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The Journal of Neuroscience, November 1, 2002, 22(21):9490-9501
Functional Dissection of Neuroanatomical Loci Regulating Ethanol
Sensitivity in Drosophila
Aylin R.
Rodan1, 3,
John
A.
Kiger Jr4, and
Ulrike
Heberlein1, 2, 3
1 Department of Anatomy and Programs in
2 Neuroscience and 3 Biological Sciences,
University of California, San Francisco, California 94143-0452, and
4 Department of Molecular and Cellular Biology, University
of California, Davis, California 95616
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ABSTRACT |
Ethanol has complex but similar effects on behavior in mammals and
the fruit fly Drosophila melanogaster. In addition,
genetic and pharmacological approaches have implicated the cAMP pathway in the regulation of ethanol-induced behaviors in both flies and rodents. Here we examine the neuroanatomical loci that modulate ethanol
sensitivity in Drosophila by targeting the expression of
an inhibitor of cAMP-dependent protein kinase (PKA) to specific regions
in the fly's brain. Expression of the inhibitor in most brain regions
or in muscle has no effect on behavior. In contrast, inhibition of PKA
in a relatively small number of cells, possibly neurosecretory cells,
in the fly's brain is sufficient to decrease sensitivity to the
incoordinating effects of ethanol. Additional brain areas are, however,
also involved. The mushroom bodies, brain structures where cAMP
signaling is required for olfactory classical conditioning, are
dispensable for the regulation of ethanol sensitivity. Finally,
different behavioral effects of ethanol, motor incoordination and
sedation, appear to be regulated by PKA function in distinct brain
regions. We conclude that the regulation of ethanol-induced behaviors
by PKA involves complex interactions among groups of cells that mediate
either increased or reduced sensitivity to the acute intoxicating
effects of ethanol.
Key words:
Drosophila; ethanol; PKA; neuroanatomy; mushroom bodies; postural control; locomotion
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INTRODUCTION |
Alcohol is among the most widely
abused drugs in the world, yet the mechanisms by which it acts are only
partially understood. Both rodents and the fruit fly Drosophila
melanogaster show behavioral responses to acute ethanol exposure
that are remarkably similar to human behaviors. Increasing doses of
ethanol elicit hyperactivity, then ataxia or incoordination, and
finally sedation (Singh and Heberlein, 2000
; Parr et al., 2001).
Importantly, some of the mechanisms that regulate these behavioral
responses also appear to be conserved. For example, genetic and
pharmacological manipulations that disrupt dopaminergic systems reduce
ethanol-induced locomotor activation in both rodents (Phillips and
Shen, 1996) and flies (Bainton et al., 2000).
Although ethanol does not act through a specific receptor, it affects
the function of certain cell surface proteins, including several ion
channels (Harris, 1999). In addition, some intracellular signaling
pathways, such as the cAMP pathway, are also affected by ethanol
(Diamond and Gordon, 1997; Tabakoff and Hoffman, 1998). A genetic
screen for Drosophila mutants with increased ethanol sensitivity identified amnesiac (Moore et al., 1998), a gene
encoding a putative neuropeptide believed to activate the cAMP pathway (Feany and Quinn, 1995). Consistent with this, flies with mutations in
the calcium/calmodulin-sensitive adenylyl cyclase rutabaga show increased ethanol sensitivity. In contrast, a mutation in the
pka-RII gene, encoding a regulatory subunit of
cAMP-dependent protein kinase (PKA), causes decreased ethanol
sensitivity (Park et al., 2000). Genetic manipulations of the cAMP
pathway in mice have been shown recently to alter ethanol sensitivity
as well (Thiele et al., 2000; Wand et al., 2001).
A complete understanding of the mechanisms by which ethanol alters
behavior requires knowledge of not only the molecules, but also the
neuronal circuits that mediate these effects. In Drosophila,
specific groups of neurons can be manipulated using the GAL4/UAS binary
expression system (Brand and Perrimon, 1993). This targeted expression
approach has been used to map neuroanatomical loci underlying behaviors
such as learning and memory (Connolly et al., 1996; Zars et al.,
2000a,b), courtship behavior (Ferveur et al., 1995; O'Dell et al.,
1995; Joiner and Griffith, 1999), and locomotion (Martin et al., 1999;
Gatti et al., 2000). In addition, chemical ablation of the mushroom
bodies (MBs), prominent central brain structures, has demonstrated
their importance in olfactory and courtship conditioning (de Belle and
Heisenberg, 1994; McBride et al., 1999).
