VAT-1: an abundant membrane protein from Torpedo cholinergic synaptic vesicles

Expression screening was used to isolate cDNA clones encoding a synaptic vesicle membrane protein, VAT-1, which is specifically expressed in the electric lobe of marine rays. The predicted protein has a molecular weight of 41,572 daltons and contains several hydrophobic regions. An antibody raised against a fusion protein synthesized in E. coli recognizes an abundant 42 kd protein that copurifies largely with synaptic vesicles. Trypsin digestion of intact and lysed vesicles as well as membrane extractions suggests that VAT-1 is an integral membrane protein. The VAT-1 RNA is localized to the electromotor nucleus, and the fusion protein antibody stains the electric organ, demonstrating that the protein is transported to nerve terminals. These studies define a novel synaptic vesicle protein that is likely to play a central role in the functions mediated by specific classes of synaptic vesicles.     

The Synaptic Vesicle-Associated G Protein o-rab3 Is Expressed in Subpopulations of Neurons

Abstract: The distribution of o-rab3—a synaptic vesicle-associated low-molecular-weight GTP-binding protein—was studied in various neural tissues of the electric ray Torpedo marmorata. o-rab3 was shown to be associated selectively with isolated cholinergic synaptic vesicles derived from the electric organ. Gel filtration of cholinergic synaptic vesicles using Sephacryl S-1000 column chromatography demonstrated a copurification of o-rab3 with the synaptic vesicle content marker ATP and with SV2—a synaptic vesicle transmembrane glycoprotein. Indirect immunofluorescence using antibodies against o-rab3 and SV2 and a double labeling protocol revealed an identical distribution of both antigens in the cholinergic nerve terminals within the electric organ and at neuromuscular junctions. An immunoelectron microscopic analysis demonstrated the presence of o-rab3 at the surface of the synaptic vesicle membrane. In the CNS immunofluorescence of o-rab3 and SV2 overlap only in small and distinct areas. Whereas SV2 has an overall distribution in nerve terminals of the entire CNS, o-rab3 is restricted to a subpopulation of nerve terminals in the dorsolateral neuropile of the rhombencephalon and in the dorsal horn of the spinal cord. Our results demonstrate that the synaptic vesicle-associated G protein o-rab3 is specifically expressed only in subpopulations of neurons in the Torpedo CNS.     

Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.     

TRANSPORT, TURNOVER AND DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLIN-ESTERASE IN THE VAGUS AND HYPOGLOSSAL NERVES OF THE RABBIT

Abstract— The transport, distribution and turnover of choline O -acetyltransferase (ChAc, EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) in the vagus and hypoglossal nerves were studied in adult rabbits. The enzymes accumulated proximally and distally to single and double ligatures on both nerves and thus indicated both a proximo-distal and retrograde flow of the enzymes. Double ligature experiments indicated that only 5–20 per cent of the enzymes were mobile in the axon. The rate of accumulation of both enzymes above a single ligature corresponded to the slow rate of axonal flow provided that all the enzymes were mobile, but to an intermediate or fast flow if only a small part of the enzymes was transported. The distribution of ChAc along the hypoglossal neurons was studied and only 2 per cent of ChAc was confined to cell bodies, 42 per cent was localized to the main hypoglossal nerve trunks and 56 per cent to the preterminal axons and axon terminals in the tongue. The ratio of AChE to ChAc was about 3 in the hypoglossal nerve and 32 in the vagus nerve.
Transection of the hypoglossal nerve was followed by a decrease in the activity of ChAc in the hypoglossal nucleus and nerve and in the axons and their terminals in the tongue. The activity of AChE decreased in the hypoglossal nucleus and nerve but not in the tongue. The half-life of ChAc in preterminal axons and terminals of the hypoglossal nerve was estimated to be 16-21 days from the results obtained on transport, axotomy and distribution of the enzyme. Intracisternal injection of colchicine inhibited the cellulifugal transport of both enzymes and led to an increase in enzyme activity in the hypoglossal nucleus.     

