Proteins rapidly transported to the synapses of a single identified neuron of Aplysia californica

The cell body of R2, a giant cholinergic neuron of Aplysia californica, resides in the abdominal ganglion, whereas its synapses are on thousands of unicellular mucus glands located in the skin. Due to the great spatial separation between the site of macromolecular synthesis and the presynaptic terminals, rapid axonal transport can be used to segregate synaptic proteins from those to be used elsewhere in the cell. The proteins of R2 were labeled by incubating the abdominal ganglion in [35S]methionine for 5 hr in a chamber separated from the rest of the isolated central nervous system. After 50 hr, 28 radiolabeled proteins were reproducibly found by one- and two-dimensional polyacrylamide gel electrophoresis to be transported to the distal regions of peripheral nerves P6, P7, and P8 that innervate the parapodia and middle body wall. We are sure that R2 is the source of these proteins since radioautography of sections taken throughout the nervous system, complemented by cobalt tracings, showed that R2 is the only neuron in the abdominal ganglion with axons in these nerves. Nine of the 28 transported proteins are glycoproteins since they were also labeled after injecting R2's cell body with [3H]-L-fucose. There is evidence that the proteins and glycoproteins are destined for R2's presynaptic terminals. For example, in experiments in which the body wall and parapodium remained attached to the nerves, the proteins were transported to the skin region that contains the glands. Moreover, analyses of the distribution of the rapidly transported proteins by qualitative radioautography and by extrusion of axoplasm indicated that none are constituents of the axolemma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Perikaryal routing of newly synthesized proteins in regenerating neurons: Quantitative electron microscopic autoradiography

Intracellular transport of newly synthesized proteins through organelles in the perikarya of regenerating goldfish retinal ganglion cells was studied using electron microscopic autoradiography. Retinas were removed 14 or 30 days after optictract cut or sham operation, pulse-labeled in [³H]proline-containing medium for 5 min, and then chase-incubated in medium containing unlabeled proline for various times up to 55 min before fixation. Fourteen days after axotomy, during rapid growth of the regenerating axons, the time course of change of relative grain density (% grains/% area) in the rough endoplasmic reticulum in regenerating cells was almost identical to that in control cells. However, the grain distribution analysis revealed an increased delivery of newly synthesized proteins to the Golgi apparatus, perikaryal plasma membrane and nucleus in regenerating cells. Thirty days after axotomy, during synaptogenesis, Golgi apparatus labeling in the regenerating cells became significantly higher than control, but the increase was delayed compared to the increase seen 14 days after axotomy. Labeling of the plasma membrane and nucleus did not rise above control in 30-day regenerating cells chase-incubated for up to 55 min. Thus the pattern of intracellular transport of newly synthesized proteins varies with the stage of axonal regeneration.     

Changes in rapidly transported proteins in developing hamster retinofugal axons

Proteins synthesized in retinal ganglion cells and conveyed to the terminals of optic tract axons in the rapid phase of axonal transport were analyzed at different developmental stages in the hamster. Animals between 2 d of age and adulthood were labeled intraocularly with 35S-methionine, and after a 4 hr survival time, the superior colliculus was dissected out, subjected to subcellular fractionation, and radiolabeled proteins in the particulate fraction analyzed by 2-dimensional gel electrophoresis and fluorography. The previously identified growth-associated phosphoprotein, GAP-43 (GAP-48, B-50, F1, pp46), was synthesized and transported at high levels in the neonate, but these levels declined precipitously after the second postnatal week. Immunohistochemical studies using a monospecific antibody showed that GAP-43 was localized along the entire length of retinal axons in the optic tract and target areas in P2 animals but was virtually absent in the adult visual pathway. By metabolic labeling, 2 proteins with molecular weights of about 230 kDa also showed a sharp decrease during development. In contrast, acidic proteins of 27 and 64 kDa, which were barely detectable in the neonate, increased steadily to become the most heavily labeled proteins of rapid axonal transport by the second postnatal week. Another group of proteins, of about 94-110 kDa, also rose to peak levels after birth but then declined. Temporal correlations between the molecular changes described here and the known anatomical events in optic tract development suggest that the synthesis and transport of particular membrane proteins may be directly related to the sequence of morphological changes.     