We used the GAL4/UAS system to drive expression of a transgene that
inhibits PKA activity in restricted brain regions and measured the
flies' sensitivity to ethanol. We find that inhibition of PKA in
discrete brain regions alters the flies' sensitivity to the acute
intoxicating effects of ethanol. We postulate that normal ethanol
responsiveness is achieved by a complex balance between loci that
increase and reduce the flies' sensitivity to the incoordinating
effects of ethanol. In addition, different brain regions seem to
regulate distinct aspects of intoxication, such as postural control and
sedation. Chemical ablation of the mushroom bodies had no effect,
suggesting that the mechanisms that regulate ethanol sensitivity and
olfactory conditioning, although sharing molecular components, rely on
separable neural structures for their manifestation.
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MATERIALS AND METHODS |
Drosophila stocks and genetics. P[GAL4] lines 201Y
(chromosome II), c522 (III), c107 (I), c747 (II), and c290 (II) as well as additional P[GAL4] lines were obtained from K. Kaiser
(University of Glasgow, Scotland, UK) (Yang et al., 1995; Manseau et
al., 1997). 3A4 and P[GAL4]MHC82 (III)
were obtained from G. Davis (University of California, San Francisco,
CA); MHC82 contains the myosin heavy chain promoter fused to GAL4
(Davis et al., 1998). Other P[GAL4] lines were obtained from L. Griffith (Brandeis University, Waltham, MA; MJ lines; see below), C. O'Kane (University of Cambridge, Cambridge, UK; OK lines; see below),
and K. Ito (National Institute for Basic Biology, Okazaki, Japan).
hsGAL4 flies were obtained from the Bloomington Stock Center at Indiana
University (Bloomington, IN). UAS-PKAinh
(III) (also called BDK33) flies were obtained from D. Kalderon (Columbia University, New York, NY). These flies carry a transgene coding for the Drosophila type I regulatory subunit of PKA
with mutated cAMP-binding sites: Gly-196 and -321 were replaced by Glu
and Asp, respectively (Li et al., 1995; D. Kalderon, personal communication). The UAS-PKAm-inh
transgene (II) (Kiger and O'Shea, 2001) contains, in addition to the
mutations carried by UAS-PKAinh, mutations
in Arg-91 and -92, which were replaced with Gly to abolish binding to
the PKA catalytic subunit. The UAS-PKAc transgene (II) (Kiger et al.,
1999) encodes a FLAG-tagged Drosophila PKA catalytic
subunit. All lines are homozygous viable but were used as heterozygotes
or hemizygotes in behavioral assays. Lines used in behavioral
experiments (with the exception of
P[GAL4]MHC82) were outcrossed for five
generations to a w1118 stock isogenic for
chromosomes II and III. Flies were raised on standard cornmeal and
molasses food at 25°C and 70% relative humidity in constant light.
For behavioral testing, flies carrying both the P[GAL4] and UAS
insertions were generated by crossing P[GAL4] virgin females to UAS
males. As controls, P[GAL4] or UAS heterozygotes or hemizygotes were
generated by crossing males to virgin females carrying attached X
chromosomes in the w1118 genetic
background. All experiments were performed with 2- to 4-d-old males,
~110 for the inebriometer and 20 for the locomotor tracking system.
All genotypes were tested on multiple days.
P[GAL4] screen. Fifty-nine P[GAL4] lines with diverse
expression patterns in the brain were initially screened by crossing to
UAS-PKAinh. Of these, 27 were not tested
behaviorally because of lethality, low viability, or other defects,
such as unexpanded wings. The remaining 32 lines were tested in the
inebriometer in the presence of
UAS-PKAinh. Lines with increased mean
elution time (MET) in the presence of
UAS-PKAinh as well as a few control lines
were also tested with UAS-PKAm-inh. An
additional 34 P[GAL4] lines driving
UAS-PKAinh or
UAS-PKAm-inh were screened. These were
chosen on the basis of expression in specific structures, such as the
fan-shaped body, ellipsoid body, mushroom bodies, pars intercerebralis
(PI), or other. Expression patterns listed in Table 1 derive from the
following published information: All c and Y lines, www.flytrap.org;
c161, 78Y, 7Y, 64Y, c561, c105, c481, c346, c819, and c232, Renn et al.
