Synaptic vesicle proteins and neuronal plasticity in adrenergic neurons.

The neurons in the superior cervical ganglion are active in plasticity and re-modelling in order to adapt to requirements. However, so far, only a few studies dealing with synaptic vesicle related proteins during adaptive processes have been published. In the present paper, changes in content and expression of the synaptic vesicle related proteins in the neurons after decentralization (cutting the cervical sympathetic trunk) or axotomy (cutting the internal and external carotid nerves) were studied. Immunofluorescence studies were carried out using antibodies and antisera against integral membrane proteins, vesicle associated proteins, NPY, and the enzymes TH and PNMT. For colocalization studies, the sections were simultaneously double labelled. Confocal laser scanning microscopy was used for colocalization studies as well as for semi-quantification analysis, using the computer software. Westen blot analysis, in situ 3'-end DNA labelling, and in situ hybridization were also employed. After decentralization of the ganglia several of the synaptic vesicle proteins (synaptotagmin I, synaptophysin, SNAP-25, CLC and GAP-43) were increased in the iris nerve terminal network, but with different time patterns, while TH-immunoreactivity had clearly decreased. In the ganglia, these proteins had decreased at 1 day after decentralization, probably due to degeneration of the pre-ganglionic nerve fibres and terminals. At later intervals, these proteins, except SNAP-25, had increased in the nerve fibre bundles and re-appeared in nerve fibres outlining the principal neurons.

Growth associated protein 43 (GAP-43) mRNA is upregulated in the rat superior cervical ganglia after preganglionic transection.

Growth-associated protein 43 (GAP-43) is a growth-associated protein which is synthesised in high amounts in neurons during neuronal outgrowth. In a previous study we have shown that GAP-43 immunoreactivity is increased in neurons in superior cervical ganglia (SCG) and in nerve terminals in the irides after preganglionic denervation. We have now examined changes in GAP-43 mRNA using in situ hybridisation. GAP-43 mRNA was seen to be constitutively expressed by principal neurons of the rat superior cervical ganglion. Expression was increased further by section of the cervical sympathetic trunk, reaching a maximum (increased by about 30%) 3 days after decentralisation. The increased GAP-43 protein seen after decentralisation thus appears to be due to an upregulation of GAP-43 mRNA in the adrenergic neurons. The results imply that GAP-43 expression in the SCG is under presynaptic control, acting at least partly by control of mRNA levels.

Cellular reactions to axotomy in rat superior cervical ganglia includes apoptotic cell death.

The cellular response to axonal injury in the superior cervical ganglion was examined by immunofluorescence at intervals from 6 h to 14 days after transection of the internal and external carotid nerves. GAP-43-immunoreactivity (IR) appeared in some neurons in the ganglia 1 day after axotomy, while neurons in control ganglia were GAP-43 negative. In 3 days axotomized ganglia GAP-43-IR structures were increased in number and intensity in nerve fiber bundles, while GAP-43-positive perikarya were restricted to the middle and caudal parts of the ganglia and showed an intensity that was stronger than at 1 day after axotomy. These GAP-43-positive neurons were also galanin positive. In the cranial part of the ganglia, S100-IR in satellite cells was weak at 18 h after axotomy. Peripheral to this area, S100-IR was stronger and co-localized with HSP-72-IR, preferentially located in satellite cells. HSP-72-IR was, however, occasionally observed also in principal neurons at 1 and 3 days after axotomy. In eosin-stained sections, neurons and satellite cells in the cranial part of 1 day axotomized ganglia were reduced in number, and a further loss was noted at 3 days. At 12 h some satellite cells in the cranial part of the ganglia were labelled by the in situ DNA 3'-end labelling method, indicating apoptosis, and at 18 h many cells were labelled. Some neuronal perikarya were also labelled in this region. Labelling was not observed at 1 day or later after axotomy, nor in control ganglia. The results may imply that not only neurons but also satellite cells react to neuronal axonal injury with apoptosis. Neurons in the middle and caudal part of the ganglia survived and showed increased content of GAP-43 and galanin, possibly a sign of regeneration/neuronal plasticity.

SNAP25 and GAP-43 behave differently in decentralized rat superior cervical ganglia.

The behaviour of synaptosome associated protein of mol. wt 25 kDa (SNAP25) in decentralized rat superior cervical ganglia (SCG) was investigated in order to observe its possible involvement in adrenergic postganglionic neuronal plasticity. Immunofluorescence and immunoblot results showed that the protein was increased in the nerve terminals in irides 8 days after operation. The intra-ganglionic nerve terminals and nerve fibres differed in their content of GAP-43 and SNAP25: GAP-43, which could not be observed at 1 day, appeared at 3 days after cutting the cervical sympathetic trunk, whilst SNAP25-immunoreactive material was still undetectable at this time. Immunoblot data also showed that SNAP25 did not reach control levels at 3 and 8 days after decentralization, in contrast to GAP-43. This observation may imply that SNAP25 is rapidly transported to its functional destinations immediately after synthesis and is possibly mainly involved in the remodelling of long projection pathways.

Clathrin light chain and synaptotagmin I in rat sympathetic neurons.

