[
Worm Breeder's Gazette,
1992]
We have recorded single channel currents from membrane patches of nerve ring neurons. To expose these neurons, we dissected pharynxes from N2 L4 worms (LA WBG, this issue) glass cover slips precoated with CELL-TAC. The dissection was done in egg salt buffer (113 mM NaCl, 40 mM KCI, 3.4 mM CaCl2 ,3.4 mM MgCl2 ,10 mM HEPES pH 7.2) by placing a no. 15 surgical blade on the worm just behind the terminal bulb of the pharynx and pressing downward. We dissected up to 50 worms per cover slip and got several good ones (most of the dissections are unsuccessful because of the activity of the worms). In a good dissection, the cuticle surrounding the pharynx shrinks up and exposes the pharyngeal terminal bulb and isthmus and the cell bodies of the nerve ring neurons. These neurons are seen with DIC or phase contrast optics and resemble a cluster of grapes. We were able to get gigaohm seals in each of our first 4 tries using 2-10 megaohms resistance pipettes filled with egg salts. While the first 3 patches contained multiple currents, the fourth patch appeared to contain only a single channel. We recorded current amplitudes over a range of command voltages in order to determine the unitary conductance of this channel. Because the current-voltage ratio was constant over the range measured, we conclude that our calculated value for the conductance, 20 picosiemens, is constant as governed by Ohm's law. The reversal potential is not informative because we do not know the resting potential of the cell. Next, we excised the patch and recorded in the inside-out cell-free patch configuration. Because the ion concentrations on the two sides of the membrane were symmetrical in this mode, all currents reversed at a command potential of zero. At the suggestion of Andy Blatz, we attempted to identify the ions that carry the current by adding 1 M KCl to the bath, producing an electrochemical driving force for both ions to flow into the pipette at a command potential of zero. Since the resulting current flowed into the pipette, we conclude that the current was carried by potassium. In summary, we have developed a dissection that is readily amenable to electrophysiological recording from nerve ring neurons. We have preliminarily characterized a cation selective channel of 20 pS conductance. We are now trying to identify the neuron from which we obtain the record by prelabeling amphid sensory neurons with a fluorescent probe in wild-type and mutant worms. [See Figure 1]
[
Worm Breeder's Gazette,
1994]
Cytology of degenerin-induced cell death in the PVM neuron David H. Hall, Guoqiang Gu+, Lei Gong#, Monica Driscoll#, and Martin Chalfie+, * Dept. Neuroscience, Albert Einstein College of Medicine, Bronx, N.Y. 10461 + Dept. Biological Sciences, Columbia University, New York, N.Y. 10027 # Dept. Molecular Biology and Biochemistry, Rutgers University, Piscataway, N.J. 08855
[
Worm Breeder's Gazette,
1992]
Pharyngeal muscle is capable of rhythmical contractions (pumps) without a nervous system (Avery and Horvitz Neuron 3:473). The function of pharyngeal neurons is to modulate the rate and rhythm of pharyngeal pumping in response to the environment. How does the environment control pharyngeal pumping rate? We know at least one environmental condition that affects pumping: food. When worms are transferred from an environment without food to one with food, their rate of pharyngeal pumping increases. We also know at least one of the neurotransmitters that regulate pumping rate: serotonin. External application of serotonin stimulates pumping. Finally, we know that there is a serotonergic neuron(s) active in the absence of food. Imipramine, a serotonin-uptake inhibitor, increases the rate of pumping in the absence of food in wild-type but not in the serotonin-deficient mutant,
cat-4 (Avery and Horvitz J Exp Zoo 253:263). What is the endogenous source of serotonin that is enhanced by imipramine? There are two plausible candidate sources for this serotonin: RIH and the NSMs. NSM is the only pharyngeal neuron type known to contain serotonin, and RIH is an extrapharyngeal serotonergic neuron with indirect inputs onto the pharyngeal neuron types I1 and M1 .To see if one or more of these neurons is necessary for the basal rate of pumping and for the stimulatory effect of imipramine, we killed the three pharyngeal neuron types I1 ,M1 ,and NSM. Indeed, in NSM-, I1 -,M1 -worms, basal pumping is almost completely abolished and there is no stimulatory effect to imipramine (20 mg/ml, [See Figure 2]). We then killed subsets of these three