Localization of cardiac parasympathetic preganglionic neurons in the medulla oblongata of pigeon, Columba livia: a study using fragment C of tetanus toxin.

The binding fragment of tetanus toxin, fragment C, was injected into several different regions of the pigeon heart. Retrogradely and/or transneuronally labeled cardiomotor parasympathetic preganglionic neurons were found in two separate nuclei within the medulla oblongata. The majority of fragment C-immunolabeled cells was confined to the caudal division of the nucleus ambiguus. This nuclear region is likely to be homologous to the ventrolateral nucleus of the external formation of the nucleus ambiguus in mammals. A smaller fraction (10-30%) of fragment C-positive cardiomotor preganglionic neurons were localized within a restricted portion of the ventrolateral subnucleus of the dorsal motor nucleus of the vagus nerve. This dual cardiac representation in an avian is very similar to the organization established in several mammalian species, and suggests that the brainstem organisation of cardiac parasympathetic efferents is evolutionarily stable across avians and mammals.

Light and electron microscopic analyses of intraspinal axon collaterals of sympathetic preganglionic neurons.

Experiments were performed in pigeons (Columba livia). Sympathetic preganglionic neurons (SPNs) in the first thoracic spinal cord segment (T1) were identified electrophysiologically using antidromic activation and collision techniques and then intracellularly labeled with horseradish peroxidase (HRP). In 6 of 10 HRP-labeled SPNs, the site of axon origin and intraspinal axonal trajectory could be specified. In 2 of the 6 HRP-labeled axons, the peripherally projecting process branched intraspinally. The presence or absence of SPN intraspinal axonal collateralization did not correlate with parent perikaryal subnuclear location or dendritic alignment. None of the collaterals were recurrent onto the SPN of origin. Light microscopically, the collateral branches appeared to end with punctate, bulbous swellings. The spinal regions of the terminal end swellings for the two axons did not overlap one another. In one instance the entire terminal field was confined within the principal preganglionic cell column (column of Terni). The other axon had collateral branches which terminated in the lateral white matter and in a ventrolateral region of lamina VII. A serial section, electron microscopic reconstructive analysis of the entire intraspinal collateral terminal field within the column of Terni revealed that: (a) the primary collateral process was unmyelinated and arose at a node of Ranvier; (b) after issuance of the collateral branch, the myelinated parent axon continued to increase its myelin wrapping throughout the spinal gray; (c) the bulbous swellings observed light microscopically corresponded to axon terminal boutons and regions of synaptic contact; (d) the axon collateral terminals were exclusively presynaptic to small caliber dendrites and formed only asymmetric specializations; and (e) the collateral terminals contained numerous mitochondria, and densely packed, electron-lucent, spherical vesicles.

Retrograde, trans-synaptic and transneuronal transport of fragment C of tetanus toxin by sympathetic preganglionic neurons.

The atoxic binding fragment of tetanus toxin, Fragment C, was injected into paravertebral ganglion 14, the avian homologue of the mammalian stellate ganglion. Postinjection survival intervals were varied from 2.5 h to 33 days. Experiments performed at the shortest survival time of 2.5 h showed that Fragment C was retrogradely transported by sympathetic preganglionic axons at a rate greater than or equal to 10 mm/h. At survival times ranging from 5 to 15 h. Fragment C-positive, retrogradely labeled sympathetic preganglionic neurons were observed within the last cervical spinal segment and throughout the first three thoracic spinal cord segments. Sporadic retrograde labeling of sympathetic preganglionic neurons was evident within the fourth and fifth thoracic spinal cord segments. Fragment C-labeled perikarya and dendrites exhibited both diffuse cytoplasmic immunostaining as well as intracellular, perinuclear accumulations of small. Fragment C-positive granules. Retrogradely labeled preganglionic neurons were found within both autonomic subnuclei within avian thoracic spinal cord; the column of Terni and the nucleus intercalatus spinalis. The distribution and numerical density of retrogradely labeled sympathetic preganglionic neurons indicated further that: (a) both myelinated and unmyelinated preganglionic axons appear to be capable of intra-axonally transporting Fragment C; and (b) it is unlikely that there is differential Fragment C labeling of a morphologically distinct population of sympathetic preganglionic neurons within or across subnuclei. Fragment C is transferred out of sympathetic preganglionic somas and dendrites into the surrounding neuropil at an aggregate rate greater than or equal to 5 mm/h. Trans-synaptic transport was evident at postinjection survival times as short as 5 h and continued to increase in density within the sympathetic preganglionic neuropil for 24 h. Fragment C-positive terminal labeling persisted for at least 20 days. At survival times greater than or equal to 1 day. Fragment C-positive puncta and weak intracellular labeling of neurons were evident in areas of the spinal gray outside of the nuclear boundaries of the column of Terni and nucleus intercalatus. The regions showing evidence of trans-synaptic and transneuronal labeling included: (a) a group of small cells dorsal to the column of Terni, (b) lamina V and (c) lamina VII. This expansion of Fragment C-labeled neuronal elements was segmental in organization and co-extensive with the retrograde labeling pattern of sympathetic preganglionic neurons. Spinal interneurons in these regions may provide segmental, monosynaptic input to sympathetic preganglionic neurons. Fragment C leaked into the systemic circulation from the site of injection in paravertebral ganglion 14.(ABSTRACT TRUNCATED AT 400 WORDS)

Light microscopic and ultrastructural localization of GABA-like immunoreactive input to retrogradely labeled sympathetic preganglionic neurons.