RETROGRADE AXONAL TRANSPORT OF RAPIDLY MIGRATING LABELLED PROTEINS AND GLYCOPROTEINS IN REGENERATING PERIPHERAL NERVES

Abstract— The redistribution of rapidly migrating [³H]leucine-labelled proteins and [³H]fucose-labelled glycoproteins was studied in ligated regenerating hypoglossal and vagus nerves of the rabbit. When regenerating and contralateral hypoglossal nerves were ligated 16 h after labelling of the nerve cell bodies, rapidly migrating proteins and glycoproteins accumulated distal to the ligatures indicating a rapid retrograde transport from the peripheral parts of the nerves within 6 h. The retrograde accumulation of both proteins and glycoproteins was greater on the regenerating side than on the contralateral side at both 1 and 5 weeks after a nerve crush. Labelled proteins and glycoproteins also accumulated proximal to the ligatures, indicating a delayed rapid anterograde phase of axonal transport. The accumulation of this phase was also greater on the regenerating side 1 week after a nerve crush for both labelled proteins and glycoproteins. One week after a crush of the cervical vagus nerve, rapidly migrating proteins and glycoproteins redistributed between he crush zone and a proximal ligature applied 16 h after labelling of the nerve cell bodies. A retrograde accumulation occurred distal to the ligature within 6 h, indicating a rapid retrograde transport from the crush zone.     

Axonal growth during regeneration: a quantitative autoradiographic study

The intraaxonal distribution of labeled glycoproteins in the regenerating hypoglossal nerve of the rabbit was studied by use of quantitative electron microscope autoradiography. 9 d after nerve crush, glycoproteins were labeled by the administration of [3H]fucose to the medulla. The distribution of transported 3H-labeled glycoproteins was determined 18 h later in segments of the regenerating nerve and in the contralateral, intact nerve. At the regenerating tip, the distribution was determined both in growth cones and in non-growth cone axons, 6 and 18 h after labeling. The distribution within the non-growth cone axons of the tips was quite different at 6 and 18 h. At 6 h, the axolemma region contained < 10% of the radioactivity; at 18 h, it contained virtually all the radioactivity. In contrast, the distribution within the growth cones was similar at both time intervals, with 30% of the radioactivity over the axolemmal region. Additional segments of the regenerating nerve also showed a preferential labeling of the axolemmal region. In the intact nerve, 3H-labeled glycoproteins were uniformly distributed. These results suggest that: (a) in this system the labeled glycoproteins reaching the tip of the regenerating axons are inserted into the axolemma between 6 and 18 h after leaving the neuronal perikaryon; (b) at the times studied, there is a fairly constant ratio between glycoproteins reaching the growth cone through axoplasmic transport and glycoproteins inserted into the growth cone axolemma; (c) the axolemma elongates by continuous insertion of membrane precursors at the growth cone; the growth cone then advances, leaving behind an immature axon with a newly formed axolemma; and (d) glycoproteins are preferentially inserted into the axolemma along the entire regenerating axon.     

Postnatal changes in [3H]fucosyl glycoconjugates axonally transported into hamster optic nerve endings

The subsynaptosomal distribution of [³H]fucosyl glycoproteins axonally transported into the optic nerve endings of neonatal and adult hamsters changed dramatically at eye-opening. In 12 day-old previsual hamsters, the highest concentration of incorporated fucose was in the axoplasmic reticulum/synaptic vesicle fraction (51%), with only 6% in the dense synaptic membrane fraction. By the end of the eye-opening period four days later proportional labeling of the dense synaptic membrane fraction had increased four-fold to 23% of total sub-synaptosomal radioactivity. Labeling of the synaptic membrane doubled again in adults (41%). Total synaptosomal radioactivity was greatest in 16 day-olds. These results imply that utilization of [³H]fucose by the retinal ganglion cells, as well as composition of the synaptic membrane, change in association with the onset of functional visual activity.     

Immunochemical characterization of brain synaptic membrane glutamate-binding proteins

Two glutamate-binding proteins (71 and 63 kDa) were previously purified from synaptic plasma membranes (Chen, J.-W., Cunningham, M.D., Galton, V., and Michaelis, E. K. (1988) J. Biol. Chem. 263, 417-426). These proteins may play a role in glutamate neurotransmission in brain. Polyclonal antibodies were raised against the denatured glutamate-binding proteins in rabbits, including sets of antibodies against each of the binding proteins. The antibodies reacted specifically against both 71- and 63-kDa proteins. The antibodies recognized the denatured form of the proteins in Western blots and the native state of the proteins in enzyme-linked immunosorbent assays and in immunoaffinity chromatography and extraction procedures. All antibodies labeled most strongly the 71-kDa protein in Western blots, but extracted both proteins from solubilized synaptic membrane preparations. These findings indicate that the two proteins are closely related immunologically but the reactivity on Western blots differs between these two proteins. Immunoextraction of the 71- and 63-kDa proteins led to a approximately 60% decrease in L-[3H]glutamate-binding activity associated with synaptic membrane proteins. Of the brain subcellular fractions examined, the isolated synaptic plasma membranes had the strongest reaction in enzyme-linked immunosorbent assays toward the antiglutamate-binding protein antisera. Electron microscopy combined with gold particle immunohistochemistry revealed the sites labeled by the antibodies as entities present either on the surface or within the postsynaptic membranes and the associated densities of brain nerve ending particles (synaptosomes). Immunohistochemical procedures of gold labeling with silver enhancement of labeled sites revealed selective neuronal labeling in brain regions enriched in glutamate neurotransmitter pathways such as the hippocampus. Labeling was along dendrites and around cell bodies of pyramidal neurons. Based on the pattern of histochemical labeling, the distribution of immune reactivity in synaptic membranes, and the extractions of a major component of membrane glutamate-recognizing proteins by the antibodies, the glutamate-binding proteins must play a role in glutamate neurotransmission.     

Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat

The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca²⁺-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca²⁺-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.     

Distribution and metabolism of glycoproteins and glycosaminoglycans in subcellular fractions of brain.

The distribution, carbohydrate composition, and metabolism of glycoproteins have been studied in mitochondria, microsomes, axons, and whole rat brain, as well as in various synaptosomal subfractions, including the soluble protein, mitochondria, and synaptic membranes. Approximately 90% of the brain glycoproteins occur in the particulate fraction, and they are present in particularly high amounts in synaptic and microsomal membranes, where the concentration of glycoprotein carbohydrate is 2-3% of the lipid-free dry weight. Treatment of purified synaptic membranes with 0.2% Triton X-100 extracted 70% of the glycoprotein carbohydrate but only 35% of the lipid-free protein residue, and the resulting synaptic membrane subfractions differed significantly in carbohydrate composition. The glycoproteins which are not extracted by Triton X-100 also have a more rapid turnover, as indicated by the 80-155% higher specific activity of hexosamine and sialic acid 1 day after labeling with [3H]glucosamine in vivo. The specific activity of sialic acid in the synaptosomal soluble glycoproteins 2 hr after labeling was greater than 100 times that of the synaptosomal particulate fraction, whereas the difference in hexosamine specific activity in these two fractions was only twofold, and by 22 hr there was little or no difference in the specific activities of sialic acid and hexosamine in synaptosomal soluble as compared to membrane glycoproteins. These data indicate that sialic acid may be added locally to synaptosomal soluble glycoproteins before there is significant labeling of nerve ending glycoproteins by axoplasmic transport. Fifty to sixty percent of the hyaluronic acid and heparan sulfate of brain is located in the various membranes comprising the microsomal fraction, whereas half of the chondroitin sulfate is soluble and only one-third is in microsomal membranes. When microsomes are subfractionated on a discontinuous density gradient over half of the hyaluronic acid and chondroitin sulfate are found in membranes with a density less than that of 0.5 M sucrose (representing a six- to sevenfold enrichment over their concentrations in the membranes applied to the gradient), whereas half of the heparan sulfate is present in membranes with a density greater than that of 0.8 M.     

Distribution and localization of pro-brain-derived neurotrophic factor-like immunoreactivity in the peripheral and central nervous system of the adult rat

The precursors for neurotrophins are proteolytically cleaved to form biologically active mature molecules which activate their receptors p75NTR and trks. A recent study showed that the precursor for nerve growth factor (NGF) can bind to p75NTR with a high affinity and induces apoptosis of neurons in vitro. Mutation in Val66Met of brain-derived neurotrophic factor (BDNF) results in reduction in hippocampal function in learning and in the dysfunction of intracellular BDNF sorting and secretion. To examine the functions of pro-neurotrophins in vivo, it is essential to know where they are expressed in the nervous system. In the present study, we have raised and characterized rabbit polyclonal antibodies against a peptide coding for the precursor region of the BDNF gene. The antibody specifically recognizes the precursor for BDNF by western blot. With the affinity purified precursor antibody, we have mapped the distribution and localization of the precursor for BDNF. The results showed that, like mature BDNF, pro-BDNF is localized to nerve terminals in the superficial layers of dorsal horn, trigeminal nuclei, nuclei tractus solitarius, amygdaloid complex, hippocampus, hypothalamus and some peripheral tissues. These results suggest that pro-BDNF, like mature BDNF, is anterogradely transported to nerve terminals and may have important functions in synaptic transmission in the spinal cord and brain.     

The phosphorylation of kinesin regulates its binding to synaptic vesicles.

