Sprouting and functional regeneration of an identified serotonergic neuron following axotomy

An identified serotonergic neuron (C1) in the cerebral ganglion of Helisoma trivolvis sprouts following axotomy and rapidly (seven to eight days) regenerates to recover its regulation of feeding motor output from neurons of the buccal ganglia. The morphologies of normal and regenerated neurons C1 were compared. Intracellular injection of the fluorescent dye, Lucifer Yellow, into neuron C1 was compared with serotonin immunofluorescent staining of the cerebral and buccal ganglia. The two techniques revealed different and complimentary representations of the morphology of neuron C1. Lucifer Yellow provided optimal staining of the soma, major axon branches, and dendritic arborization. Immunocytochemical staining revealed terminal axon branches on distant targets and showed an extensive plexus of fine fibers in the sheaths of ganglia and nerve trunks. In addition to C1, serotonin-like immunoreactivity was localized in approximately 30 other neurons in each of the paired cerebral ganglia. Only cerebral neurons C1 had axons projecting to the buccal ganglia. No neuronal somata in the buccal ganglia displayed serotonin-like immunoreactivity. Observations of regenerating neurons C1 demonstrated: Actively growing neurites, both in situ and in cell culture, displayed serotonin-like immunoreactivity; severed distal axons of C1 retained serotonin-like immunoreactivity for up to 28 days; axotomized neurons C1 regenerated to restore functional control over the feeding motor program.     

Innervation of the anococcygeus muscle of the rat

Summary The innervation of the anococcygeus muscle of the rat was investigated with regard to the histochemical features of nerve fibers within the muscle and to the location of the postganglionic autonomic neurons which are the source of these fibers. Acetylcholinesterase-positive fibers and catecholaminergic fibers are abundant in the anococcygeus as well as the related retractor penis muscle. Neuronal somata, either between muscle bundles of the anococcygeus or in the connective tissue sheath, are also acetylcholinesterase-positive. Nerve fibers and a minority of the ganglion cells in the anococcygeus and retractor penis muscles are immunoreactive for vasoactive intestinal polypeptide. Injection of the retrogradely transported dye Fluorogold into the anococcygeus muscle filled neurons in the abdominopelvic sympathetic chain, pelvic plexus and a small number of neurons in the inferior mesenteric ganglion. In the pelvic plexus, some neurons were located in the major pelvic ganglion but most were found along the main penile nerve and its branches to the anococcygeus muscle. Immunocytochemistry of these identified neurons indicates that about one half of them are positive for vasoactive intestinal polypeptice. These results raise the possibility that both acetylcholine and vasoactive intestinal polypeptide are important neurotransmitters in autonomic nerves to the anococcygeus muscle.     

Immunocytochemistry of GABA and glutamic acid decarboxylase in the thoracic ganglion of the crab Eriphia spinifrons

We have used specific antisera against protein-conjugated -aminobutyric acid (GABA) and rat-brain glutamic acid decarboxylase (GAD) in immunocytochemical preparations to study the distribution of putatively GABAergic neurons in the fused thoracic ganglion of the crab Eriphia spinifrons. In the thoracic neuromeres, about 2000 neurons with somata arranged in clusters or located singly in the cell cortex exhibited both GABA-like and GAD-like immunoreactivity. In addition, more than a hundred cells showed only GABA-like immunoreactivity. Fibrous immunoreactive staining to GAD and GABA was distributed throughout the neuropil of the thoracic ganglion, and several fiber tracts contained immunoreactive processes. Sets of serially homologous neurons exhibited GABA-like and GAD-like immunoreactivity in the thoracic neuromeres. Especially prominent were one medial and four ventro-lateral clusters of somata, together with thirteen individually recognized cells in each neuromere. Six of these cells in the ventro-medial cell cortex may be the somata of inhibitory motoneurons. The leg nerves contained three immunoreactive fibers, corresponding to the previously described common inhibitory motoneuron and the two specific inhibitors. The results present further evidence for GABA being the neurotransmitter of all inhibitory leg motorneurons, and suggest its presence and role as a neurotransmitter in a considerable number of interneurons in the thoracic ganglion of the crab.     