(1999); 78Y and 7Y, Martin et al. (1999); c309, c747, and OK348,
Connolly et al. (1996); MJ126a and MJ162a, Joiner and Griffith (1999); MZ423, Ito et al. (1997); c302 and c739, Yang et al. (1995); MHC82, Davis et al. (1998); 17D, Martin et al. (1998); and Okt30, Feb204, Feb170, Kurs6, Jan129, Mai301, Jan229, Kurs21, Mai179, Kurs58, and
Sep54, Siegmund and Korge (2001).
Behavioral assays. Inebriometer assays were performed as
described previously (Moore et al., 1998; Singh and Heberlein, 2000). Inebriometers were equilibrated to 20°C and an ethanol/humidified air
mixture of ~50/40 U. Flies were equilibrated for 1 hr at 20°C before being loaded into the inebriometer. Elution was quantified in 3 min intervals using a monitor (Trikinetics, Waltham, MA), and mean
elution times were calculated from the resulting distribution. One-way
ANOVAs were performed in Statistica (StatSoft, Tulsa, OK). To maintain
an experiment-wide error rate of 
= 0.05, the adjusted error
rates were p = 0.0167 for the 3 subsequent planned pair-wise comparisons in Figures 1 and 3. In Figure 5, two-way ANOVA
was performed in Statistica (StatSoft) with post hoc
Newman-Keuls testing.
Locomotor tracking assays were performed as described previously
(Scholz et al., 2000), with an ethanol/humidified air mixture of 40/25
U. Flies were exposed to humidified air for 6 min, filmed in air for 2 min, and then filmed in the presence of ethanol vapor for an additional
18 min. Ten second movie clips were captured on an Apple G4 computer
using Adobe Premiere. The flies' locomotion was tracked using the
dynamic image analysis system (DIAS) (Solltech, Oakdale, IA).
Average speed over the interval was calculated by dividing the flies'
total path length by time. One-way ANOVAs across each of 16 time points
were performed in Statistica (StatSoft). To maintain an experiment-wise
error rate of = 0.05, the critical p value was
adjusted to = 0.003. For time points showing a main effect of
genotype, post hoc Newman-Keuls tests were performed to
determine whether P[GAL4]+UAS-PKAinh was
significantly different from both P[GAL4] and UAS controls at
= 0.05.
Ethanol absorption. Absorption assays were performed as
described previously (Moore et al., 1998). Thirty flies of each
genotype were exposed in triplicate to an ethanol/humidified air
mixture of 50/45 U for 30 min. The alcohol concentration in extracts
was measured using an alcohol dehydrogenase assay (Sigma, St.
Louis, MO). To calculate the ethanol concentration in the flies, we
estimated the volume of one fly to be 2 µl. The values for
201Y+UAS-PKAinh and 201Y were corrected for the total amount of
protein, because 201Y+UAS-PKAinh flies are
slightly smaller than 201Y flies. Protein was measured using Coomassie
blue reagent (Pierce, Rockford, IL).
Histology. Staining for 
-galactosidase expression in CNSs
of 5- to 7-d-old P[GAL4]/UAS-lacZ males was done as described
previously (Scholz et al., 2000). Samples were incubated in
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal;
Labscientific, Livingston, NJ) solution at 37°C for 20-30 min
(c747), 1-2 hr (c522 and c107) or overnight (c290, 201Y, and c747).
Multiple specimens were observed for each genotype.
For anti-tau immunohistochemistry, larval brains were dissected in cold
PBS, fixed in 4% paraformaldehyde for 50 min at room temperature, and
washed in PBS with 0.3% Triton X-100. Specimens were incubated with
anti-tau antibody (Sigma) diluted 1:500, and with a secondary
HRP-coupled goat anti-mouse antibody (Vector Laboratories, Burlingame,
CA) diluted 1:100. The Vectastain Elite ABC kit was used for DAB
staining (Vector).