Membrane organella are transported bidirectionally in cells, and the axonal transport system has provided an ideal model system for studying this bidirectional transport. Kinesin and cytoplasmic dynein were identified as candidates for the motor molecules of fast axonal transport, which transport organella along microtubules anterogradely and retrogradely. However, the mechanism that controls this bidirectional transport is unknown. Our previous work revealed that kinesin in axons was associated abundantly with anterogradely transported membranous organella, most of which are believed to be precursors of synaptic vesicles and axonal plasma membranes, while the fractions bound to retrogradely transported ones were very small (Hirokawa, N., Sato-Yoshitake, R., Kobayashi, N., Pfister, K. K., Bloom, G. S., and Brady, S. T. (1991) J. Cell Biol. 114, 295-302). Here we demonstrated in vitro that the binding of kinesin to synaptic vesicles was concentration-dependent and saturable and could be released by high salt concentration. When kinesin was phosphorylated by cAMP-dependent protein kinase, its binding to symaptic vesicles was significantly reduced. By motility assay and by statistical analysis using electron microscopy, we further revealed that synaptic vesicles preincubated with phosphorylated kinesin associated less frequently with microtubules than synaptic vesicles preincubated with unphosphorylated kinesin. The phosphorylation of kinesin should therefore play an essential role in regulating the direction of fast axonal transport by inhibiting its binding to membrane organella, thus releasing it from membrane organella at nerve terminals.     

Neurochemical properties of the synapses in the pathways of orofacial nociceptive reflexes

The brainstem premotor neurons of the facial nucleus (VII) and hypoglossal (XII) nucleus can integrate orofacial nociceptive input from the caudal spinal trigeminal nucleus (Vc) and coordinate orofacial nociceptive reflex (ONR) responses. However, the synaptoarchitectures of the ONR pathways are still unknown. In the current study, we examined the distribution of GABAergic premotor neurons in the brainstem local ONR pathways, their connections with the Vc projections joining the brainstem ONR pathways and the neurochemical properties of these connections. Retrograde tracer fluoro-gold (FG) was injected into the VII or XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the Vc. Immunofluorescence histochemical labeling for inhibitory/excitatory neurotransmitters combined with BDA/FG tracing showed that GABAergic premotor neurons were mainly distributed bilaterally in the ponto-medullary reticular formation with an ipsilateral dominance. Some GABAergic premotor neurons made close appositions to the BDA-labeled fibers coming from the Vc, and these appostions were mainly distributed in the parvicellular reticular formation (PCRt), dorsal medullary reticular formation (MdD), and supratrigeminal nucleus (Vsup). We further examined the synaptic relationships between the Vc projecting fibers and premotor neurons in the VII or XII under the confocal laser-scanning microscope and electron microscope, and found that the BDA-labeled axonal terminals that made asymmetric synapses on premotor neurons showed vesicular glutamate transporter 2 (VGluT2) like immunoreactivity. These results indicate that the GABAergic premotor neurons receive excitatory neurotransmission from the Vc and may contribute to modulating the generation of the tonic ONR.     

Characterization of Axonally Transported Glycoproteins in Regenerating Garfish Olfactory Nerve

Abstract: This study examined changes in composition and concanavalin A (Con A) binding of axonally transported glycoproteins and their pronase-generated glycopeptides in regenerating garfish olfactory nerve. A previous study had demonstrated a regeneration-related increase in the proportion of [³H]glucosamine label in lower-molecular-weight Con A-binding glycopeptides derived from transported glycoproteins. Further analysis of carbohydrate composition shows that these molecules resemble mannose-rich oligosaccharides in composition and are increased in absolute amount in regenerating nerve. Subcellular analysis shows that the Con A-binding glycopeptides are enriched in membrane subfractions, particularly in a high-density fraction that morphologically resembles isolated cell surface coat. Regeneration-related changes in intact axonally transported glycoproteins were also detected. Sodium dodecyl sulfate gel electrophoresis of transport-labeled glycoproteins disclosed growth-correlated increases in radioactivity associated with 180–200K, 105–115K, and 80–90K components, while a 150–160K molecular weight class of glycoproteins was diminished in relative labeling. Intact glycoproteins displaying an affinity for Con A were also augmented in regenerating nerve, the increases occurring primarily in molecules in the 50–140K range.     

Use of fluorescent carbocyanine dyes in the study of the organization of the pathways in autopsy material of the human brain

Our report provides evidence that fluorescent carbocyanine dyes (diI and diO) can be used in experimental anatomical studies of the fixed autopsy human brain. The dyes transported in both anterograde and retrograde directions, providing labeling of axons with collaterals and neurons including dendrites. To study the retrograde labeling of pyramidal neurons and anterogradely labeling of afferent fibers in human motor cortex, we applied diI and diO to the white matter, I and III layers of cortex. During 2 months there was no evidence of passive diffusion from labeled fibers and neurons to other neurons or glia. This method will be useful for identifying alterations of neuronal connections associated with neurological and psychiatric disorders.     

