Worm Breeder's Gazette 9(2): 55

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Identified GABAergic Neurons and the Immunocytochemical Identification of Mutations Affecting Axonal Outgrowth and GABA Expression in C. elegans

S.L. McIntire, H.R. Horvitz, and J.G. White

We have previously described the GABA immunoreactive neurons of C.  
elegans and the immunocytochemical identification of mutants affecting 
VD and DD axonal outgrowth or GABA expression (CSH Abstracts, 1985).  
Through further screening of existing locomotory mutants, we have 
identified additional genes affecting these processes.  We summarize 
our results 
Putative GABAergic Neurons Antibodies to GABA label six embryonic 
and thirteen postembryonic cell bodies in the ventral nervous system, 
a corresponding set of commissures, and the ventral and dorsal nerve 
cords.  The morphology of these cells and their positions in the nerve 
cord, as ascertained by double labeling with DAPI in adults and 
embryos, have led us to conclude that they are VD and DD motorneurons. 
Notable swellings occur at lateral and sublateral positions along the 
commissures.  An additional set of four embryonic neurons just 
anterior to the nerve ring are also labelled.  Based on their location 
and the disposition of their processes, we strongly suspect these to 
be the RME neurons.  Analogous sets of GABA immunoreactive neurons 
have been described in Ascaris (See Gazette 8(2), p.33).  A single 
postembryonic neuron, which is likely to be DVB, is labelled in the 
dorsal rectal ganglia of the hermaphrodite.  Our results with certain 
mutants (described below) are consistent with this assignment.  Two 
unpaired postembryonic neurons occur in the ventral ganglia.  We 
suspect these to be the RIR and RIS neurons.  A faintly labelled cell 
has been noted in the lateral ganglia just posterior to the nerve ring.
Likely candidates are AVL or RID.  A number of additional 
unidentified cells are labelled in the male tail.
Mutants with Abnormal VD or DD Axonal Outgrowth We have identified 
ten genes that affect the outgrowth of VD and DD axons.  Mutations in 
four genes [unc-5(e53), unc-6(e78), 69), and unc-69(
e587)] result in a complete failure of the dorsal extensions of these 
neurons to reach the dorsal midline.  Labelled processes appear in 
various lateral positions where they frequently interconnect or 
reenter the ventraI cord.  Ectopic branches are observed.  The 
disposition of labelled processes in the ventral cord can also appear 
abnormal in these mutants.  The normal spacing between immunoreactive 
cell bodies in the ventral cord is variably disrupted and the DD or VD 
cell bodies have been observed in positions dorsal or lateral to the 
ventral cord.  unc-5 was previously identified as being 'dorsal 
cordless.' Hedgecock et al.  have identified aberrant amphid, phasmid, 
and PDE processes in unc-51.  (Developmental Biology 111, 158 (1985)) 
Partial reconstructions of unc-6 have revealed a general 
disorganization of the dorsal cord and displacement of the excretory 
cell canal in three of nine cut animals (Leon Nawrocki, personal 
Mutations in another four genes [unc-34(e315), unc-40(e271), 
11), and unc-62(e644)] result in less complete 
defects in commissure formation.  In these mutants, the dorsal 
extensions of DD and VD processes often run laterally for considerable 
distances before turning again to meet the dorsal cord.  The 
commissures occasionally branch before reaching the dorsal midline and 
ectopic extensions are observed.  Cell bodies can again appear dorsal 
or lateral to the ventral cord.  Processes from these cell bodies tend 
to reach the dorsal midline by an atypical path.  The placement of 
labelled processes in the ventral and dorsal cords can also appear 
abnormal.  Axonal growth defects have been visualized in other neurons 
in unc-76 by FITC uptake Hedgecock et al., Dev.  Bio.  111, 158 (1985)
Mutations in two genes, unc-71(e541) and unc-73(e936), result in 
frequent (25% and 35% respectively) formation of commissures on the 
incorrect side of the animal.  Again, the labelled processes in the 
ventral cord appear disorganized.  The commissures themselves are 
generally normal although they occasionally run anteriorly or 
posteriorly in a lateral position before continuing to the dorsal 
Mutants with Altered GABA Expression   We have identified possible 
defects in GABA expression in four genes with a shrinker phenotype.  
In unc-25(e156), there appears to be no immunoreactivity in any of the 
neurons that are normally labelled with antisera to GABA.  Double 
labelling with Hoechst or DAPI reveals a normal number of neuron-like 
nuclei in the ventral cord.  In unc-30(e191), there is normal staining 
in the head and tail of the hermaphrodite.  The VD and DD motorneurons,
however, consistently fail to stain.  EM reconstructions of unc-30 
had shown that the placement of VD and DD processes is abnormal; other 
ventral cord neurons appear unaffected (J.  White, personal 
communication).  In the shrinker, unc-47(e307), the intensity of 
labelling appears considerably enhanced which may represent a defect 
in the release of GABA from nerve terminals.  Finally, in unc-43(e408),
the VD neurons have a reduced intensity of labelling, larvae move 
better than adults in this mutant.
Lineage Mutants   We looked at several lineage mutants, many of 
which were suspected to involve lineages generating the GABAergic 
neurons.  The mutation lin-4(e912) results in reiterations of K, which 
generates the postembryonic DVB neuron.  In approximately 50% of lin-4 
hermaphrodites, multiple labelled cells occurred in the position of 
DVB and had a similar morphology.  We also noted from one to three 
additional labelled cell bodies in varying positions of the ventral 
cord in approximately 15% of lin-4 hermaphrodites.  Additional DAPI 
stained nuclei were observed in the ventral cords of these animals.  
In unc-86(e1416), two RIR like cells were observed in 70% of adults 
and L1s.  (RIR is the sister of the interneuron AVG.) One to two 
additional stained nuclei were observed in the tail of 60% of unc-86 
hermaphrodites.  Finally, enlarged stained cell bodies have been 
observed in unc-59, cesses of 
these cells were not, however, definable.
Further analysis of some of the uncoordinated mutants discussed here 
may prove useful to an understanding of axonal guidance and the 
metabolism of GABA or neurotransmission at GABAergic synapses in C.