genetic analysis of chemosensory pathway

Proc, Indian Acad. Sci••Vol. 87B (Experimental Biology-J), No.7, July 1978, pp. 147-160,@ printed in India

Genetic analysis of chemosensory pathway *

VERONICA RODRIGUES and 0 SIDDIQIMolecular Biology Unit, Tata Institute of Fundamental Research, Bombay 400 005

MS received 15 December 1977; revised 24 June 1978

Abstract. Some olfactory and gustatory mutants of Drosophila melanogaster are described. The olfmutants are insensitive to the repellent, benzaldehyde or the attractantethyl acetate or simultaneously to both. The gust mutants are unable to taste quinineor NaCI or sucrose. Electrophysiological tests show that one of the sugar non-tastershas an altered primary chemoreceptor response to sucrose.

Keywords. Neurogenetics; behavioural genetics; chemosensory mutants; olfaction;gustation.

I. Introduction

Behavioural mutants of Drosphila melanogaster provide rich source material for investigating the organisation and development of the nervous system (Benzer 1971, 1973).Among the many neurological mutants that exist, some are defective in primaryneurophysiological functions such as impulse conduction or synaptic transmission(Siddiqi and Benzer 1972, 1976; Ikeda et a11976;Jan et al 1977); others carry lesionsin sense organs or exhibit abnormalities of complex behaviour controlled by centralnervous system (Benzer 1973).

Thanks to the pioneering work of Dethier, Hodgson, Morita and their associateson the larger flies, a great deal is known about the gustatory behaviour and chemosensory physiology of dipterans (Dethier 1955, 1968; Hodgson 1955; Morita andYamashita 1959; Tateda and Morita 1959). Kikuchi (1973) found a mutant ofD. melanogaster whose olfactory preferences for acetic acid and some other chemicals were altered; Falk and Atidia (1975) have described taste mutants that are notrepelled by NaCl. Tompkins and Sanders (1977a, b) have reported a numberof Xvlinked gustatory mutants defective in responses to NaCI and quinine in thelarvae and imago of Drosophila. A combination of genetic and physiologicalmethods, therefore, seems to offer an attractive approach to the study of chemosensory mechanisms. We describe here some recent experiments in our laboratoryon a set of chemosensory mutants of D. melanogaster unable to taste or smell aselected set of attractants or repellents.

*A lecture given at the Accademia Dei Lincei, Rome on ]st November, 1977

147P. (B)-l

148 Veronica Rodrigues and O·Siddiqi

2. Methods

2.1. Isolation ofmutants

Canton Special (CS) males werefed with 0'025M ethylmethyl sulphonate in 1%sucrosefor 24 h and mated to attached X tester females. Pure lines ofmutagenised X chromosome were set up with single F1 males over tester females. These lines were individually screened for behavioural deficits. Putative mutants were retested for 2subsequent generations. The 13 olfactory and 6 gustatory mutants listed in tables1 and 2 were found among 1054 mutagenised lines examined. The yield of mutantsin this experiment was unexpectedly high and the status ofsome ofthe isolates remainsto be confirmed. A fuller genetic study of the isolates is under 'Way.

2.2. Behavioural tests

The olfactory responses of the flies were measured in an olfactometer designed by us(see section 3). The exact conditions of measurement are somewhat different forattractants and repellents and will be described in greater detail elsewhere. Gustatory thresholds were measured by the proboscis extension test developed by Dethieret al (1955, 1968) for larger flies.

2.3. Electrophysiology

The methods of recording the electrical responses of primary chemoreceptor units inflies have been described by Hodgson et al (1955). The labellar hairs were excitedat the tip by a solution of the appropriate stimulant in a pipette of 50 J-t tip diameterwhich also served as a recording electrode. The signals were led through a highimpedance amplifier to the oscilloscope and permanently recorded by an oscillographic camera.

