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.

Quantitative analysis of immunofluorescence and lipofuscin distribution in human cortical areas by dual-channel confocal laser scanning microscopy.

A method for quantitative analysis of confocal laser scanning microscopy (CLSM) images of immunofluorescence and lipofuscin in human brain cortical areas is described. Indirect immunofluorescence was used to show the distribution and density of a synaptic vesicle protein--synaptophysin (p38). Dual channel CLSM was used for imaging immunofluorescence and autofluorescence of lipofuscin granules. Special software was developed for quantitative analysis of the CLSM images. The method has been tested in studies of frontal, temporal, motor, visual, and entorhinal cortices in normal and pathological human brains (Rett syndrome and autism). Using this technique the intensity and amount of p38 immunoreactivity was quantified after subtraction of lipofuscin fluorescence. In normal cortex no statistically significant differences were found for p38 antigen between layers II, III and V. The highest concentration of p38 immunoreactivity was found in the frontal cortex. In Rett syndrome and autistic patients, the p38 immunofluorescence in the investigated cortex areas appeared reduced compared with normal. The software is also suitable for quantitative analysis of double-labelled immunofluorescent specimens in animals without lipofuscin.

Fast and slow axonal transport-different methodological approaches give complementary information: contributions of the stop-flow/crush approach.

This 'minireview' describes experiments in short term crush operated rat nerves, to study endogenous substances in anterograde and retrograde fast axonal transport. Immunofluorescence was used to recognize transported antigens, and cytofluorimetric scanning was employed to quantitate different antigens which had accumulated proximal and distal to the crushes. Vesicle membrane components p38 (synaptophysin) and SV2 accumulated on both sides of a crush. This was expected from a number of studies from different laboratories. Surface associated molecules, however, like synapsins and rab3a, have been studied by other groups with biochemical methods, and suggested to be transported with slow transport. The crush method, however, revealed that a considerable fraction of these two substances are transported with the fast transport system, and, thus, associated with fast transported organelles in the living neuron. Evidently, more than one technique is required to give a more complete picture of intraneuronal transport related events.

Micromapping of the human brain: Three-dimensional imaging of immunofluorescence and dendritic morphology using dual-channel confocal laser scanning microscopy.

A strategy for investigating neuronal networks at the microscopic level is described. The Lucifer Yellow microinjection technique was combined with immunofluorescence on human brain material, which was then studied in a confocal laser scanning microscope with dual-channel scanning and equipped with an argon/krypton laser. The three-dimensional architecture of the Lucifer Yellow-injected neurons was investigated after transfer of the scanned frames in file format to a Silicon Graphics IRIS computer, using VoxelView software from Vital Images Inc. Microinjection of Lucifer Yellow revealed the dendritic morphology of various types of cells in different brain areas. Indirect immunofluorescence, with Texas Red as the secondary label, was used to determine the distribution of various categories of macromolecules (enzymes, receptor protein, and synaptic vesicle proteins) in the brain slices. We used single- as well as dual-channel confocal laser scanning microscopy for imaging these double-stained fluorescent specimens. Using these techniques in combination, we have created and saved three-dimensional confocal images of detailed morphology (axons and dendrites with spines and varicosities) of individual cells, together with the localization of immunofluorescence. These three-dimensional confocal images will be collected in a database for probable future use in human brain mapping. © 1994 Wiley-Liss, Inc.

Distribution of GAP-43 in relation to CGRP and synaptic vesicle markers in rat skeletal muscles during development.