Adenovirus-mediated WGA gene delivery for transsynaptic labeling of mouse olfactory pathways

Detailed knowledge of neuronal connectivity patterns is indispensable for studies of various aspects of brain functions. We previously established a genetic strategy for visualization of multisynaptic neural pathways by expressing wheat germ agglutinin (WGA) transgene under the control of neuron type-specific promoter elements in transgenic mice and Drosophila. In this paper, we have developed a WGA-expressing recombinant adenoviral vector system and applied it for analysis of the olfactory system. When the WGA-expressing adenovirus was infused into a mouse nostril, various types of cells throughout the olfactory epithelium were infected and expressed WGA protein robustly. WGA transgene products in the olfactory sensory neurons were anterogradely transported along their axons to the olfactory bulb and transsynaptically transferred in glomeruli to dendrites of the second-order neurons, mitral and tufted cells. WGA protein was further conveyed via the lateral olfactory tract to the olfactory cortical areas including the anterior olfactory nucleus, olfactory tubercle, piriform cortex and lateral entorhinal cortex. In addition, transsynaptic retrograde labeling was observed in cholinergic neurons in the horizontal limb of diagonal band, serotonergic neurons in the median raphe nucleus, and noradrenergic neurons in the locus coeruleus, all of which project centrifugal fibers to the olfactory bulb. Thus, the WGA-expressing adenovirus is a useful and powerful tool for tracing neural pathways and could be used in animals that are not amenable to the transgenic technology.     

Localization of Epstein-Barr virus envelope glycoproteins on the inner nuclear membrane of virus-producing cells.

Epstein-Barr virus-producing cells were used as a model to analyze, with a fracture-immunolabel technique, the distribution, behavior on fracture, and extent of glycosylation of viral transmembrane glycoproteins at the inner nuclear membrane. Surface and fracture immunolabeling with two monoclonal antibodies directed against the carbohydrate or polypeptide portions of the major viral envelope glycoproteins gp350/220 showed the following. (i) The glycoproteins present on the inner and outer nuclear membranes were labeled only with the monoclonal antibody directed against the polypeptide chain, whereas over the surface of virus-producing cells and on mature virions the labeling was dense and uniformly distributed with both monoclonal antibodies. (ii) The glycoproteins were nonuniformly distributed only over the inner nuclear membranes; at the sites of viral budding, the glycoproteins showed a preferential partition with the protoplasmic face. Since fully glycosylated glycoproteins were not present on the nuclear membranes, our observations support the proposed model of herpesvirus maturation. The peculiar distribution and partition on fracture of the envelope glycoproteins on the inner nuclear membrane are similar to those of Sindbis virus envelope glycoproteins on the plasma membrane of infected cells. Therefore, our results suggest that inner nuclear membranes may behave like plasma membranes during viral assembly.     

A novel in vivo method for isolating antibodies from a phage display library by neuronal retrograde transport selectively yields antibodies against p75NTR

The neurotrophin receptor p75^NTR is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75^NTR antibody or phage scFv library pre-panned against p75^NTR are internalized by neurons expressing p75^NTR; (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75^NTR antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75^NTR expression is upregulated in motor neurons in response to injury and in disease, the p75^NTR antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier.     

Anatomical relationship between neuropeptide-containing fibers and efferent vagal neurons projecting to the rat corpus.

Injections of the retrograde tracers into the posterior surface of the stomach at the greater curvature resulted in labelling of the right half of the dorsal motor nucleus of the vagus. Whereas injections into the anterior and posterior surfaces of the corpus resulted in bilateral labelling in the medulla. Immunocytochemical staining of the labelled sections using antisera to substance P was confined to a dense network of fibers within the dorsal motor nucleus of the vagus and the nucleus tractus solitarius with no cell bodies being detected. Calcitonin gene-related peptide-immunoreactivity was detected in nerve fibers in the nucleus tractus solitarius and cell bodies of the hypoglossal nucleus. Finally, neuropeptide Y-immunoreactivity was confined to nerve fibers within the vagal complex. Of the neurons labelled by the retrograde tracers injected into the corpus all were in close spatial contact with fibers containing substance P-immunoreactivity. A smaller number were associated with neuropeptide Y-containing fibers with a few adjacent to calcitonin gene-related peptide-immunoreactive fibers. These results indicate that substance P and neuropeptide Y may directly regulate efferent neurons controlling gastric motility and acid secretion. Calcitonin gene-related peptide, however, is unlikely to directly modulate the cell bodies of the neurons in the dorsal motor nucleus but may modulate the dendrites from these neurons in the nucleus tractus solitarius.