PKA activity assay. Fifty male
UAS-PKAinh, hs-GAL4,
hs-GAL4+UAS-PKAinh, or
hs-GAL4+UAS-PKAm-inh flies were reared at
25°C and collected under CO2 anesthesia as for
a behavioral assay. One day after collection, flies were heat-shocked
for 1 hr at 37°C in a water bath and then returned to 25°C.
Non-heat-shocked controls were kept at 25°C. Twenty-four hours after
the beginning of heat shock, flies were transferred to Eppendorf tubes
with brief CO2 anesthesia and frozen by placing the tubes on dry ice. Whole flies were homogenized in 600 µl of buffer (in mM: 10 sodium phosphate, pH 6.8, 1 EDTA, 0.5 EGTA, 2.5 -mercaptoethanol, 25 benzamidine, and 1 PMSF) and
centrifuged for 5 min at 14,000 rpm; the pellet was discarded. Protein
concentration in the extracts was measured with Coomassie blue reagent
(Pierce). Eight micrograms of total protein were assayed with the
Colorimetric PKA assay kit, Spinzyme format (Pierce) according to the
manufacturer's instructions. Each extract was assayed in duplicate,
and the experiment was repeated three times. Two-way ANOVA was
performed in Statistica (StatSoft) with post hoc
Newman-Keuls testing.
Hydroxyurea ablation of mushroom bodies. Hydroxyurea
ablation was performed according to the method of de Belle and
Heisenberg (1994). 201Y virgin females were crossed to UAS-lacZ or
UAS-lacZ;UAS-PKAinh males. Eggs were
collected on apple juice plates at 25°C in 1 hr intervals and kept at
25°C for 23.5 hr (this time was determined empirically to result in
the most efficient MB ablation). The newly hatched first instar larvae
were transferred to a microcentrifuge tube containing a paste of
heat-killed yeast with or without 50 mg/ml hydroxyurea (Sigma) for 4 hr
at 25°C. At this time, larvae were washed and transferred to regular
food bottles; adult males, 2-4 d old, were tested in the inebriometer.
Males eluting from each inebriometer run were collected and stained for
-galactosidase expression (between 20 and 50 from
hydroxyurea-treated groups and ~10 from each control group). Ablated,
partially ablated, and unablated mushroom bodies were observed. For
201Y+UAS-lacZ, the percent complete ablation values observed in
individual inebriometer runs were 58, 78, 82, and 85%, with an average
of 76%. For 201Y+UAS-lacZ+UAS-PKAinh, the
percent complete ablation values were 68, 70, 84, and 95, with an
average of 79%, which is not significantly different from the percent
ablation seen in 201Y+UAS-lacZ flies (Student's t test). No
significant correlation was found between percent ablation and MET:
201Y+UAS-lacZ, p = 0.604;
201Y+UAS-lacZ+UAS-PKAinh,
p = 0.328. No mushroom body ablation was observed in
untreated flies. METs were subjected to two-way ANOVA using Statistica (StatSoft).
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RESULTS |
PKA inhibitor expression in specific brain regions decreases
ethanol sensitivity
A collection of Drosophila strains, in which the
transcription factor GAL4 is expressed in various discrete regions of
the CNS under the control of endogenous enhancers, was used to express a PKA inhibitor in a spatially restricted manner. Inactive PKA holoenzyme consists of a dimer of regulatory subunits bound to two
catalytic subunits. PKA activation occurs when cAMP binds to regulatory
subunits, causing dissociation of the holoenzyme and release of active
catalytic subunits (Taylor et al., 1990). The PKA inhibitory transgene
PKAinh (Li et al., 1995) encodes a
Drosophila type I regulatory subunit with mutated cAMP
binding sites; it therefore remains bound to the endogenous catalytic
subunit when cAMP levels rise, inhibiting activation in a dominant
manner (Fig. 1A). As a
control we used a transgene encoding an inactive PKA inhibitor,
PKAm-inh, which is identical to
PKAinh with the exception of two
additional mutations in amino acids needed for binding to the catalytic
subunit (Kiger and O'Shea, 2001). Both transgenes are positioned
downstream of UAS sites that allow transcriptional regulation by GAL4.