Clathrin light chain (clathrin LC) and synaptotagmin I in sympathetic neurons in rat superior cervical ganglia (SCG) were studied using immunofluorescence and confocal microscopy. The distributions of clathrin LC and synaptotagmin I were compared with that of tyrosine hydroxylase (TH) and neuropeptide Y (NPY) in double label experiments. The influence of preganglionic regulation on the expression of clathrin LC and synaptotagmin I in post-ganglionic adrenergic neurons was investigated after cutting the cervical sympathetic trunk. In SCGs and irides of control animals, the calthrin LC- and synaptotagmin-I-positive structures were present in a granular pattern in nerve fibers and varicose terminals. In principal neurons, the two proteins were present in a perinuclear network (the Golgi complex). After decentralization, the synaptotagmin-I- and clathrin LC-positive granules normally present in preganglionic nerve terminals outlining the neuronal somata were no longer observed on day 1, but reappeared, and were increased above control in number and intensity, in axon bundles in the ganglia, on day 3 and up to day 28 post-decentralization. In irides, the fluorescence intensity and density of clathrin LC- and synaptotagmin-I-positive nerve terminals in the dilator plate, were semi-quantified using the confocal microscopy software. It was found that both proteins increased shortly after decentralization. Immunoblot data confirmed the immunohistochemical/confocal microscopy observations. Fast axonal transport of clathrin LC- and synaptotagmin I in preganglionic sympathetic neurons was demonstrated in crush-operated cervical sympathetic trunk. Both proteins rapidly accumulated proximally as well as distally to the crush, demonstrating fast anterograde and retrograde axonal transport (recycling). Thus, clathrin LC and synaptotagmin I are normally present in pre- as well as post-ganglionic sympathetic neurons. The colocalization of clathrin LC with synaptotagmin I in the Golgi complex of the adrenergic neurons may imply that clathrin participates in the synthesis/sorting of the fast transported materials in these neurons. Possible explanations for the increase of the two proteins after decentralization are discussed.

Synaptic vesicle proteins in cells of the sympathoadrenal lineage.

The cells of sympathoadrenal lineage display different characteristics after differentiation, although they stem from the same neural crest precursor during embryonic development. In the present study we compared the distribution patterns of several synaptic vesicle proteins in the superior cervical ganglion (SCG) and the adrenal medulla. Using indirect immunofluorescence combined with confocal laser scanning microscopy, it was observed that antisera against integral synaptic vesicle membrane proteins (SV2, synaptotagmin I, synaptobrevin II and synaptophysin) induced strong immunoreactivities in these cells, but anti-synaptobrevin I caused only a faint fluorescence. Immunoreactivities of the synaptic vesicle-associated proteins Rab3a and SNAP25 were also observed in the cells. Synapsin-Ia-reactive material appeared absent from chromaffin cells but present in small amounts in sympathetic neurons in the SCG and iris terminals. On the other hand, synapsin IIa immunoreactive material was strong in most SCG neurons and in adrenergic iris terminals. The neural specific clatrin light chain was detected in the SCG cells and in ganglion cells of the adrenal, but only weak traces could be observed in chromaffin cells. One of the vesicular monoamine transmitter transporters, VMAT2, which is expressed in catecholamine neurons in the brain stem, was observed in most cells in the SCG and also in groups of cells in the adrenal medulla, where the VMAT2-positive small chromaffin cells were PNMT-negative. SIF cells in the SCG contained most of the synaptic vesicle proteins investigated. The results show that after differentiation, sympathetic neurons, SIF cells and adrenal chromaffin cells still share many vesicle proteins even though their physiology is different.

Distribution of Rab3a in rat nervous system: comparison with other synaptic vesicle proteins and neuropeptides.

In the present study we have investigated the distribution of Rab3a in rat peripheral nervous system and compared it with the distribution of other synaptic vesicle proteins (synaptophysin, synapsin I), neuropeptides (CGRP, SP, NPY) and tyrosine hydroxylase (TH). Rab3a immunoreactivity (-IR) was always colocalized with synaptophysin-IR and synapsin I-IR in nerve terminals of the spinal cord and peripheral nerve endings. In many cases, Rab3a-IR was also present in the same axons and terminals as peptides. In crushed sciatic nerve axons, Rab3a was colocalized, proximal to the crush, with synaptophysin-IR, synapsin I-IR, CGRP-IR, and TH-IR, but only partially co-localized with NPY-IR and SP-IR. In the area distal to the crush, Rab3a-IR was very weakly positive in a few thin axons, while larger amount of synaptophysin, CGRP, NPY and SP immunoreactivities were detected. The subcellular distribution of peptides and Rab3a differed in that peptides were observed mainly in large granular structures, while Rab3a-IR was observed mainly as diffuse, finely granular immunoreactivity, in addition to a few exceptional large granules present in some axons. The results demonstrate that Rab3a is widely distributed in different types of neurons, i.e. motor, sensory, autonomic adrenergic and cholinergic neurons, and colocalized with other synaptic vesicle proteins, suggesting that Rab3a may play an essential role in neuronal function. Furthermore, Rab3a is present in many peptide containing axons and terminals, but with an apparently different subcellular distribution, being affiliated mostly with small synaptic vesicles and only occasionally with large vesicles, that may represent peptide contained vesicles.