GAP 43 in nerve terminal structures of rat skeletal muscles, was investigated during postnatal development using immunofluorescence and confocal laser scanning microscopy. Comparison with synaptophysin, synapsin, SV2, CGRP, SP and NF was done in double immunoincubation studies. GAP 43-like immunoreactivity (LI) was demonstrated in preterminal axons and motor endplates in all age groups (from E18 to adult), although the intensity of immunofluorescence was considerably higher in the younger rats. The outgrowing nerve sprouts in E18 muscles were strongly GAP 43-positive. The intensity decreased with increasing age, but even in adult animals GAP 43-LI was present in some p38- or SV2-positive endplates. GAP 43-LI was also present in muscle spindles and preterminal nerve branches, and likewise decreased with age. Perivascular nerve terminals (around arteries mainly) were, however, strong in GAP 43-LI during both development and adulthood. GAP 43-LI was strong, and present in both small and large granules. SP-LI was observed in a few thin, presumably sensory, axons around vessels, which also contained a few GAP 43-positive large granules. Most of the strongly GAP 43-positive terminals around vessels were probably autonomic postganglionic terminals. The results suggest that GAP 43, in addition to development and regeneration, may play a significant role also in normal adult rats, especially in perivascular nerve terminals, possibly connected with a high potential for plasticity in this kind of nerve terminals.

Sabeluzole administration does not enhance fast axonal transport in normal adult rat sciatic nerve.

Sabeluzole (R58735, Janssen Research Foundation) increased rates of axonal transport in short term tissue culture experiments and in rats with streptozotocin-induced diabetes. The drug was tested for its subacute (3 days) net effect on axonally transported substances in motor, sensory, and adrenergic axons of normal adult rats. Sabeluzol was given once daily for 3 days, 1 or 10 mg/kg/day intraperitoneally. Immunofluorescence was used to identify transported material. Three or 6 hr after crushing the sciatic nerves, to interrupt anterograde and retrograde intraaxonal transport, cytofluorimetric scanning was used to quantitate accumulated immunoreactive material. Compared with vehicle treated control rats, no clear differences in the net amounts of accumulated material, or in rates of accumulation, were detected in any axonal type. Since the short-term crush procedure interrupts ongoing axonal transport, the accumulation pattern reflects the transport characteristics in the crushed axons. The absence of clear increases in transport of several substances in this study indicates that sabeluzole did not enhance net axonal transport above control levels in peripheral axons of normal adult rats. Possible reasons for the discrepancy with earlier observations on the effect of sabeluzole on fast axonal transport is discussed.

Development of calcitonin-gene-related peptide, chromogranin A, and synaptic vesicle markers in rat motor endplates, studied using immunofluorescence and confocal laser scanning.

The presence of calcitonin-gene-related peptide (CGRP) and chromogranin A was investigated in the developing rat (E18-adult) motor system, using immunofluorescence and confocal laser scanning, and compared with synaptic vesicle markers, synaptophysin and synapsin I. In lumbar motor perikarya CGRP-LI and Chr A-LI were present in high intensities in E18 and P1 perikarya in the anterior horn. With increasing age immunoreactivity decreased. Chr A-LI was sparse in the adult. In peroneal endplates, p38-LI and SYN I-LI were present in all stages, including E18. Peptide-LI was very weak or absent in early stages (E18 and P1), but abundant in P8 and P18, especially CGRP-LI, and decreased again in P32 and adult animals. These observations indicate that the peptides have precise functions during certain developmental stages, possibly related to synapse maturation, receptor concentration, and reduction of supernumerary endplates. Both peptides are rapidly transported anterogradely in adult motor axons, and may serve physiological functions also in the adult.

Organelles in fast axonal transport. What molecules do they carry in anterograde vs retrograde directions, as observed in mammalian systems?