Allocation of newly synthesized proteins in spinal motoneurons

When vinblastine sulfate is used in vitro to block slow and fast transport of proteins within frog lumbar spinal motoneurons, the amounts of both newly synthesized protein and total protein increase in motoneuronal perikarya. Analyses of motoneurons isolated from control and vinblastine-treated spinal cords show that 1) about 55 p. 100 of the newly synthesized protein is exported from motoneuron cell bodies during a 4 h incubation period; 2) only about 5 p. 100 of the total perikaryal protein is exported during the same period; and 3) less than 10 p. 100 of the labeled protein is exported by fast axonal transport. Thus, a substantial amount of the newly synthesized protein is quickly and preferentially exported from the cell body. It is not known how much of this exported protein reaches the axon by slow transport. However, when interpreted in conjunction with the studies of Schubert, Kreutzberg and Lux (Brain Res. 47, 331-343, 1972), the above data strengthen the possibility that substantial amounts of the new protein made by a motoneuron may be committed to its dendrites.     

Localization of axonally transported [H]glycine in vesicles of identified neurons

A specific association of axonally transported, free [³H]glycine with vesicle in the identified neurons R3–R14 ofAplysia is demonstrated by high resolution autoradiography. The association of glycine with vesicles, the first such finding in any animal for a neuroactive amino acid, adds to evidence that glycine may be utilized as a neurochemical messenger by R3–R14.     

Specific changes in rapidly transported proteins during regeneration of the goldfish optic nerve

Double labeling methods were used to identify changes in the complement of proteins synthesized in the retinal ganglion cells and transported down the optic nerve during the process of axonal regeneration. Eight to 62 days after goldfish underwent a unilateral optic nerve crush, one eye was labeled with [3H]-, the other with [14C]proline. Control and regenerating optic nerves were dissected out and homogenized together after 5 hr, a time which allowed us to examine selectively membrane-bound components which migrate in the rapid phase of axoplasmic transport. Proteins from the two sides were so-purified and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the 3H and 14C incorporation patterns along the gels revealed a radical shift away from the normal labeling spectrum during regeneration, with selective changes in labeling at particular molecular weights varying over a 3-fold range. Eight days after crushing the optic nerve, the greatest increases in labeling were seen for material with apparent molecular weights of 24,000 to 27,000, 44,000, and 210,000 daltons. These peaks declined thereafter, and on days 29 to 39, the most prominent increases were at 110,000 to 140,000 daltons. These studies indicate a continuously changing pattern in the synthesis and/or degradation of proteins that are rapidly transported down the optic nerve during regeneration and point to molecular species potential significance in the establishment of the visual map upon the brain.     

Ca-activated protease activity in frog sciatic nerve: Characterization and effect on rapidly transported axonal proteins

Protease activity was studied in the frog sciatic nerve. The activity was measured as the release of TCA-soluble radioactivity from either³H-labelled proteins transported by rapid axonal transport (AXT) or³H-labelled ganglionic proteins.In nerve homogenates containing transported substrates, protease activity exhibited two peaks, one around pH 5 and one around pH 8. Ca²⁺ at 100 μM or higher concentrations only stimulated the latter, which was inhibited by 1 mM parachloromercuric benzoate, a sulphydryl reagent, but unaffected by ATP (1 mM). The proteolytic activity was recovered in the 10⁵ g supernatant of the homogenate.In desheathed nerves containing³H-labelled transported proteins, the protease activity could be activated by exposing the nerve to a Ca²⁺-ionophore, X-537 A, or to an elevated Ca²⁺-concentration (50 mM). These conditions were also shown to increase the influx and efflux of⁴⁵Ca²⁺ in the nerves.The results indicate the presence within axons of a Ca²⁺-activated soluble protease, which degrades rapidly transported proteins. The finding that the protease degraded ganglionic soluble proteins to about the same extent suggests a broad substrate specificity. The present system should be useful for further characterization of protease activity during various physiological conditions.     

Changes in rapidly transported proteins in the auditory nerve after hair cell loss

In an effort to further characterize the proteins of the auditory nerve, the effects of hair cell loss on rapidly transported proteins of the auditory nerve were studied. The effects were studied in the waltzing guinea pig, a genetically deaf animal which displays an age-dependent loss of sensory cells, and in normal guinea pigs treated with neomycin. In both cases hair cell loss is followed by a slow degeneration of spiral ganglion cells and corresponding auditory nerve fibers. Rapidly transported proteins in the cochlear nucleus were analyzed by two dimensional electrofocusing/electrophoresis 3 h after cochlear injection of [35S]methionine. In both the waltzing guinea pig and the neomycin-treated animals, a significant increase in labeling of two series of polypeptides (average molecular weights of 27,000 and 36,000 daltons) was apparent. Quantitation of the 36,000 dalton protein by extracting from dried gels showed a 2-fold increase in the 10-day-old waltzing guinea pig and a 6-fold increase in the 80-day-old waltzing guinea pig. Further analysis shows these proteins to be membrane-associated glycoproteins.     