3. Results

3.1. Chemosensory behaviour of Drosophila

The olfactoryresponses of D. melanogaster can beconveniently measured in an olfactometer, several variants of which have been described (Hirsch 1965; Fuyama 1974).The apparatus designed by us is shown in figure 1. It is a simple V-maze made ofglass tubing. The three arms of the Yare connected by adapters to glass vials. Airis withdrawn at the junction causing a controlled and symmetric inflow into the uppervials, one of which is connected to the source of smell. About 100 flies are startedat the bottom of the lower tube. Being negatively geotatic, the fliesclimb up and aredistributed in the two-vials. At the end of the' rUD, the flies in the upper vials arecounted and the difference between the smell side (S)and the control side (C) is usedto measure the olfactory response. The measure of response is the response index(RI) defined as S-C;S+C. RI can vary between +1 and -1. The former represents total attraction and the latter total repulsion; zero on this scale corresponds

Genetic analysis of chemosensory pathway 149

Figure 1. Drosophila olfactometer: Flies distribute in the upper vials in response toa smell cue. The excess of flies on the smell side. divided by the total number in thetwo vials gives the response index (RI). Positive values of RI denote attraction andnegative values denote repulsion.


to indifference. Responses are measured against varying concentrations of a substance and a plot of RI against the logarithm of concentration yields curves of thetype shown in figure 2. These curves describe the olfactory behaviour of a population of flies against a given attractant or repellent. From such data one can easilydetermine the threshold for response as well as the concentration at which theresponse saturates. The strongest available concentration of the stimulant istaken as 1.

The responses of wild type strain Canton Special (CS) to a few stimulants are givenin figure 2. Well-fermented Drosophila medium is a strong attractant with a concentration threshold of 10-7 and a saturating response index of +0'8. Ethyl acetateacts as an attractant at lower concentrations but above 10-7 it becomes a repellent.Formaldehyde and methyl benzoate similarly attract weakly at very low concentrations but at higher concentrations become repellents. Benzaldehyde and acetic acidare strong repellents, the former being the strongest of the repellents we have testedso far.

The complex responses to ethyl acetate, formaldehyde or methyl benzoate in thecurves of figure 2 do not necessarily mean a change in the fly's preference with changing concentrations of these chemicals per se. It is possible that a contaminatingchemical is present which can be detected at lower dilutions of the test chemical.Ethyl acetate, for instance, is likely to be hydrolysed to acetic acid which is a repellent.In the experiments described here we are primarily concerned with distinguishing

OLFACTORY RESPONSES OF WILO TYPE

DROSOPHiLA

+I·Or----------, +1·0...-----------, +- 10 r---------------,FOOD

o·01---,r----.6----------1

0·6

004

0·2

0·8

-0-6

-0·4

-0,2

-0·8

METHYLBENZOATE

-0,4

0'0

-6 -4 -2

-O·B

-0·2

-0,6

- 8 -6 -4 - 2

-0-6

-0,8

oe

Q) 0enc&. -0'2enQ)

a:: -0-4

Log concentration

Figure 2. Olfactory responses of normal Drosophila to attractants and. repellents :The strongest available concentration of the stimulant is taken to be 1. This 1S su~cessively diluted in steps of 10. Standard deviations are obtained from repeated independent trials.

152 Veronica Rodrigues and 0 Siddiqi

Table 1. Responses of olfactory mutants of D. melanogaster

Strain Response index Response indexfor benzal-for ethyl acetate dehyde

es +0'81 -0,85olfx1 +0'76 -0'02olfx6 +0'80 -0'10OlfX6 +0'81 -0'12Olfxll +0'84 -0'09olfx7 +0'71 -0'48Olfx13 +0'80 -0,39Olfx4* +0'85 +0'02Olfx9 +0'15 -0'06ol/x'!. +0'18 -0'80olfx8 +0'07 -0,83olfx6 -0'20 -0'89olfx1O +0'40 -0'71Olfx1 2 +0'31 -0,75

*X4 is a temperature-sensitive developmental mutant. The flies reared at 28°C exhibit olf phenotype but when grown at 200 e they are normal.

the smell-defective olf mutants from the normal flies and the olfactory responsecurves are to be taken merely as descriptions of the average behaviour of a strainunder specified conditions.