Structures with GABA-like and GAD-like immunoreactivity in the cervical sympathetic ganglion complex of adult rats

Summary The distribution of gamma-aminobutyric acid (GABA)-like and glutamate decarboxylase (GAD)-like immunoreactivity was studied in the cervical sympathetic ganglion complex of rats, including the intermediate and inferior cervical ganglia and the uppermost thoracic ganglion. GABA-positive axons may enter the ganglion complex via its caudal end. Others apparently arise from small GABA-positive cell bodies which are scattered among principal neurons, within clusters of SIF cells and in bundles of GABA-negative axons. The majority of these cells is located in the lower half of the ganglion complex. Principal neurons did not react with antibodies against GABA or GAD. An unevenly distributed meshwork of GABA-immunoreactive axons was seen in each of the ganglia. Immunoreactive axons formed numerous varicosities. Some of them were aggregated in a basket-like form around a subpopulation of GABA-negative principal ganglion cell bodies. Electron-microscopic immunocytochemistry revealed that GABA-positive nerve fibers establish asymmetric synaptic junctions with dendritic and somatic spines of principal neurons, whereas postsynaptic densities are inconspicous or absent on dendritic shafts and somata. The results suggest that in the cervical sympathetic ganglion complex principal neurons are not GABAergic, but are innervated by axons which react with both antibodies against GAD and/ or GABA antibodies and originate from a subpopulation of small neurons.     

Metamorphosis of wing motor system in the silk moth,Bombyx mori L. (Lepidoptera: Bombycidae): Anatomy of the sensory and motor neurons that innervate larval mesothoracic dorsal musculature,stretch receptors,and epidermis

Anatomy of dorsal mesothoracic structures, such as muscles, sensory organs, and innervation, was studied in the silkworm, Bombyx mori L. (Lepidoptera : Bombycidae), and compared with the adult wing motor system. Musculature and nerve innervation were investigated by dissection and electron micrograph; and central projection of sensory fibers and morphology of somata and dendrites of motor neurons by cobalt back-filling, followed by silver intensification. There are 23 muscle bundles (DLM) and 2 stretch receptors (SR). The DLMs, SRs, and epidermis are innervated by a branch of the dorsal nerve trunk emerging from the mesothoracic ganglion (MSG). The branch bifurcates into a dorsal sensory branch of about 300 sensory fibers and a dorsal motor branch of 14 fibers. The sensory fibers project mainly to a longitudinal portion near the mid line in the ventral neuropil of MSG and the metathoracic ganglion. Several fibers extend into the prothoracic ganglion (PG) and a few into the subesophageal and 1st abdominal ganglia. At least 13 (probably 14) motor neurons send axons to DLMs: 9 (probably 10) in PG, and 4 in MSG. Their dendrites are located mostly on the dorsoipsilateral side of the neuropil, but several branches cross the mid line and give rise to many fine branches on the contralateral side. Comparison between the larval (present study) and adult motor system shows a significant similarity in the musculature, peripheral nerve pattern, and motor neurons with some peculiarities.     

Glutamate, GABA and acetylcholine signaling components in the lamina of the Drosophila visual system