As shown below, expression of PKAinh or
PKAm-inh had the expected effect on PKA
catalytic activity.
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Figure 1.
Expression of
PKAinh in specific brain regions alters ethanol
sensitivity in the inebriometer. A, Inactive PKA
holoenzymes consist of two regulatory (R) and two
catalytic (C) subunits. Binding of cAMP to R
subunits results in their dissociation from C and hence C activation.
PKAinh lacks cAMP binding ability and remains bound
to C even on increases in cellular cAMP, thereby inhibiting C
activation. PKAm-inh is unable to bind cAMP or C and
therefore should not have an inhibitory effect. B,
PKAinh expression under the control of 201Y, c107,
and c522 resulted in increased MET. One-way ANOVA revealed a
significant effect of genotype for all three P[GAL4] lines: 201Y
(F(2,24) = 24.2; p < 0.0001), c107 (F(2,15) = 14.8;
p < 0.0001), and c522
(F(2,15) = 28.1; p < 0.0001). Pair-wise planned comparisons, with the critical
p value adjusted to = 0.0167, revealed
significant differences between P[GAL4]+UAS-PKAinh
and both P[GAL4] and P[GAL4]+UAS-PKAm-inh
(p < 0.001 for all comparisons). Pairwise
planned comparisons did not reveal significant differences between
P[GAL4] and P[GAL4]+UAS-PKAm-inh: 201Y
(p = 0.966), c107
(p = 0.346), and c522
(p = 0.061). For each P[GAL4] line,
n is the same for P[GAL4],
P[GAL4]+UAS-PKAinh, and
P[GAL4]+UAS-PKAm-inh: 201Y (n = 9), c107 (n = 6), c522 (n = 6), c747 (n = 8), c290 (n = 5),
MHC82 (n = 7), UAS-PKAinh
(n = 59), and UAS-PKAm-inh
(n = 20). Asterisks denote
significant differences. In all figures, error bars indicate SEM. In
this and subsequent figures, flies were heterozygous for
autosomal insertions and hemizygous for X-linked insertions (see
Materials and Methods for chromosomal location of insertions).
C, The MET of c747, c290, and MHC82
P[GAL4] lines was not altered by the presence of
UAS-PKAinh or UAS-PKAm-inh.
One-way ANOVA comparing P[GAL4],
P[GAL4]+UAS-PKAinh, and
P[GAL4]+UAS-PKAm-inh revealed no significant
effect of genotype for c290 (F(2,12) = 1.35; p = 0.30) and MHC82
(F(2,18) = 2.93; p = 0.078). For c747, there was a weak effect of genotype
(F(2,21) = 4.0; p = 0.038). Planned pair-wise comparisons, with the critical
p value adjusted to = 0.0167, revealed a
marginally significant difference between
c747+UAS-PKAm-inh and c747
(p = 0.012) but not between
c747+UAS-PKAm-inh and
c747+UAS-PKAinh (p = 0.061) or between c747+UAS-PKAinh and c747
(p = 0.459). D, Expression of
PKAinh under the control of c107 and c747 resulted
in normal MET. t tests with the critical
p value adjusted to = 0.025 revealed no
significant difference between c107+c747+UAS-PKAinh
and c747+UAS-PKAinh (p = 0.05) but did reveal a significant difference between
c107+c747+UAS-PKAinh and
c107+UAS-PKAinh (p < 0.0001). The UAS-PKAinh MET is from
B. n = 10 for
c747+UAS-PKAinh and
c107+UAS-PKAinh; n = 8 for
c107+c747+UAS-PKAinh.
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Flies carrying UAS-PKAinh and individual
P[GAL4] insertions were screened for sensitivity to ethanol in the
inebriometer. This apparatus consists of a 4-foot-long vertical column
fitted with obliquely oriented mesh baffles on which sober flies will
stand (Cohan and Hoffman, 1986; Weber, 1988). When exposed to ethanol vapor, flies become uncoordinated, lose postural control, and fall
through the column. The MET of a population of flies is a measure of
their sensitivity to ethanol (increased MET equals decreased sensitivity).