For measuring taste reactions we use the method developed by Dethier (1955) forlarger flies. If the tip of a chemosensory hair, either on the tarsus or the labellum,is touched with a droplet of an attractant solution, the fly extends its proboscis.The concentration threshold (Gl/2) of the attractant is the molar concentration of asubstance which elicits an extension response in 50%of the trials. Sucrose has athreshold of 2 X 10-oM. Detection of repellents by chemosensory hairs can bemeasured by estimating the concentration of the substance required to inhibit proboscis extension against a prescribed concentration of attractant. NaCI (5X lO-lM)for instance, inhibits the response to IO-3M sucrose in wild type Drosophila (table 2).Sincethirsty fliesextend proboscis to water, these tests must be carried out on watersatiated flies.

Table 2. Responses of gustatory mutants of D. melanogaster

Response index InhibitoryStrain threshold forfor quinine

NaClt (M)

CS -0-95 5 x 10-1

gust x 1 -0'90 t 4 x 10-1

gust x4 -0,89 t 5 x 10-1

gustxf> -0,91 >1'0gust x2 -0'12 t >1'0*gustx3 -0'04 2 x to-I*gustx 6 -0'11 5 x 10-1

Thresholdfor sucrose

(M)

2 X 10-6

>2'0>2'08 x 10-6

>2'01 x 10-4

9 X 10-4

*Reared at zs-cteoncentration at which thirsty flies rejected 10-3M sucrose.

Genetic analysis ofchemosensory pathway

3.2. Olfactory mutants

153

The responses of 13 olfactory mutants (olfXl to olfxta) are summarised in table 1.Although these mutants were at first isolated using food as an attractant and benzaldehyde as a repellent, it turned out that those unable to smell food were also indifferentto ethyl acetate. In subsequent characterizations, ethyl acetate was employed as anattractant and benzaldehyde as a repellent.

The olfmutants fall into 4 different phenotypic groups. The first group containing6 alleles (xl, x6, x8, xl l, x7 and x13) is insensitive to benzaldehyde but respondsnormally to ethyl acetate (figure 3). Conversely, the last five (x2, x3, x5, xl0 andx12) are insensitive to ethyl acetate but respond normally to benzaldehyde(figure 4). The lone member of the third group OlfX9 is indifferent to bothethyl acetate and benzaldehyde. olfxtl is a conditional mutant; the flies reared at20°C are normal but, when reared at 28°C, they do not respond to benzaldehyde(figure 5).

The olfmutants are, thus, either singly insensitive to an attractant or a repellentor doubly insensitive to both. The latter class might carry a lesion in a more centralstep in the olfactory pathway than the former. The conditional mutant oljX4 isspecially interesting from a developmental point of view. The dependence of its

)(1Olfactory response of off

1·0Ethyl acetate

0·8

0·6

0.4

xQ) 0·2'Q

.sQ) 0'"c

~'H"0

g. -0·2Q)

a:

-0.4 \-0.6

oJ-0.8 ~

Benzaldehyde-1·0

-8 -6 -4 -2 0Log concentration

Figure 3. A benzaldehyde-insensitive mutant: ol/xl responds to ethyl acetate but itis repeJled weakly by benzaldehyde. 6. and 0 normal flies; .. and ., mutants.


Olfactory response of olf x 10

-1·0

0.8

0.6

0.4

x-8 0.2£•fit&:: -0·2C[

-0.4

-0.6

-8 -6 -4 -2 0Log concentrotion

Figure 4. A mutant unable to detect food and ethyl acetate: olfx10 is repelled bybenzaldehyde just like the normal flies but cannot detect attractants. b. and o.normal flies; • and •• mutant.

phenotype on temperature ofgrowth suggests that, at the non-permissive temperature,the normal development of the olfactory network is blocked.

3.3. Gustatory mutants

The six mutants defective in taste belong to four groups; their responses to sucrose,NaCl and quinine are given in table 2. Response to quinine was measured in aY-maze with one arm of the Ytube painted with quinine sulphate.

gust X1 and gustx4. are allelic mutants which can detect quinine and NaCI but do notrespond to sucrose. In sucrose non-tasters, inhibition threshold of NaCI was measured in thirsty flies which respond to water alone. gustX5 is not repelled by NaClbut responds to quinine and sucrose normaIIy. gustX5 can detect neither quinine norsalt nor sucrose, it seems to be entirely taste blind. The last two mutants gustXS andgustX6 are insensitive to quinine but are repelled by NaCI. These two are also developmentally temperature-sensitive, that is to say, the mutant phenotype is evident onlyif the flies are reared at the non-permissive temperature (table 3).