neurons. Neither NSM nor M1 kills alone had a significant effect on the basal rate of pumping or on imipramine stimulation. Killing I1 alone or together with NSM resulted in an obvious phenotype. I1 -worms seldom pumped in the absence of food with or without imipramine [See Figure 2]. Figure 1 represents our current view of the rate-control circuit. I1 has output onto MC, a sensorimotor neuron type that is known to be necessary for the increased pumping rate in response to food. Hence, it is likely that I1 is excitatory onto MC which, in turn, is excitatory onto pharyngeal muscle. I1 is the major recipient of input from the extrapharyngeal nervous system and has an indirect connection with RIH, a serotonergic neuron. Therefore, we suggest that in the absence of food, RIH activity dictates the rate of pumping via I1 .Imipramine enhances the effect of serotonin secreted by RIH only in the presence of I1 . Surprisingly, exogenous serotonin stimulates pumping in worms that lack I1 [See Figure 3] Is serotonin bypassing I1 and acting directly onto MC to stimulate pumping? Consistent with this possibility, we find that the response of MC- worms to exogenous serotonin is attenuated [See Figure 3]. An alternative possibility is that serotonin does not affect MC directly but that MC is partially necessary for the response to serotonin. At the moment, we cannot distinguish between these possibilities. Serotonin may have a small MC-independent effect on pumping rate, in addition to the larger MC-dependent effect [See Figure 3]. We propose that there are two neural circuits that control the pumping rate, a pharyngeal one and an extrapharyngeal one [See Figure 1]. The pharyngeal circuit exerts the main control on rate. In response to food, pharyngeal neurons can increase the pumping rate up to ten-fold. However, they can only do so if they taste the food in the pharyngeal lumen. In order to get food into the lumen, a basal rate of pumping is necessary. This rate is controlled by input from the extrapharyngeal nervous system. According to this scheme, the pair of NSM neurons secrete serotonin only when activated by bacteria in the lumen. Therefore, NSM- worms are not different from intact worms in basal pumping or in the response to imipramine. In the presence of food, NSM could be redundant with other pharyngeal neurons. In addition to NSM, I1 and MC and six other neuron types have endings that could be mechanosensory (Albertson and Thomson Phil Trans R Soc 257: 299). Therefore, NSM- worms respond normally to food (Avery and Horvitz Neuron 3: 473).
[
Worm Breeder's Gazette,
1992]
The
unc-5 gene encodes a novel cell adhesion receptor of the immunoglobulin super family which is required to guide dorsal migrations of neuron growth cones on the epidermis (UNC-5 ;Neuron 4:61; Leung-Hagesteinj et al.,submitted).
unc-5 has been shown genetically to require
unc-6 ,which encodes a putative epidermal guidance cue, for its guidance functions (Neuron 4:61). The neurons mostly affected by
unc-5 mutations are the motor neurons DA, DB, DD, AS, and VD.
unc-5 has been shown to act cell autonomously in these cells (WBG 12[1]:32). We wanted to test whether
unc-5 expression in neurons that do not normally grow dorsally on the epidermis would suffice to steer them in a dorsal direction The six touch receptor neurons normally extend axons ventrally or longitudinally on the epidermis. These trajectories are not affected by
unc-5 mutations. The mec- 7 tubulin gene promoter has been shown to express lacZ specifically in the touch neurons (WBG 11[3]:40; Hamelin et al., in press). A fusion gene was made with the
mec-7 promoter and the entire
unc-5 coding region. This
mec-7 -
unc-5 construct was injected in the germline of wild type animals, along with a mec- 7-lacZ construct (WBG 11[3]:40), and pRF4 (WBG 11[1]:18). Transgenic animals were stained with X-Gal to reveal their touch neuron axons. Virtually all detectable axons (376/378) were found to be dorsally re-directed. The cell bodies were often displaced, mostly dorsally, but sometimes anteriorly (AVM, ALMs, PLMs) or posteriorly (PVM). Most transgenics were also touch insensitive. If UNC-5 induces dorsal trajectories in the touch neurons by utilizing its normal guidance functions, then these abnormal trajectories should depend on UNC-6 .In a null mutant of
unc-6 carrying the
mec-7 -
unc-5 transgene, the touch neuron axons were no longer found to be misguided Together, these results indicate that UNC-5 can act cell-autonomously, and is sufficient to steer dorsally the growth cones of neurons that normally don't.
