John B. Cabot

Pincher, a pinocytic chaperone for nerve growth factor/TrkA signaling endosomes

Acentral tenet of nerve growth factor (NGF) action that is poorly understood is its ability to mediate cytoplasmic signaling, through its receptor TrkA, that is initiated at the nerve terminal and conveyed to the soma. We identified an NGF-induced protein that we termed Pincher (pinocytic chaperone) that mediates endocytosis and trafficking of NGF and its receptor TrkA. In PC12 cells, overexpression of Pincher dramatically stimulated NGF-induced endocytosis of TrkA, unexpectedly at sites of clathrin-independent macropinocytosis within cell surface ruffles. Subsequently, a system of Pincher-containing tubules mediated the delivery of NGF/TrkA-containing vesicles to cytoplasmic accumulations. These vesicles selectively and persistently mediated TrkA-erk5 mitogen-activated protein kinase signaling. A dominant inhibitory mutant form of Pincher inhibited the NGF-induced endocytosis of TrkA, and selectively blocked TrkA-mediated cytoplasmic signaling of erk5, but not erk1/2, kinases. Our results indicate that Pincher mediates pinocytic endocytosis of functionally specialized NGF/TrkA endosomes with persistent signaling potential.

Avian bulbospinal pathways: anterograde and retrograde studies of cells of origin, funicular trajectories and laminar terminations.

Publisher Summary This chapter reviews the anterograde and retrograde studies of the cells of origin, funicular trajectories, and laminar terminations. Although a considerable amount of efforts are directed toward the study of the descending spinal pathways in mammals and reptiles, both with the older degeneration techniques and the more current horseradish peroxidase (HRP) and autoradiographic techniques. Very little published information is available on the anatomical organization of the descending pathways to the spinal cord in birds. To determine, as completely as possible, the full set of brain structures giving rise to descending spinal projections, several birds received multiple unilateral injections of HRP into the spinal cord at high cervical levels. Some of these cells extend dorsally from nucleus ruber along the midline and are seemingly in continuity with retrogradely labeled cells located in the interstitial nucleus of Cajal situated ipsilaterally to the injection site. The identity of these cells along the midline dorsal to nucleus ruber is unclear, but they may correspond to the prerubral field.

Effects of spinal lesions on substance P levels in the rat sympathetic preganglionic cell column: Evidence for local spinal regulation

Substance P has been localized to the neuropil of sympathetic preganglionic neurons in light and electron microscopic studies. Two recent reports have suggested that the majority of substance P in the rat intermediolateral cell column was contained in synaptic terminals of bulbospinal axons. However, previous investigations in our laboratory indicated the presence of major substance P spinal-sympathetic preganglionic neuron circuitry in pigeon. The present study used radioimmunoassay and immunohistochemistry to examine substance P levels in rat intermediolateral cell column following various spinal lesions in order to assess the relative contributions of bulbospinal and intraspinal substance P neurons to the substance P content of the intermediolateral cell column. The results from these experiments support the existence of both bulbospinal and intraspinal substance P-containing projections to the rat intermediolateral cell column. In addition, characterization of spinal cord substance P-like immunoreactivity by combined high performance liquid chromatography and radioimmunoassay, revealed that substance P in rat intermediolateral cell column was indistinguishable from synthetic substance P. Following transection of thoracic spinal cord, substance P-immunoreactive staining was still evident in the intermediolateral cell column caudal to the lesion. These substance P-positive fibers were studded with bouton-like swellings and appeared normal. Following high cervical hemisection, depletion of substance P (radioimmunoassay measurements) was bilateral and equal in the intermediolateral cell column: 25% depletion was observed after 7 days and 35% depletion after 14 days. However, rats which were hemisected at low cervical and/or mid-thoracic levels contained normal or elevated amounts of substance P in the intermediolateral cell column. Since substance P remains in the intermediolateral cell column following total transection, substance P spinal-sympathetic preganglionic neuron circuitry must exist. Additionally, depletion of substance P following high cervical hemisection suggests the existence of a substance P-containing, bilateral bulbospinal pathway to the intermediolateral cell column. The observation that substance P levels were normal or elevated following low cervical lesions raises the possibility that intraspinal substance P neurons can compensate for loss of substance P in the spinal cord. Sprouting or altered substance P metabolism and/or release by intraspinal substance P neurons could be responsible, suggesting an important homeostatic mechanism for maintaining substance P content within the intermediolateral cell column.

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.

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.

Chapter 3 Some principles of the spinal organization of the sympathetic preganglionic outflow

Publisher Summary This chapter reviews the spinal cord organization of the sympathetic motor component. It attempts to formalize some general principles of spinal sympathetic organization. The opportunity to attempt such an undertaking is particularly timely, as recently reported anatomical data have provided some critically missing links central to interpreting long-standing physiological observations. Together, the anatomical and physiological data accentuate and highlight the limitations of reflex circuitry intrinsic to spinal cord. Sympathetic preganglionic neurons are frequently referred to as autonomic “final common pathway neurons”. The analogy with spinal somatomotor system is obvious. One thematic undertone of this review is that perhaps the analogy is too imprecise or maybe overemphasized. The chapter emphasizes that spinal autonomic circuits in isolation are, in fact, remarkably rudimentary. Intrinsic spinal circuits cannot, for example, generate patterned sympathetic responses which typically accompany the expression of the fear, orienting or flight behaviors. It aims to discuss four broad principles of organization.

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.

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

Ultrastructural localization of the binding fragment of tetanus toxin in putative ?-aminobutyric acidergic terminals in the intermediolateral cell column: A potential basis for sympathetic dysfunction in generalized tetanus

Tetanus toxin (TeTx) causes sympathetic hyperactivity, a major cause of mortality in generalized tetanus, apparently by obstructing the inhibition of sympathetic preganglionic neurons (SPNs). Neuroanatomic tracing and immunohistochemistry were used to investigate whether axon terminals in the intermediolateral cell column (IML) that synapse on SPNs and use the inhibitory neurotransmitter γ‐aminobutyric acid (GABA) may be infected transsynaptically with TeTx. The binding fragment of TeTx (TTC; an atoxic surrogate of TeTx) and the cholera toxin B subunit (CTB; a retrograde tracer) were injected into the rat superior cervical ganglion and, over 16–48 hours, were transported to the ipsilateral IML in the caudal half of the last cervical and first three thoracic spinal cord segments. With light microscopy, diffuse CTB immunolabeling extended throughout SPN perikarya and dendrites. Punctate TTC and GABA immunolabeling were accumulated densely in the neuropil between and surrounding SPN processes. With electron microscopy, 54% of the axon terminals in the IML (n = 1,337 terminals) were TTC immunolabeled (TTC+), and 25% contained putative neurotransmitter levels of GABA immunolabeling (GABA+). On average, GABA+ terminals had a 76% chance of also being TTC+ and a 62% greater chance of being TTC+ than GABA− terminals (P < 0.000001). Axon terminals were just as likely to be TTC+ and/or GABA+ regardless of whether the dendrites they synapsed on were large (>1 μM) or small in cross‐sectional area or were labeled retrogradely. Sympathetic hyperactivity in tetanus may involve 1) retrograde and transsynaptic transport of TeTx by SPNs and 2) at least in part, an infection of GABAergic terminals in the IML. J. Comp. Neurol. 419:471–484, 2000.