Changes in rapidly transported proteins associated with development of abnormal projections in the diencephalon.

The development of the hamster visual system is accompanied by striking changes in the pattern of proteins that are synthesized in retinal ganglion cells and rapidly transported to their nerve terminals. To determine whether any of these protein changes are regulated by interactions between the developing nerve endings and the cells with which they form synapses, we induced retinofugal axons to form abnormal projections in the lateral posterior (LP) nucleus of the thalamus and dense patches of hyperinnervation in the lateral geniculate nucleus (LGN) by removing their principal target, the superior colliculus (SC), the day after birth. Under these experimental conditions, two rapidly transported proteins, including the neural cell adhesion molecule, NCAM, showed significant changes in their time course of expression. NCAM, identified here using a monospecific antibody, is normally synthesized and transported at high levels at early stages of development and then declines during the second and third postnatal weeks. However, this decline was delayed when optic fibers were re-routed. A second rapidly transported protein, M(r) = 67 kDa, pI = 4.7, normally shows a rise in its synthesis and transport during terminal arbor formation and a subsequent decline, but it also remained elevated for a prolonged period when the SC was absent. These findings cannot be accounted for by a simple delay in the retinal ganglion cells' program of axonal growth, since other rapidly transported proteins, including the growth-associated protein GAP-43, showed a normal developmental time-course when the SC was removed. Target interactions therefore appear to influence the retinal ganglion cells' expression of different proteins in a specific fashion.     

Movement of newly synthesized membrane by fast transport along the axon of an identified Aplysia neuron

In order to ascertain the form in which newly synthesized membrane is moved by fast axonal transport, we examined the distribution of label along an axon of the identified giant serotoninergic neuron (GCN) in the cerebral ganglion of Aplysia californica. Membrane glycoproteins were labeled by intrasomatic injection of 3H-fucose, and segments of GCN's axon in the posterior lip nerve containing the transported organelles were examined at 1, 5, 15, and 24 hours by quantitative electron-microscopic autoradiography. To show that membrane which is rapidly transported is contained only in discrete organelles rather than in continuous sheets of axoplasmic reticulum, we systematically varied conditions of fixation. We found that we could distinguish vesicles from axoplasmic reticulum most reliably in axons fixed with 2% formaldehyde and 2% glutaraldehyde in cacodylate buffer. At short times after intrasomatic injection of 3H-fucose, dense-cored vesicles and multivesicular tubules were the only axonal organelles labeled.     

Temperature-dependent blockade of nucleocytoplasmic transport of newly synthesized RNA in neurons.

This study evaluates the temperature sensitivity of transport of recently synthesized RNA from the nucleus to the cytoplasm (nucleocytoplasmic transport) in CNS neurons. Rat hippocampal slices were incubated with [3H]uridine for 1 h to label recently synthesized RNA. Slices were then fixed immediately or maintained at 27 degrees C or 37 degrees C for chase intervals of 3, 4.5, and 6 h to allow for nucleocytoplasmic transport of recently synthesized RNA. The time-dependent translocation of recently synthesized RNA was evaluated autoradiographically. At the end of the 1 h pulse at either 27 degrees C or 37 degrees C, the label was localized exclusively over nuclei. In slices maintained at 37 degrees C, labeling expanded to cover the cell body and proximal dendrites. However, in slices that were labeled and maintained at room temperature, labeling remained confined to the nucleus. In slices that were pulse-labeled at room temperature, and then transferred to 37 degrees C medium, cytoplasmic labeling increased as a function of time. Nucleocytoplasmic transport of RNA in cultured rat hippocampal neurons showed a comparable temperature sensitivity. The inhibition of nucleocytoplasmic transport of RNA at room temperature provides an opportunity to evaluate neuronal function when no new RNA molecules can reach the cytoplasm.     

A calcium-activated neutral protease in the frog nervous system which degrades rapidly transported axonal proteins

A Ca2+-activated protease (CANP) was prepared from a soluble extract of the frog nervous system and separated from an endogenous high-molecular weight inhibitor by ion exchange chromatography. Its proteolytic action was tested on rapidly transported [3H]leucine-labelled proteins from sensory axons of the sciatic nerve. The enzyme was activated at neutral pH by Ca2+ at millimolar level, and was inhibited by leupeptin and the endogenous inhibitor. Its most pronounced degradative action was exerted towards two prominent rapidly transported peaks corresponding to 82 kDa and 90 kDa proteins. Proteolysis was accompanied by the appearance of mainly 16 kDa degradation products. When newly synthesized soluble proteins of the spinal dorsal ganglia were used as substrates, the presence of CANP resulted in degradation of proteins in a broad molecular-weight range (40-200 kDa).     