Synaptic connections of neurons in the Drosophila lamina, the most peripheral synaptic region of the visual system, have been comprehensively described. Although the lamina has been used extensively as a model for the development and plasticity of synaptic connections, the neurotransmitters in these circuits are still poorly known. Thus, to unravel possible neurotransmitter circuits in the lamina of Drosophila we combined Gal4 driven green fluorescent protein in specific lamina neurons with antisera to gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, a GABA(B) type of receptor, L-glutamate, a vesicular glutamate transporter (vGluT), ionotropic and metabotropic glutamate receptors, choline acetyltransferase and a vesicular acetylcholine transporter. We suggest that acetylcholine may be used as a neurotransmitter in both L4 monopolar neurons and a previously unreported type of wide-field tangential neuron (Cha-Tan). GABA is the likely transmitter of centrifugal neurons C2 and C3 and GABA(B) receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABA(A) receptor subunit RDL may be expressed by L4 neurons and a type of tangential neuron (rdl-Tan). Strong vGluT immunoreactivity was detected in alpha-processes of amacrine neurons and possibly in the large monopolar neurons L1 and L2. These neurons also express glutamate-like immunoreactivity. However, antisera to ionotropic and metabotropic glutamate receptors did not produce distinct immunosignals in the lamina. In summary, this paper describes novel features of two distinct types of tangential neurons in the Drosophila lamina and assigns putative neurotransmitters and some receptors to a few identified neuron types.     

The distribution of glutamate-like immunoreactivity in the thoracic and abdominal ganglia of the locust (Schistocerca gregaria)

The distribution of glutamate-like immunore-activity in the thoracic and abdominal ganglia of the locust was studied using two polyclonal antibodies against glutamate. Because glutamate is a precursor of the inhibitory transmitter -amino butyric acid (GABA) the distribution of immunostaining by antibodies against glutamate and GABA was closely compared in adjacent serial sections. When the antibodies were used at optimal dilutions there was no overlap in the distribution of immunostaining for glutamate and GABA. In the pro- and mesothoracic ganglia 360–400 somata are immunoreactive for glutamate, while in the metathoracic ganglion about 600 somata were stained. These range in diameter from 10–100 m in diameter and include the majority of the large somata in these ganglia. Bundles of primary neurites emerging from these large somata can be traced through the neuropile. Most of the bundles correspond to the known paths of motor neurone primary neurites. In addition the T-tract is also immunolabelled. The free abdominal ganglia each contain 80–100 somata ranging in size from 10–45 m while the terminal ganglion contains about 250 somata, 10–60 m in diameter.     

Immunocytochemistry of GABA in the brain and suboesophageal ganglion ofManduca sexta

Summary We have used specific antisera against protein-conjugated-aminobutyric acid (GABA) in immunocytochemical preparations to investigate the distribution of putatively GABAergic neurons in the brain and suboesophageal ganglion of the sphinx mothManduca sexta. About 20000 neurons per brain hemisphere exhibit GABA-immunoreactivity. Most of these are optic-lobe interneurons, especially morphologically centrifugal neurons of the lamina and tangential neurons that innervate the medulla or the lobula complex. Many GABA-immunoreactive neurons, among them giant fibers of the lobula plate, project into the median protocerebrum. Among prominent GABA-immunoreactive neurons of the median protocerebrum are about 150 putatively negative-feedback fibers of the mushroom body, innervating both the calyces and lobes, and a group of large, fan-shaped neurons of the lower division of the central body. Several commissures in the supra- and suboesophageal ganglion exhibit GABA-immunoreactivity. In the suboesophageal ganglion, a group of contralaterally descending neurons shows GABA-like immunoreactivity. The frontal ganglion is innervated by immunoreactive processes from the tritocerebrum but does not contain GABA-immunoreactive somata. With few exceptions the brain nerves do not contain GABA-immunoreactive fibers.     

Excitatory neural control of posterograde heartbeat by the frontal ganglion in the last instar larva of a lepidopteran, Bombyx mori