.4OLFACTO'h RESPONSE OF 011

GROW!'t AT 20·( GROWN AT 28·c

.,·O,------------r-----------,

BENZALDEHYDE

ETHYL ACETATE

0·8

-0-8

0·4

-0,6

-0·4

)(

~ 0·2c

QJenco0.

:c -0· 2a::

-8 -6

Figure S. A conditional behavioural mutant: olfx4 flies grown at 200 e respond normally to attractant and repellent. When grown at 28°C they smell ethyl acetate butnot benzaldehyde, 6. and O. normal flies; A and •• mutant.

Table 3. Responses of conditional developmental gust mutants to quinine

Response index for quininewhen flies were grown at

200e 28°e

gust x 3

gustX 8

-0,95

-0,86

-0-15

-0-09

In all of the gustatory mutants examined so far, the responses to labetlar and tarsalstimulation are simultaneously deficient.

3.4. Receptor physiology of gustatory mutants

One of the principal advantages of working with the insect chemosensory system isthe fact that the primary response of sensory neurons can be recorded from the


sensillum. Using electrophysiological methods first developed by Hodgson et al(1955), we are currently examining the chemoreceptors of the gustatory mutants.

The response of the normallabellar hair to increasing concentrations of NaCI isshown in figure 6. At extremely low concentrations of NaCI, the chemoreceptorresponse consists of small, about 0·5 millivolt spikes which are apparently generatedby a neuron sensitive to water (W cell). As NaCI concentration is increased to 0·1Mthe 0·5 millivolt spikes are depressed and somewhat larger spikes 0·9 millivolt inamplitude appear (L1 cell). At yet higher concentrations of NaCl, the W spikes arecompletely inhibited and a second salt-sensitive cell (L2) with a spike amplitude of1·9 millivolt begins to fire. Finally with 1M NaCI, the LI response is also inhibitedand L2 predominates. The fact that three discrete classes of spikes are observedsuggests that we are dealing with three different sensory neurons. The firing frequency of W, Ll and L2, in response to increasing concentrations of NaCI is plottedin figure 7.

_.~0·001 M No CL

20cs

""~ 10

~20

O'IMNaCI U

(1) 10en'-Q)

a.

~,.~enOJ 0·5 M NoeL

..x::

.0. 20

""~U)

10

1·0 M NoeL

~20

1M

NaCl 10

Amplitude ( rnv l

Figure 6. Electrophysiological responses of labellar receptors of Drosophilato NaC!.The kymograph tracings on the left show sample responses, whose amplitude distributions are given on the right. The prominent deflections at the beginning of each tracemark the contact of the chemoreceptor tip with the salt solution contained in a micropipette which also serves as a recording electrode,

Genetic analysis of chemosensory pathway

Effect of Noel on Wand L cells

157

80

ol».x'Q.m

20

L2

/~

Figure 7. The response of chemosensory neurons to NaCl: L2 responds to high concentrations of NaCI which inhibit Wand Lt.

The response of labellar hair to sucrose is presented in figure 8. Only two classesof spikes are observed, spikes of 0·5 millivolt amplitude (W) which are inhibited bysucrose and 0·8 millivolt spikes that increase with increasing sucrose (8 cell).

The chemoreceptor responses of Drosophila to NaCl and sucrose closely resemblethe responses of other dipterans (Dethier 1968; Dethier and Goldrich-Rachman1976). Here too, as in the larger flies, there seem to be four different neurons present;the W cell that is excited by water and inhibited by NaCI and sucrose, the S cell thatis excited by sucrose, and the two L cells that measure increasing salt. The inferencethat the discrete classes of spike amplitudes come from different neurons is stronglyreinforced by the distinctive adaptation characteristics of the spikes (figure 9).