[
Worm Breeder's Gazette,
1987]
We have recently been attempting to assign functions to various neurons, many of which are located in the head ganglia near the nerve ring. We use a Laser Sciences Inc. nitrogen-pumped laser (described in WBG 9(2), 1986, p. 110) to kill specific neurons identified on the basis of position in L1 worms, then let the animals grow to adulthood and test behavior. In combination with the complete wiring diagram ( White et al., 1986), this method provides a powerful approach to understanding the function of the nervous system. We can envision at least four potential pitfalls with this approach. First, we may fail to eliminate functionally the neuron that is killed. Second, we may cause damage to neighboring cells or processes, especially those located near the site of the laser strike. Third, the cell that we identify on the basis of position might not invariably be the same cell; for example, it might occasionally be switched with some neighboring cell. Fourth, the cell in a particular position might not be correctly correlated with the wiring diagram. Of these pitfalls, we have collected some information about the first three. The fourth problem is more difficult to address, and largely depends on the assignments by Sulston et al. (1983). The evidence so far suggests that killing a neuron with the LSI laser results in functional elimination of the target cell. First, when we kill a neuron (by this we mean all members of a class of neurons) with a previously described function, we reliably obtain the expected phenotype for loss of the neuron's function (based on genetic studies and/or embryonic kills; Chalfie et al., 1986; M. Chalfie, C. Desai, personal communications). Such cells include AVA (backward Unc; 6), RIP (loss of Mec inhibition of pumping; 2), HSN (egg-laying defective; 3), PLM (tail Mec; 5), ALM (reduced head touch sensitivity; 3), and PVC (tail Mec; 3). (Parenthetical remarks after each neuron indicate the behavioral phenotype and the number of animals analyzed.) In other cases, we have observed a behavioral defect associated with killing a particular neuron. When we kill this neuron in more than one animal we consistently observe the same behavioral defect. These cells include ASH (12), ASJ (2), PVQ (3), and M4 (150). (For the functions of ASH, ASJ, and PVQ see the accompanying Thomas and Horvitz newsletter entry.) A related concern is how fast a killed neuron loses function. The best evidence for this time course is for the pharyngeal motorneuron M4. This neuron is required for peristalsis of the pharyngeal isthmus (Avery and Horvitz, WBG 9(2), 1986, p. 57), a function that can be assayed at any stage of development. There is a pronounced, but not always complete, deficit in M4 function 5 hours after laser killing in young L1 larvae, and M4 is invariably nonfunctional after 24 hours. Similarly, when PVC (3), AVA (6), or PLM (5) is killed during the early L1 stage, animals acquire the loss- of-neuron-function phenotype within 24 hours (the earliest time tested) . These results suggest that laser killing in the early L1 typically eliminates neuron function well before adulthood. We also have evidence that laser damage to cells or processes near the target cell is not generally a problem. Even when we kill neurons in the head ganglia, where neuron cell bodies and processes are closely packed, we do not observe effects on other behaviors. For example, no kill (other than AVA) has caused a backward Unc phenotype, characteristic of AVA animals. The most extensive analysis has been done for the cell ASH, which is required for normal osmotic avoidance ( see accompanying Thomas and Horvitz newsletter entry). Neurons with cell bodies that surround ASH on all sides (ADF (2), AWC (3), AUA (2), AIB (1), and ASE (3)) have been killed with no effect on osmotic avoidance. In addition, many neurons with a process that runs alongside the ASH process in the amphid bundle (ADL (2), AWC (3), and ASK (4)) or in the nerve ring (AIB (l) and ADF (2)) or both (ASE (3)) have been killed with no discernible effect on ASH function. These results should be taken with a grain of salt, since most C. elegans neurons (including AVA and ASH) are bilateral. Since probably only one of the pair need function for normal behavior, we might require damage of both cells to see a phenotype. However, this redundancy also works to our advantage, since only damage to both sides will produce spurious results. The same set of laser experiments show that, for several neurons in the head, position in the early L1 is sufficient to identify the same cell in