The present minireview describes experiments carried out, in short-term crush-operated rat nerves, using immunofluorescence and cytofluorimetric scanning techniques to study endogenous substances in anterograde and retrograde fast axonal transport. Vesicle membrane components p38 (synaptophysin) and SV2 are accumulating on both sides of a crush, but a larger proportion of p38 (about 3/4) than of SV2 (about 1/2) is recycling toward the cell body, compared to the amount carried with anterograde transport. Matrix peptides, such as CGRP, ChRA, VIP, and DBH are recycling to a minor degree, although only 10-20% of surface-associated molecules, such as synapsins and kinesin, appear to recycle. The described methodological approach to study the composition of organelles in fast axonal transport, anterograde as compared to retrograde, is shown to be useful for investigating neurobiological processes. We make use of the "in vivo chromatography" process that the fast axonal transport system constitutes. Only substances that are in some way either stored in, or associated with, transported organelles can be clearly observed to accumulate relative to the crush region. Emphasis in this paper was given to the synapsins, because of diverging results published concerning the degree of affiliation with various neuronal organelles. Our previously published results have indicated that in the living axons the SYN I is affiliated with mainly anterogradely fast transported organelles. Therefore, some preliminary, previously unpublished results on the accumulations of the four different synapsins (SYN Ia, SYN Ib, SYN IIa, and SYN IIb), using antisera specific for each of the four members of the synapsin family, are described. It was found that SYN Ib clearly has a stronger affiliation to anterogradely transported organelles than SYN Ia, and that both SYN IIa and SYN IIb are bound to some degree to transported organelles.

The axonal transport motor 'kinesin' is bound to anterogradely transported organelles: quantitative cytofluorimetric studies of fast axonal transport in the rat.

Monoclonal antibodies to the axonal transport ATPase kinesin were used in an immunofluorescent study on mammalian nerves. Following crushing of the sciatic nerve and the ventral roots of adult rats, immunoreactive material was found to accumulate rapidly, mainly proximal to a crush but also, to some degree, distal to a crush. The strongest immunofluorescence was observed after incubation with the H2 antibody against the heavy subunit of kinesin. Using the cytofluorimetric scanning (CFS) procedure, the accumulated amounts were quantified and it was found that the retrogradely accumulating kinesin-like immunoreactivity (IR) was about 4-12% of the anterogradely transported kinesin-IR. The results were compared to the vesicle marker p38 (synaptophysin), which was found to accumulate to a significant extent on both sides of the crush. Cytofluorimetric scanning measurements indicated that nearly 50% of the anterogradely accumulated p38-IR was recycling to the cell body. The results demonstrate that kinesin in the living axon is affiliated with anterogradely transported organelles. Retrogradely transported organelles appeared to carry very little kinesin-IR, suggesting that kinesin may be subject to turnover, distinct from that of p38, in the distal regions of the axon.

Rapid axonal transport as a chromatographic process: the use of immunocytochemistry of ligated nerves to investigate the biochemistry of anterogradely versus retrogradely transported organelles.

The distribution and axonal transport of cholinergic organelles has been studied in the rat motor system, using immunofluorescence methods and a cytofluorimetric technique for quantification of immunoreactive material. Crush-operated spinal roots and sympathectomized sciatic nerves were sectioned longitudinally and incubated with antisera against p38, SV2, CGRP, chromogranin A (Chr A), synapsin I (SYN I), and with RASVA (rabbit anti-synaptic vesicle antiserum). Motor endplates were also studied. It was observed that proximally accumulating organelles--i.e., organelles which were in transport distally in the axons--contained RASVA-like immunoreactivity (LI) p38, SV2, CGRP-LI, Chr A-LI, and SYN I-LI. Retrogradely transported organelles, however, contained only p38 and SV2 in addition to RASVA-LI, but virtually no CGRP-LI, ChrA-LI, or SYN I-LI. It is suggested that the rapid axonal transport mechanism operates in the nerves like a chromatographic process, which allows the concentration in the axons, proximal or distal to the crush, of organelles in anterograde or retrograde transport, respectively. The technique of nerve crushes in combination with immunocytochemistry can therefore be used to investigate the biochemical composition of organelles in transit along the axon, and give information on neurobiological events occurring in these long processes leading to the nerve endings. In this study, biochemical differences between anterogradely and retrogradely transported cholinergic organelles in the motor neuron of the rat have been observed, and were related to suggested events in the endplate.