We have examined the chemoreceptor responses of some of the gust mutants toNaCI and sucrose. The mutant, gustX1 has a greatly reduced 8 cell response tosucrose. Simultaneously the W cell in this mutant has become insensitive to inhibition by sucrose (figure 10). The response of the two L cells as well as the W cell toNaCI remains unchanged. It is likely that, in this mutant, a surface receptor forsucrose common to Wand S cells has been affected.

4. Conclusions

The experiments described here are still at an early stage. It is, nevertheless, evidentthat the olfactory and gustatory nutants of Drosophila provide a convenient way ofexploring the organisation of the chemosensory pathway. These mutations blockdifferent steps in the sequential processing and transfer of information from peripheral receptors to central parts of the nervous system.


~:~;~105

M SUCROSE20 CS

to

103 M SUCROSECS

~20

103M U

SUCROSE: OJ 10cs

~(/)

1-OJo,

'0-2 M SUCROSE

'O'M~(/) CSQ)

20~

0.

~RO::~Cf)

10

~'M ~10-2 M SUCROSE

20gUlf Jl 23

::;.••o:!~10

0-2 0·6 1·0 1'4

Amplitude (mV)

Figure 8. The responses of labellar chemoreceptors to sucrose: In the normal fly(upper three responses), increasing concentrations of sucrose inhibit the W cell andstimulate the S cell. In the mutant gust xl the S cell fails to respond to to- 11M sucroseand the W cell is not inhibited.

o 2Time after stimulation (sec I

z

Figure 9. Adaptation characteristics of chemosensory neurons: Wand S ceIlsadaptrapidly to continuous stimulation, Ll adapts after an initial lag while L2 shows leastadaptation.


Response of Wand 5 cellsto sucrose

XI

h9ust

-----/rw

o -8 -4 -2 0Log molar concentration

sucrose

40

30 rHi/

u~vt~

C» 20e,vtQ).¥·a(/)

10

Figure 10. Sugar response of Wand S cells in normal and mutant Drosophila: Increasing concentrations of sucrose in normal flies stimulate the S cell CA) and inhibit theW cell (0). In the mutant gust xl neither S nor W cells respond to sucrose.

Conditional behavioural mutations affecting normal development of sensory-motorcapabilities are a particularly useful tool for analysing the ontogeny of neuralcircuits. The approach is not restricted to the chemosensory pathway alone. Themany possible applications of the powerful technique of conditional lesions to thedevelopmental genetics of nervous system, with the help of such mutants, remain tobe explored.

Acknowledgements

We thank Dr L Tompkins and Dr T G Sanders for informing us about their recentunpublished work on gustatory mutants of Drosophila.

References

Benzer S 1971 J. Am. Med. Assoc. 118 1010Benzer S 1973 Sci. Am. December p. 24Dethier V G 1955 Q. Rev. Biol. 30 348


Dethier V G 1968 Science 161 389Dethier V G and Goldrich-Rachman N 1976 Proc, Natl. Acad. Sci. USA 73 3315F'alk Rand Atidia J 1975 Nature (London) 254 325Fuyama Y 1974 Drosophila In/ormation Service 51 142Hodgson E S 1955 Q. Rev. BioI. 30 331Hodgson E S, Lettvin J Y and Roeder D K 1955 Science 122417Hirsch J 1965 in Readings in Animal Behaviour ed. T E McGill (Holt Rinehart and Winston)Ikeda K, Ozawa Sand Hagiwara S 1976 Nature (London) 259 289Jan Y N, Jan Y and Dennis M J 1977Proc. Roy. Soc. (London) B198 87Kikuchi T 1973 Nature (London) 243 36Morita H and Yamashita S 1959 Science 130 922Siddiqi 0 and Benzer S 1972 Caltech. Biol, Ann. Rep. p. 38Siddiqi 0 and Benzer S 1976Proc. Natl. Acad. Sci. USA 733253Tateda H and Morita H 1959J. Cell. Camp. Physiol. 54 171Tompkins L and Sanders T G 1977a Genetics 86 Suppl, S64Tompkins L and Sanders T G 1977b Genetics 86 Suppl, S63

genetic analysis of chemosensory pathway

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