued loss of neurons if the administration was delayed by 33 days. The peripheral processes of auditory neurons in cochleae that received neurotrophins were in greater number and had larger diameters compared with the untreated cochleae. Localized regions of resprouting peripheral processes were observed in deafened cochleae and were enhanced in response to neurotrophin treatment, occurring across wider regions of the cochlea. These findings have significant implications for an improvement in the performance of the cochlear implant and for future therapies to restore hearing to the deaf.     

Satellite microglia show spontaneous electrical activity that is uncorrelated with activity of the attached neuron

Microglia are innate immune cells of the brain. We have studied a subpopulation of microglia, called satellite microglia. This cell type is defined by a close morphological soma‐to‐soma association with a neuron, indicative of a direct functional interaction. Indeed, ultrastructural analysis revealed closely attached plasma membranes of satellite microglia and neurons. However, we found no apparent morphological specializations of the contact, and biocytin injection into satellite microglia showed no dye‐coupling with the apposed neurons or any other cell. Likewise, evoked local field potentials or action potentials and postsynaptic potentials of the associated neuron did not lead to any transmembrane currents or non‐capacitive changes in the membrane potential of the satellite microglia in the cortex and hippocampus. Both satellite and non‐satellite microglia, however, showed spontaneous transient membrane depolarizations that were not correlated with neuronal activity. These events could be divided into fast‐rising and slow‐rising depolarizations, which showed different characteristics in satellite and non‐satellite microglia. Fast‐rising and slow‐rising potentials differed with regard to voltage dependence. The frequency of these events was not affected by the application of tetrodotoxin, but the fast‐rising event frequency decreased after application of GABA. We conclude that microglia show spontaneous electrical activity that is uncorrelated with the activity of adjacent neurons.     

Neuropeptide diversity in Ascaris: an immunocytochemical study

An immunocytochemical method was used for localization of various peptide-like substances in the Ascaris nervous system. Out of 45 antipeptide antisera, 12 demonstrated immunoreactivity in different subsets of neurons; these 12 antisera were raised against luteinizing hormone-releasing hormone (LHRH), Aplysia peptide L11 (L11), Aplysia peptide 12B (12B), small cardioactive peptide B (SCPB), neuropeptide Y (NPY), FMRFamide, gastrin-17, cholecystokinin octapeptide (CCK-8), alpha-melanocyte stimulating hormone (alpha MSH), calcitonin gene related peptide (CGRP), corticotropin releasing factor (CRF), and vasoactive intestinal peptide (VIP). Several peptide-like substances were colocalized to the same neuron. Our results suggest that Ascaris, like other organisms, contains multiple peptidergic systems.