Ca2+-activated protease activity in frog sciatic nerve: characterization and effect on rapidly transported axonal proteins

Protease activity was studied in the frog sciatic nerve. The activity was measured as the release of TCA-soluble radioactivity from either 3H-labelled proteins transported by rapid axonal transport (AXT) or 3H-labelled ganglionic proteins. In nerve homogenates containing transported substrates, protease activity exhibited two peaks, one around pH 5 and one around pH 8. Ca2+ at 100 microM or higher concentrations only stimulated the latter, which was inhibited by 1 mM parachloromercuric benzoate, a sulphydryl reagent, but unaffected by ATP (1 mM). The proteolytic activity was recovered in the 10(5) g supernatant of the homogenate. In desheathed nerves containing 3H-labelled transported proteins, the protease activity could be activated by exposing the nerve to a Ca2+-ionophore, X-537 A, or to an elevated Ca2+-concentration (50 mM). These conditions were also shown to increase the influx and efflux of 45Ca2+ in the nerves. The results indicate the presence within axons of a Ca2+-activated soluble protease, which degrades rapidly transported proteins. The finding that the protease degraded ganglionic soluble proteins to about the same extent suggests a broad substrate specificity. The present system should be useful for further characterization of protease activity during various physiological conditions.     

Serotonin is not synthesized, but specifically transported, in the neurons of the hypothalamic dorsomedial nucleus

A small group of neurons in the hypothalamic dorsomedial nucleus (DMN) have been reported to contain serotonin after pharmacological treatments enhancing brain serotonin levels. This study aimed at elucidating whether these neurons are able to synthesize serotonin de novo, and whether they possess a specific serotonin transport mechanism. Serotonin content in these neurons was raised by administration of l -tryptophan and pargyline. Double immunostaining for serotonin and tryptophan hydroxylase (TpOH), the serotonin synthesizing enzyme, revealed that none of the serotonin-containing neuronal somata expressed TpOH. Intracerebro- ventricular colchicine treatment did not result in TpOH-IR in these neurons. Fluoxetine, a specific serotonin transport inhibitor, prevented the accumulation of serotonin in these neurons. The present results thus indicate that the serotonin-containing DMN neurons are not able to synthesize serotonin. Instead, they take up exogenous serotonin via a specific serotonin transport mechanism. As serotonin and DMN are associated with various physiological functions, such as regulation of food intake and modulation of fear and anxiety, the mechanisms revealed in the present study may participate in these clinically important brain functions.     

Modification of a rapidly transported protein in regenerating nerve

From 1 to 28 days after frog sciatic nerve damage, dorsal root ganglia were incubated with [35S] methionine, and the labeled, rapidly transported proteins at various points along the nerve were analyzed on two-dimensional gels. The results show a dramatic increase in the labeling of a protein, which we have designated as A25, only after the arrival of the rapidly transported proteins at regenerating nerve tips. This effect is first seen 3 to 5 days after injury. On gels from regenerating nerves, A25 appears as a series of intense spots with an apparent molecular weight of 70,000. A25 is retrogradely transported from the regenerating nerve tip regions. Since labeled A25 increases only after the rapidly transported proteins reach regions of nerve containing regenerating axons, we conclude that it most likely arises from post-translational modification of a transported protein. Various experiments were conducted to rule out alternative sources of A25 labeling at the nerve periphery.     

Specific in vitro synaptogenesis between identified Lymnaea and Helisoma neurons.

We tested the ability of identified neurons from two different families of pulmonate molluscs to form specific connections in vitro. The presynaptic neuron chosen for this study was the giant dopamine cell of Lymnaea stagnalis and Helisoma trivolvis which is known to synapse upon specific visceral and parietal ganglion neurons in both species. Here we show that the giant dopamine cells can reform specific connections in vitro on follower neurons from both species. Thus the mechanisms that determine synapse specificity are conserved between two different families of molluscs.     

In vivo release of newly synthesized [H]GABA in the substantia nigra of the rat: relative contribution of GABA striato-pallido-nigral afferents and nigral GABA neurons