The frontal ganglion of the silkworm (Bombyx mori) gives rise to a visceral nerve, branches of which include a pair of anterior cardiac nerves and a pair of the posterior cardiac nerves. Forward-fill of the visceral nerve with dextran labeled with tetramethyl rhodamine shows the anterior cardiac nerves innervate the anterior region of the dorsal vessel. Back-fill of the anterior cardiac nerves with Co²⁺ and Ni²⁺ ions and the fluorescent dye reveals that the cell bodies of two motor neurons are located in the frontal ganglion. Injection of 5, 6-carboxyfluorescein into the cell body of an identified motor neuron shows that the neuron gives rise to an axon running to the visceral nerve. Unitary excitatory junctional potentials (EJPs) were recorded from a myocardial cell at the anterior end of the heart. They responded in a one-to-one manner to electrical stimuli applied to the visceral nerve, or to impulses generated by a depolarizing current injected into the cell body. EJPs induced by stimuli at higher than 0.5 Hz showed facilitation while those induced at higher than 2 Hz showed summation. Individual EJPs without summation, or a train of EJPs with summation, caused acceleration in the phase of posterograde heartbeat and heart reversal from anterograde heartbeat to posterograde heartbeat. It is likely that the innervation of the anterior region of the dorsal vessel by the motor neurons, through the anterior cardiac nerves is responsible for the control of heartbeat in Lepidoptera, at least in part.     

Nitric oxide synthase-containing nerve fibers and neurons in the genital tract of the female mouse

Nitric oxide (NO) is generated intracellularly from L-arginine by the action of the enzyme nitric oxide synthase (NOS). The present investigation demonstrates immunoreactivity against NOS and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity in nerve cells and fibers of the reproductive system of the female mouse. The density of nerve fibers staining for NOS varied among different genital organs. The ovary and Fallopian tube were devoid of NOS-positive nerves. The uterine horns received sparse innervation by NOS-containing nerve fibers. The most abundant NOergic innervation was found in the uterine cervix and vagina, where the nerve fibers ran parallel to the smooth muscle bundles and beneath the epithelium; they also accompanied intramural blood vessels. The vaginal muscular wall contained single or groups of NOS-reactive nerve cells. Clusters of NOS-containing neurons were located in Frankenhäuser's ganglion at the cervico-vaginal junction. NO may therefore act as a transmitter in the nervous control of the female reproductive tract.     

Disruption of peripheral target contact influences the development of identified central dendritic branches in a leech motor neuron in vivo

Retrograde signaling from target tissues has been shown to influence many aspects of neuronal development in a number of developmental systems. In these experiments using embryonic leeches (Hirudo medicinalis), we examined how depriving a neuron of contact with its peripheral target affects the development of the cell's central arborization. We focused our attention on the motor neuron cell 3, which normally stimulates dorsal longitudinal muscle fibers to contract. At different locations in the periphery and in embryos of several different stages, we cut the nerve containing the growing axon of cell 3. This surgery led to dramatic overgrowth of cell 3's central dendritic branches, which normally accept synaptic contacts from other neurons, including the inhibitory motor neuron cell 1. When cell 3's peripheral axon was cut relatively early in development, its overgrown central branches eventually retracted. However, cells that were disrupted later in development retained their overextended branches into adulthood. In addition, if the axon was cut close to the ganglion early in development, depriving the cell of contact with any dorsal tissues, the central branches failed to retract and were instead retained into adulthood. Unlike cell 3, the central branches of cell 1, which has the same peripheral target muscles as cell 3, remained unchanged following all axotomy protocols. These results suggest that in at least some neurons contact with peripheral targets can influence development of the central processes that normally mediate synaptic contacts.     

Substance P-like immunoreactivity in the stomatogastric nervous systems of the crab Cancer borealis and the lobsters Panulirus interruptus and Homarus americanus

Summary The distribution of substance P-like immunoreactivity in the stomatogastric nervous systems of three decapod crustacean species, Cancer borealis, Homarus americanus, and Panulirus interruptus, was studied. The stomatogastric ganglion showed dense staining in the neuropil, but none in the somata. A single neuron stained in the esophageal ganglion. Lucifer yellow backfills and intracellular injections followed by incubation with the substance P antibody showed that the axons of this neuron project into the inferior esophageal nerves towards the paired commissural ganglia. The commissural ganglia showed a pronounced projection from a large bundle of fibers in the anterior medial portion of the circumesophageal connective. Additionally, less dense neuropil and stained somata were seen in the commissural ganglia. Staining was completely blocked by preabsorption with authentic substance P, physalaemin, eledoisin, and substance K. These data suggest that in the nervous system of crustacean species a molecule with C-terminal homology to substance P and other tachykinins is released as a neuroregulator in the stomatogastric ganglion.     

