Worm Breeder's Gazette 14(1): 46 (October 1, 1995)

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.

The expression pattern of eat-4 and its requirement for AVM mediated touch sensitivity support its role in glutamatergic neural transmission

Raymond Lee1, Beth Sawin2, Leon Avery1, Bob Horvitz2

1 Department of Biochemistry, UT-Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9038
2 HHMI, Department of Biology, MIT, 77 Massachusetts Ave., Cambridge, MA 02139

Loss-of-function mutations in eat-4 (aka. not-1) lead to abnormal
behaviors like defective feeding (Eat; Avery, Genetics 133: 897),
insensitivity to light touch on the nose (Not), abnormal foraging
(Fab) (Kaplan, et al., WBG 12.3: 105, J Kaplan, pers. comm.),
defective chemotaxis (Che; C Bargmann, pers. comm.), and defective
thermotaxis (Ttx; Mori, et al., WBG 12.5:73).  At least two of these
phenotypes (Eat and Not) may be attributed to loss of glutamatergic
neurotransmission. Therefore, it was proposed that eat-4 may be
necessary for all glutamatergic neurotransmission (Avery, et al., WBG
13.4:72). eat-4 has been cloned. It shares 46% amino acid sequence
identity with a neural-specific Na-Pi cotransporter found in rat brain
(Lee and Avery, WBG 13.5:36).

To learn whether eat-4 is expressed in the neurons that it affects and
if there are other cells where it might have a function, we have
started characterizing eat-4::GFP and eat-4::lacZ translational fusion
expression. eat-4::lacZ and eat-4::GFP are expressed in more than 20
extrapharyngeal neurons. In addition, we could detect lacZ expression
in the pharyngeal M3 and NSM neurons. (We were unable to see GFP
expression in the pharynx, probably because the expression level is
too low.) The expression in M3s is consistent with our hypothesis that
eat-4 is necessary for M3 synaptic transmission. NSM, however, is
known to be serotonergic. We don't yet know the significance of NSM

The extrapharyngeal eat-4::GFP expressing neurons include the
mechanosensory touch neurons ALM, PLM, and AVM. The fact that eat-4 is
expressed in the mechanosensory touch neurons suggests that eat-4
might be required for touch cell function. However, eat-4 animals are
sensitive to gentle touch to the body, indicating that the touch cell
circuits in eat-4 animals are able to mediate the touch response. In
adults the anterior touch response is mediated by two neural pathways
that function redundantly (Chalfie et al., J. Neurosci 5: 956). One
pathway depends upon the gap junctions between ALM and AVD. The second
pathway depends upon the outputs of AVM which makes chemical synapses
to AVB, BDU, PVC and gap junctions to AVD (White et al.,
Philos. Trans. R. Soc. Lond. B314:1). Chalfie et al. reported that
adults lacking either the AVD or the AVM neurons remained touch
sensitive but that animals lacking both classes of neurons were touch
insensitive. This result suggests that either the gap junction pathway
from ALM to AVD or the chemical pathway from AVM can mediate anterior
touch sensitivity. This feature of the circuitry allowed us to test
the model that eat-4 was defective in chemical transmission from AVM.
We created animals in which anterior touch senstivity was mediated
only by the AVM-dependent pathway by killing AVD with a laser
microbeam. To confirm that we were successfully killing the
appropriate neurons, we first performed an experiment similar to that
of Chalfie et al., killing AVD, AVM or AVD and AVM in L2 animals and
measuring the touch response 2 days later by scoring the animals'
response to anterior touch. As shown below, we found that ablation of
AVD or AVM caused a decrease in the percentage of trials in which the
animal backed in response to touch. However, animals lacking both AVM
and AVD were more defective than animals lacking only one class of

      genotype/cells killed    # animals    #trials     % of touches
                                                        resulting in
(1)   N2/none                      23          157          96
(2)   N2/AVD                       10           66          62
(3)   N2/AVM                        8           55          76
(4)   N2/AVD+AVM                    7           47          23

We used the same approach to test the function of AVM in eat-4
animals. As shown below, mock ablated eat-4 animals were slightly less
sensitive to touch than N2, and eat-4 animals lacking AVD responded
very poorly.

      genotype/cells killed    # animals    #trials    % of touches
                                                       resulting in
(5)   eat-4(n2474)/none            8           42           86
(6)   eat-4(ky5)/none             13          100           86
(7)   eat-4(n2474)/AVD             8           42           19
(8)   eat-4(ky5)/AVD              11           84           24

These results can be summarized as follows: intact eat-4 mutants have
anterior touch sensitivity equivalent to AVM- wild type (compare 5&6
to 3), and AVD- eat-4 have anterior touch sensitivity equivalent to
AVM-AVD- (compare 7&8 to 4) wild type. That is, an eat-4 mutation has
the same effect as eliminating the chemical synapse-dependent touch
response pathway, consistent with the hypothesis that eat-4 is
necessary for chemical transmission from AVM.

We further tested the effect of an avr-15 mutation on the AVM pathway
since avr-15, like eat-4, is required for normal M3 function (Avery,
et al., WBG 13.4:72). As shown below, we found that avr-15(ad1051)
shows AVM defects similar to those seen in eat-4, but slightly weaker.

      genotype/cells killed    # animals    #trials    % of touches
                                                       resulting in
(9)   avr-15(ad1051)/none          12          89           98
(10)  avr-15(ad1051)/AVD           12          87           31

Although eat-4 appears to express in both ALM and AVM, the laser
ablation experiments argue that eat-4 specifically affects the
chemical transmission. Since avr-15 has been implicated in the
glutamatergic transmission in the pharynx and since avr-15 is required
for AVM synaptic transmission, it seems likely that AVM is also
glutamatergic. Taken together, our results suggest that eat-4 may
indeed be necessary and possibly specific for glutamatergic neuron

We thank Marty Chalfie and Andy Fire for providing GFP and lacZ vectors.