Effects of decentralization on the levels of GAP-43 and p38 (synaptophysin) in sympathetic adrenergic neurons: a semi-quantitative study using immunofluorescence and confocal laser scanning microscopy.

The distribution of GAP-43 in superior cervical ganglion (SCG) and iris were studied in normal animals and following decentralization using immunofluorescence and confocal laser scanning microscopy (CLSM). GAP-43-like immunoreactivity (LI) was compared with p38 (synaptophysin)-LI, and tyrosin hydroxylase (TH)-LI. In the control SCG, GAP-43-LI and p38-LI were mainly localized in nerve terminals around the principal neurons. The neuronal perikarya were negative for GAP-43, but positive for p38 in a perinuclear zone, as well as positive for TH. SIF cells (Small Intensely Fluorescent cells, ganglionic interneurons) were positive for GAP-43, TH and p38. One day after decentralization, GAP-43-LI and p38-LI in nerve terminals around principal neurons had disappeared. Some of the principal neurons showed a weak GAP-43-immunoreactivity. Three days post-decentralization, GAP-43- and p38-positive nerve terminals around the neurons had reappeared in considerable numbers and the intra-ganglionic nerve bundles were positive for both antibodies. In the control irides, GAP-43-LI and p38-LI were distributed in a varicose pattern in the nerve bundles, around blood vessels and in the network of terminals. Double labelling studies showed that GAP-43-LI was colocalized with TH-LI and p38-LI. The network of terminals in the dilator plate of the irides was quantified by measuring the fluorescence intensity of randomly selected areas, using CLSM. Three days after decentralization the intensity of GAP-43-LI and p38-LI had significantly increased. TH-LI had decreased 8 days after decentralization. The results indicate that GAP-43-LI and p38-LI are normally present in the nerve fibers and terminals of both pre- and post-ganglionic neurons in adult rats. The expression of GAP-43-LI and p38-LI in post-ganglionic neurons is preganglionically regulated, as indicated by the increased expression after decentralization. The expression of p38 in these neurons is probably regulated via mechanisms that are separate from those which regulate GAP-43, since it showed a different time course than that of GAP-43-LI.

GAP-43 and its relation to autonomic and sensory neurons in sciatic nerve and gastrocnemius muscle in the rat.

The presence of the growth-associated protein, GAP-43, in rat sciatic nerve and gastrocnemius muscle was studied, using indirect immunofluorescence, in lumbar sympathectomized and sham-sympathectomized rats. To study fast axonal transport and accumulation of immunogenic GAP-43, the sciatic nerves were crush operated, 6 h before perfusion fixation. In sections of normal, crushed sciatic nerve GAP-43-like immunoreactivity (LI) rapidly accumulated, on both sides of the crushes, in medium and thin sized axons. In double immunostaining experiments, GAP-43-LI was mainly colocalized with tyrosine hydroxylase (TH)-LI, or neuropeptide Y (NPY)-LI, markers of sympathetic nerves. In some axons, GAP-43-LI was colocalized with Substance P (SP)-LI. In perivascular nerve terminals in the gastrocnemic muscle, strong GAP-43-immunofluorescence was observed, in most cases colocalized with TH-LI, but in some terminals with SP-LI. Three days after lumbar sympathectomy (removal of the L1-L4 sympathetic ganglia bilaterally), TH-LI and NPY-LI positive axons in the sciatic nerve were reduced in number by more than 90%. GAP-43-LI positive axons were reduced by about 50%. In the gastrocnemic muscle, some GAP-43-LI positive terminals, but very few TH-LI positive nerve fibres, were found around blood vessels. No further changes were seen 8 days after sympathectomy. SP-LI in axons in the sciatic nerve and in perivascular nerve terminals of the gastrocnemic muscle, did not change after sympathectomy; most of these axons also contained GAP-43-LI. S-100-LI was present periaxonally in the sciatic nerves, but it did not colocalize with GAP-43, indicating that Schwann cells contained no GAP-43-LI in these experiments. The results show that, in normal adult rats, GAP-43-LI is mainly present in sympathetic and sensory nerve fibers in sciatic nerve and in perivascular nerve terminals. The peptide is axonally transported, mainly in sensory and adrenergic axons.

[Comparison of the development between the satellite cells of ALD and PLD muscles of adult chicken in vitro].

Satellite cells isolated from the anterior (slow) and posterior (fast) latissimus dorsi of adult chick were cultured separately in the absence of neural factors. The cells were observed under an inverted phase contrast microscope every day. The morphological changes of satellite cells, described by other workers, were seen in these cultures. Furthermore, the difference in the time course of development of the satellite cells from different muscles was found. That is, the transformation of the satellite cells of PLD into the myoblasts and the fusion of the latter delayed about 24 h as compared with ALD. The results support the idea that there may be an intrinsic developmental tendency in the myogenic cells of bird, since it proved possible to see the difference of the myogenic cells even in mononuclear phase.