The release of [3H]gamma-aminobutyric acid ([3H]GABA) continuously formed from [3H]glutamine has been measured with a push-pull cannula implanted in the substantia nigra of the rat anesthetized with ketamine. Consistent with the high density of GABA terminals coming from both the striato-pallido-nigral afferents, and from GABA nigrofugal neurons, our results showed that a large amount of [3H]GABA was spontaneously released in the reticulata, about 4 times higher than in the compacta. In the absence of calcium the spontaneous [3H]GABA release was reduced (-30%), as well as the K(+)-induced release of [3H]GABA (-66%). Bicuculline (10(-4) M) did not affect the K(+)-evoked release of [3H]GABA, suggesting that autoreceptors on GABA afferent fibers are distinct from the GABAA subtype. Partial lesions of striato- and pallido-nigral GABA neurons with kainic acid (1.2 micrograms) decrease by 40% the glutamic acid decarboxylase (GAD) activity in the ipsilateral SN without decreasing the spontaneous release of [3H]GABA; even following extensive lesions with kainic acid (2.5 micrograms), GAD activity (-72%) and spontaneous [3H]GABA release (-83%) were not completely abolished. These results suggest that a non-negligible contribution of GABA nigral neurons accounts for the spontaneous GABA release measured in the substantia nigra. This is further supported by the decrease (-20%), and the increase (+40%) of [3H]GABA release produced by the local application of glycine (10(-6) M), and bicuculline (10(-4) M), which respectively, inhibits and activates the nigral neuron activity. The contribution of nigral GABA neurons to the amount of [3H]GABA release from the substantia nigra, is likely linked to their high spontaneous firing rate.     

Intracellular translocation of newly synthesized myelin proteins in the rat brain stem slices

Brain stem slices from actively myelinating rats were incubated 30 min with [³H]leucine, then the labeling of proteins was halted by a chase with cold amino acid and slices incubated for an additional 30 min. Proteins from 10 particulate fractions of the tissue dispersion were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bands corresponding to five major myelin proteins were analyzed. All proteins appeared in myelin after an initial time lag and the labeling continued after the chase, although distinct differences between integral and peripheral myelin proteins regarding the kinetics of entry were observed. Labeling of proteins in a subcellular fraction in the chase experiment was used to determine the localization of the fraction along the intracellular route of myelin protein flow. The data support the contention of precursor-product relationships between myelin subfractions and myelin-like membranes. The involvement of other particulate fractions in processing myelin proteins is considered.     

Modulation of peptide release from single identified Aplysia neurons in culture.

Aplysia neurons B1 and B2 contain large amounts of the neuropeptides SCPA and SCPB. When grown in culture, individual B1 and B2 cells incorporate 35S-methionine into the SCPs, which can be released in a stimulus- and calcium-dependent fashion (Lloyd et al., 1986). We now show that single cells can be stimulated in a manner to evoke release of the SCPs that declines only slightly with repeated stimulation. This has allowed us to examine the ability of several physiologically relevant agonists to modulate the stimulus-evoked release of the SCPs. Bath application of either FMRFamide or 5-HT resulted in a significant decrease in the amount of SCPs released by intracellular stimulation of B1 or B2. The action of 5-HT was dose dependent with an inhibition of release of approximately 70% at a concentration of 100 microM. SCPA did not significantly affect release. The bath application of several compounds that are expected to elevate intracellular levels of cAMP were also found to depress release. To investigate the possibility that the agonists inhibited the release of the SCPs via a hyperpolarization of membrane potential (and perhaps a loss of spikes in the neurites), we examined the actions of 5-HT, FMRFamide, and SCPA on several electrophysiological parameters intended to monitor the level of cell excitability. Surprisingly, even though 5-HT depressed the release of the SCPs from both cells, it depolarized and increased the excitability of B1, and hyperpolarized and decreased the excitability of B2. Furthermore, in contrast to the effects seen in culture, 5-HT depolarized both B1 and B2 in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of GAP-43, a rapidly transported growth-associated protein, and class II beta tubulin, a slowly transported cytoskeletal protein, are coordinated in regenerating neurons

GAP-43 is a membrane-associated phosphoprotein enriched in elongating axons (Meiri et al., 1986; Skene et al., 1986). After an axon has been interrupted by cutting or crushing a nerve (axotomy), the portion of the axon disconnected from the cell body (distal stump) degenerates and is replaced by the outgrowth (elongation) of regenerating sprouts arising from the proximal stump. Previous studies have shown that increased amounts of pulse-labeled GAP-43 undergo fast axonal transport in regenerating neurons (Benowitz et al., 1981; Skene and Willard, 1981 a, b). Using hybridization with a cloned cDNA probe, I now show that mRNA levels for GAP-43 increase in lumbar sensory neurons of rat after regeneration is initiated by crushing the sciatic nerve; the relatively high levels of GAP-43 mRNA in regenerating neurons are comparable to those in the developing neurons of 5-d-old animals. I further demonstrate that the induction of GAP-43 expression in regenerating neurons coincides temporally with an increase in mRNA levels for class II beta tubulin (Hoffman and Cleveland, 1988), suggesting that the expression of these proteins is closely coordinated during axonal elongation.