Mechanisms of the inhibitory effect of the stellate ganglion on cardiac activity

The mechanisms of cardiac activity inhibition caused by stimulation of the stellate ganglion were studied in acute experiments on 28 dogs and 37 cats and chronic experiments on 12 cats. It was shown that inhibition of cardiac activity is caused by stimulation of the parasympathetic fibers of the vagus, anastomozing with stellate ganglion branches and ingoing as part of these fibers to the heart. The hypothesis of change over of the sympathetic nerve fibers to the intracardial cholinergic neurons and the hypothesis of the cholinergic component in the mechanism of catecholamine release by the sympathetic nerve terminals was not confirmed. Therefore, the known Dale's principle as to that one neuron exerts its efferent effect with the aid of one transmitter is quite just. alpha-Adrenoreceptors does not produce any noticeable effect on cardiac activity.     

Expression of TrkB and BDNF in human cochlea-an immunohistochemical study

Surgical human cochlear specimens were obtained during the removal of large posterior cranial fossa meningioma by a transcochlear approach in which the cochlea was removed for maximal exposure of the tumor and protection of important structures, such as the brainstem, cranial nerves, and pivotal blood vessels. The cochlear tissue was fixed and cryo-sectioned for tyrosine kinase receptor B (TrkB) and brain-derived neurotrophic factor (BDNF) immunohistochemistry. TrkB receptor protein was expressed in both neuronal somata and the processes of human spiral ganglion neurons (SGNs). In the human organ of Corti, TrkB immunoreactivity was mainly present in nerve fibers underneath outer hair cells. BDNF expression was found neither in the organ of Corti nor in the spiral ganglion of human cochlea. For antibody specificity and for control and comparative purposes, TrkB immunocytochemistry was performed in primary cultures of cochlear neuron/glia from adult guinea pig. Confocal laser scanning microscopy showed that TrkB was homogeneously distributed in the cytoplasm of both neuronal somata and axons. Knowledge of the expression of TrkB receptor in human cochlea should help to determine the target structures for neuron preservation in hearing-impaired patients. Our results indicate that the regeneration of SGNs under pathological conditions can be enhanced with BDNF/TrkB-based pharmaceutical or genetic strategies.     

Development of the glutamate system in rabbit retina

We have investigated two characteristics of the glutamate system in the developing rabbit retina. 1) Glutamate immunoreactivity was observed at birth within developing processes of four cell types; two of which, photoreceptors and ganglion cells, are known to be glutamatergic in the adult. Two other cell types, type A horizontal cells and amacrine cells, are immunoreactive to both glutamate and GABA at birth, suggesting that endogenous pools of glutamate in GABAergic neurons serve as precursor for GABA synthesis. Thus it appears that endogenous glutamate pools are present within neurons prior to synaptogenesis as part of the early expression of either the glutamate or GABA transmitter phenotype. 2) Analysis of³H-glutamate metabolism during retinal development showed that rapid conversion of glutamate to glutamine does not occur until the second postnatal week, coincident with the expression of Muller (glial) cell activity. In the absence of glial metabolism in the neonate, extracellular concentrations of glutamate remain relatively high and are likely to have major effects on neuronal maturation.Special issue dedicated to Dr. Frederick E. Samson     

Immunohistochemistry of biogenic polypeptides in nerve cells and fibres of the guinea pig inferior mesenteric ganglion after perturbations