The organization of gamma-aminobutyric acid-like immunoreactive (GABA-LIR) processes was studied within the sympathetic preganglionic neuropil of male Sprague-Dawley rats and pigeons (Columba livia). Sympathetic preganglionic neurons were retrogradely labeled following horseradish peroxidase (HRP) injections into either the adrenal medulla or superior cervical ganglion in rats or into the avian homologue of the mammalian stellate ganglion (paravertebral ganglion 14) in pigeons. GABA-LIR staining was visualized using peroxidase-antiperoxidase (PAP), avidin-biotin complex (ABC), or post-embedding immunogold methods. The pigeon preganglionic neuropil contained a dense network of GABA-LIR processes with punctate swellings that encircled sympathetic preganglionic perikarya within the principal preganglionic cell column (column of Terni) and the nucleus intercalatus spinalis. GABA-LIR spinal neurons were intermingled among HRP-labeled sympathetic preganglionic neurons within the column of Terni and throughout the zona intermedia. In the rat the density of the GABA-LIR processes within the four thoracic sympathetic preganglionic nuclei was less than that observed in the pigeon. Nevertheless, GABA-LIR profiles distinctively dotted preganglionic perikarya within the nuclei intermediolateralis pars principalis and pars funicularis, nucleus intercalatus spinalis and the central autonomic nucleus. GABA-LIR neurons were rarely observed within the nucleus intermediolateralis pars principalis, but were numerous in the zona intermedia and area X. No GABA-LIR spinal neurons in either vertebrate were retrogradely labeled with HRP. The ultrastructural arrangements of GABA-LIR processes within the sympathetic preganglionic neuropils of pigeons and rats were similar. GABA-LIR boutons formed symmetrical synaptic contacts and contained small round electron-lucent vesicles (50 nm) and one to several larger dense-core vesicles (80 nm). GABA-LIR terminals contacted HRP-labeled sympathetic preganglionic perikarya in all spinal nuclear regions in both vertebrates. More frequently, GABA-LIR boutons synapsed on dendrites. Occasionally, axo-axonic configurations were observed; each time only one of the axonal elements was GABA-LIR. Numerous unmyelinated and some thinly myelinated GABA-LIR axons coursed through the sympathetic preganglionic neuropils of both vertebrates. Synapses between GABA-LIR processes were present within the sympathetic preganglionic neuropil of both vertebrates. GABA-LIR dendrites were contacted by unlabeled terminals (predominantly small spherical vesicles with asymmetric synaptic specializations) and GABA-LIR terminals on GABA-LIR dendrites were similar in appearance to those synapsing on sympathetic preganglionic cell bodies and dendrites.

Intraspinal substance P-containing projections to the sympathetic preganglionic neuropil in pigeon, Columba livia: high-performance liquid chromatography, radioimmunoassay and electron microscopic evidence.

The present study uses quantitative and electron microscopic methods to investigate the hypothesis that intraspinal substance P-sympathetic preganglionic neuron circuitry exists in vertebrates. Radioimmunoassay and high-performance liquid chromatography were used to: (1) characterize the chemical nature of the substance P-like immunoreactivity in the sympathetic preganglionic neuropil; and (2) quantify the relative contributions of brain stem, primary sensory and intraspinal neurons to the substance P content within the sympathetic preganglionic neuropil. Electron microscopic observations on the localization of substance P-like immunoreactivity within the preganglionic neuropil caudal to complete thoracic spinal cord transections are also reported. High-performance liquid chromatographic analyses demonstrate that pigeon substance P-like immunoreactivity co-migrates with synthetic substance P, suggesting that the substance P-like material is authentic substance P content within the sympathetic preganglionic neuropil. Electron microscopic observations on the localization of substance P-like immunoreactivity within the preganglionic neuropil caudal to complete preganglionic cell column (inclusive of intermediate spinal laminae V and VII as well as preganglionic neurons located within nucleus intercalatus spinalis); (2) cutting the dorsal rootlets entering the last cervical (C14) and first two thoracic (T1, T2) spinal segments resulted in massive depletion of substance P content in dorsal horn of T1, but no detectable losses within the preganglionic cell column or ventral horn of T1; and (3) total mid-thoracic (T3-4) spinal cord transection significantly depleted the substance P content in the preganglionic cell column (T3-4) as well as in the dorsal (T1-4) and ventral horns (T2-4). Ultrastructural examination of the sympathetic preganglionic neuropil caudal to spinal transections (survival times of 3-14 days) revealed the presence of numerous, intact, normal appearing substance P-like immunoreactive terminals. Immunolabeled terminals formed asymmetric contacts on medium-sized and small caliber dendrites. Extensive degeneration was evident in this material as well. The ultrastructural features of degenerating processes were distinctive and quite dissimilar in appearance from those exhibiting substance P-like immunoreactive staining. No evidence for damage-induced sequestration of substance P-like material into glial elements was found. The above observations are consistent with earlier findings in rat and pigeon, and provide new quantitative and qualitative evidence to support the hypothesis that intraspinal substance P-containing interneurons contribute t

Light microscopic observations on the morphology of sympathetic preganglionic neurons in the pigeon, Columba livia.