different animals. These cells are RIP (2), ASH (12), ASJ (2), and AVA (6). For each of these cases, when the cells in these positions are killed in different animals they cause the same behavioral defects in the adult. We have also tested cells in the tail ganglia (PLM (5), PVC (3), and PVQ (3)) with the same result. This evidence indicates that the same functional cell type occupies the same position during the L1 stage in different animals. This rule may not apply to all neurons, but as more cells are assigned invariant positions, the potential variability of the remaining neurons becomes more restricted. Although we cannot directly address the final possible pitfall ( correlation of the killed neuron with the wiring diagram), we can point out that the behavioral phenotype observed for each kill fits well with the target cell's assigned connectivity. Finally, we offer some general comments on the usefulness of this method. The position of most neurons in the head ganglia and elsewhere can be learned fairly easily by careful inspection of a number of animals and frequent comparison with the excellent diagrams found in Sulston et al. (1983). Some neurons are easier to identify than others, and a few (around the posterior bulb of the pharynx) may not be possible to identify because of their variable positions (as noted in Sulston et al., 1983). However, the general impression one gets, after sufficient observation time to learn the patterns, is the remarkable reproducibility of the relative positions of most neurons. It is quite reasonable, for a cell one knows well, to kill a particular type of neuron and confirm the kill in more than 30 animals in one day.
[
Worm Breeder's Gazette,
1997]
We have begun reconstructing serially-sectioned embryos in order to understand how the nerve ring is formed, in particular how the orderly relationship between the GLRs, muscle arms, and axons is established. We have focused on two embryos: one at a stage before axons enter the nerve ring proper (350') and the other when the nerve ring contains many processes (430'). In both embryos, we have identified all of the non-neuronal cells outside the pharynx. And in the 430 minute embryo, we have identified all neurons except those in the anterior ganglion, primarily using Fig. 8 of Sulston et al., 1983. This later stage is useful as a reference for cellular relations in the relatively mature nerve ring and as an aid in identifying cells in the younger embryo. In the 350' embryo, the first axons are establishing the amphid commissures and the sublateral pathway. We are beginning to understand how head and neck muscles connect to the nerve ring. In mature worms, it appears as if muscles must extend long processes from their somas, which are next to the hypodermis, to the inner nerve ring (see White et. al., 1986). However, early in development, head and neck muscles directly surround the pharynx. During development then, muscle cell bodies themselves must migrate peripherally to the hypodermis leaving behind a process (arm) next to the pharynx. In addition, the processes left behind from the neck muscles must grow anteriorly to reach the nerve ring, since the neck muscles are posterior to the GLRs in these young embryos. Therefore, the formation of muscle arm connections to the nerve ring may be quite different from the extension of muscle arms from body wall muscles to the ventral and dorsal nerve cords. The relation between muscles and the developing amphid commissural pathway is also interesting (see diagram below). In the 350' embryo, muscles do not separate lateral ganglia from the ventral ganglion where the commissures are forming. Instead muscles are separated from the hypodermis by a large neuron (spanning 90 thin sections) forming a substrate for early sublateral and amphid commissural axons (neuron 122 on LHS and neuron 113 on RHS in section #402). Anterior (e.g. secn #386) and posterior (e.g. secn# 522) to these nascent pathays, muscles are attached to the hypodermis. We do not know whether earlier in development, muscles were in contact with the hypodermis along the anterior-posterior length and the neuron migrated between the tissues or whether the muscles differentially set down upon the hypodermis. Our tentative identification of the neuron that provides the substrate for the early commissures is SMBD (neurons 122
and113 in digram below). We are beginning to process more embryos of intermediate stages to fill in the developmental gaps, and are hoping that by working our way back from the older 430 ' embryo, we can become more certain of cell identifications.