Immunohistochemistry of peptide- and dopamine-beta-hydroxylase-(DBH)-containing varicose nerve fibres and ganglion cells, respectively, in the guinea pig inferior mesenteric ganglion was investigated following a) transsection of mesenteric (colonic) branches, b) transsection of central (lumbar splanchnic, intermesenteric and hypogastric) branches, and c) transplantation into the spleen. The findings indicate that pathways of different opioid peptides are not identical. Met-enkephalin- and met-enkephalin-arg-phe- (cleavage products from pre-proenkephalin) containing fibres course in central branches to make contact in the inferior mesenteric ganglion. Dynorphin- and alpha-neo-endorphin- (deriving from pre-prodynorphin) containing fibres as well as leu-enkephalin- (included in the dynorphin sequence) fibres appear to rise not only from central and from enteric somata, but also from intraganglionic noradrenergic neurons. Similar pathways seem to be used by VIP- and by neurotensin-immunoreactive fibres, although intraganglionic neurotensin-immunoreactive cell bodies are rare. Practically all substance P- and most CGRP-immunoreactive fibres enter the ganglion via central branches and, to a large extent, traverse it, but some CGRP-immunoreactive influx appears to come from the intestine. The origin of intraganglionic substance P- and CGRP-immunoreactive fibres after ganglion transplantation remained unidentified. Somatostatin- and neuropeptide Y-immunoreactive fibres predominantly have an intraganglionic origin as have DBH-immunoreactive noradrenergic fibres. The demonstrated alterations in neuropeptide immunoreactivity of intraganglionic and periganglionic nerve fibres following the applied transsection procedures contribute to the present knowledge on origin and destination of peptidergic transmitter segments in the guinea pig inferior mesenteric ganglion. Moreover, the present study provides evidence that intrinsic participation in intraganglionic fibre supply is more extensive than hitherto believed.     

Synaptic connections of dopamine-immunoreactive neurons in the antennal lobes of Periplaneta americana. Colocalization with GABA-like immunoreactivity

Dopamine-like immunoreactivity was demonstrated histochemically in about ten local interneurons in the antennal lobe of Periplaneta americana. The somata of these neurons are within the ventrolateral group of cell bodies. Additional immunohistochemical tests revealed that the same neurons also have a GABA-like immunoreactivity. Immunohistochemical dopamine staining (preembedding) of preparations in which the antennal receptor fibers had been caused to degenerate showed that in the glomerular neuropil these antennal fibers form output synapses on dopamine-immunoreactive neurons. The latter form output synapses on unstained neuron profiles.     

Observations on the ultrastructure of ganglion cells in the circumvallate papilla of rat and mouse

Ganglion cells in the circumvallate papilla of adult rodents are described as typical autonomic neurons. Some neurons are aggregated to form a discrete structure in the base of the papilla; others are scattered through the core, along the nerve bundles, and particularly near the dome. The term "circumvallate ganglion" is applied to the entire population. Satellite cells completely ensheathe each neuron. Preganglionic fibers, containing clear vesicles, synapse on the soma and stumpy dendrites of the neurons. Axons, containing dense-cored vesicles, are observed in close proximity to the neurons. However, these fibers do not establish true morphological synaptic contacts with the neurons. We have not observed serial or reciprocal synapses on or in the vicinity of the ganglion cells. The hypothesis that the axons of the circumvallate ganglion neurons act as parasympathetic vasodilators is indicated by the proximity of the two structures and by nerve terminations on the arteriole muscle cells. Direct modulation of taste transduction by these neurons is ruled out.     

Morphology of soma and dendrites of the giant fiber system in the sixth abdominal ganglion of the cockroach

This study, using the cobalt chloride technique, clarifies the origin of the giant axons in the cockroach, Periplaneta. Each giant axon in the ventral nerve cord arises from a single cell body located in the sixth abdominal ganglion. The position of the soma is always contralateral to the giant axon; it projects anteriorly. In six giant neurons, the axonic and dendritic branches are ipsilateral while the somata are contralateral. In two neurons, both the soma and the dendritic branches are ipsilateral while the axons are contralateral. The dendritic arborizations of the giant neurons form a dense and compact mass of neuropile in each half of the posterior and middorsal part of the ganglion where sensory fibers, primarily from the cercal nerves terminate. The relation of these findings to earlier electrophysiological studies is discussed.