Experiments were performed in anesthetized, immobilized, artificially respirated pigeons (Columba livia). Extracellular recordings from 56 antidromically activated and collided sympathetic preganglionic neurons were obtained. Eleven cells were intracellularly labeled with horseradish peroxidase and reconstructed at the light microscopic level. Electrophysiologically there were no statistical differences between labeled and unlabeled neurons. Four different somatic shapes were observed: fusiform, pyriform, multipolar and stellate. Nine of 11 cells were located within the principal preganglionic cell column (column of Terni), the other two were within nucleus intercalatus spinalis. Principal column neurons exhibited planar, horizontally aligned dendritic arbors with major extensions directed rostrocaudally. Unexpectedly, the majority of these cells also had dendritic branch projections which spanned the entire width of the ipsilateral zona intermedia. Contralateral dendritic terminal arborizations were evident in seven neurons. Intercalatus neurons were multipolar-shaped and exhibited a notably different dendritic arrangement from principal column preganglionic cells. The dendrites of intercalated cells coursed obliquely within the transverse spinal cord axis, giving rise to major dendritic extensions into the base of the dorsal horn, the dorsolateral funiculus, and the dorsal aspects of the ventral horn. Irrespective of somatic subnuclear location, the morphology of preganglionic dendrites was similar: (1) Largely primary, secondary, and tertiary processes were smooth. (2) Fine caliber proximal and distal elements appeared beaded or "varicose." (3) Distal processes gave rise to thin-stalked, spine-like appendages. The axons of preganglionic neurons arose from cell bodies as well as primary and secondary dendrites. The axons of two cells branched intraspinally. The present findings provide detailed descriptions of the somatic structures and accompanying dendritic trees of preganglionic neurons within nucleus intercalatus. The observations also include anatomical evidence showing the intraspinal collateralization of sympathetic preganglionic axons. In general, avian sympathetic preganglionic neurons located within the principal cell column appear to be structurally homologous to their mammalian counterparts within the intermediolateral cell column of thoracic spinal cord.

Alpha-2-adrenergic receptors in avian spinal cord: increases in apparent density associated with the sympathetic preganglionic cell column.

Vertebrate spinal cord receives a dense and diversified catecholaminergic innervation from brainstem and diencephalon. Within the spinal gray, the densest terminations appear to be within the neuropil surrounding sympathetic preganglionic neurons (SPNs) in thoracic spinal cord. Results of recent iontophoresis investigations showed that several catecholamines and clonidine, an alpha-2 agonist, uniformly inhibited the maintained discharge activity of SPNs [19]. These experiments raised the possibility that the inhibitory effects might be mediated by activation of an alpha-2-adrenergic receptor. The present series of ligand binding studies provide biochemical evidence suggesting the presence of alpha-2-adrenergic receptors in the SPN cell column. Total specific binding (Bmax) of the radiolabeled agonists clonidine (CLO) and para-amino-clonidine (PAC) (at concentrations above and below apparent KDS) was significantly greater in thoracic spinal cord in comparison with cervical spinal cord (P less than 0.001). The elevated levels in thoracic spinal cord were entirely accounted for by increases in apparent receptor density in dorsal horn and the SPN cell column (inclusive of the adjoining intermediate spinal laminae) (P less than 0.005). Adrenergic receptor subtype specificity of [3H]PAC was tested in competitive inhibition experiments. The results confirmed that [3H]PAC is a preferential alpha-2 agonist in thoracic and cervical spinal cord, and indicated the following rank order of potency for its displacement: norepinephrine = yohimbine much greater than prazosin greater than propranolol.

Distribution of enkephalin-like immunoreactivity in the rat main olfactory bulb.

Enkephalin-like immunoreactivity was localized within the main olfactory bulb of the rat using immunohistochemical techniques. These studies utilized well characterized antisera directed to either leu5- or met5-enkephalin. Specificity was established by absorption of the antisera with either 10 microM synthetic leu5- or met5-enkephalin. Specific enkephalin-like immunoreactivity was observed within several different cell populations including (1) periglomerular cells, (2) granule cells and their processes within the external plexiform layer and (3) occasional short-axon (horizontal) cells within the granule and external plexiform layers. The granule cell layer contained the greatest number of immunoreactive cells. Only a limited number of immunoreactive cells were found in both the periglomerular and granule cell layers, suggesting the enkephalin-containing neurons represent a sub-population within each layer. The absence of immunoreactive processes in the periventribular white matter, as well as the morphologies of immunoreactive bulbar neurons, indicates that enkephalin is found exclusively within intrinsic olfactory bulb neurons.