Of 64 P[GAL4] lines tested, 42 displayed normal ethanol sensitivity
in the presence of UAS-PKAinh (Table
1). These included lines with expression
in a variety of structures, including the MBs, ellipsoid body (EB),
fan-shaped body (FSB), central complex small-field neurons, and
antennal lobes (ALs). These structures are composed of multiple
different cell types (Hanesch et al., 1989; Crittenden et al., 1998;
Renn et al., 1999), and GAL4 expression in the lines tested may be restricted to only certain subsets of cells. Therefore, we cannot completely rule out the possibility that PKA signaling in these structures contributes to the modulation of ethanol sensitivity. An
additional 19 P[GAL4] lines resulted in altered MET in the presence
of either PKAinh or
PKAm-inh, presumably because of an effect
of protein overexpression (Table 1). In total, then, 61 of 64 lines
screened did not result in a specific effect of
PKAinh expression on ethanol
sensitivity.
In contrast, expression of PKAinh under
the control of three P[GAL4] lines, 201Y, c107, and c522, led to a
decrease in ethanol sensitivity (resistance) in the presence of
UAS-PKAinh but not
UAS-PKAm-inh (Fig. 1B).
The lack of effect of UAS-PKAm-inh
indicated that the inhibitory activity of
PKAinh (the ability to bind to the PKA
catalytic subunit) is required for the altered behavior. Thus,
expression of the inhibitor in a subset of CNS cells caused a specific
reduction in ethanol sensitivity.
Three examples of lines with normal sensitivity when driving
expression of PKAinh are shown in
Figure 1C. Expression of PKAinh
in postembryonic muscle, under the control of
P[GAL4]MHC82 (Davis et al., 1998), did
not affect ethanol sensitivity (Fig. 1C). Line c290 shows
little or no expression in the CNS, whereas c747 is expressed broadly
and at high levels in the CNS (Fig. 2).
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Figure 2.
Expression patterns of P[GAL4] lines.
-Galactosidase expression under the control of each P[GAL4]
was visualized by staining whole-mount nervous systems with X-gal. Some
planes are out of focus in the photographs. A, In the
brains of 201Y males, expression is observed in a subset of MB neurons,
in the DGIs, and in neurons in the PI, vSEG, and lateral protocerebrum.
PI neurons are easily dissociated from the brain during dissection and
are not observed in this sample (but see Fig.
4D). In c107, expression is seen in small-field
neurons projecting to the FSB and EB of the central complex
(CC), in the DGIs and optic lobes (OL),
and elsewhere. c522 is expressed in parts of the central complex
(subsets of FSB and EB neurons), in the ALs, antenno-mechanosensory
center (AMC), and SEG. c290 has little expression in the
CNS. c747 is widely and highly expressed. A short (20-30 min) exposure
in X-gal solution reveals high levels of expression in the MBs, PI, EB,
AL, and OL (Zars et al., 2000a). Longer X-gal exposure (overnight)
reveals widespread expression. E, Esophagus;
LPC, lateral protocerebrum. B, Ventral
nerve cord (VNC) expression is observed in all P[GAL4]
lines except c290. 201Y has very limited expression in a small number
of cell bodies in the abdominal ganglion, two longitudinal fibers, and
one transverse fiber. c107, c522, and c747 are expressed in thoracic
and abdominal neuromeres. c107 expression is seen in a median
longitudinal tract of the VNC, which appears to either give rise to or
derive from tangles of fibers at each of the three levels of the
thoracic ganglia. There is additional staining in the abdominal
ganglion of the VNC. In c522, prominent longitudinal tracts in the VNC,
which are continuous with tracts in the ventral part of the brain,
resemble the median tract of the dorsal cervical fasciculus and the
dorsal lateral tract of the ventral cervical fasciculus described by
Power (1948). In addition, transverse fiber tracts are also
observed, as well as additional cell bodies. In c747, some tracts
appear to overlap with those observed in c522, specifically the median
tract of the dorsal cervical fasciculus.
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Given the widespread expression of GAL4 in line c747, the normal
ethanol sensitivity of c747+UAS-PKAinh was
surprising. We reasoned that if expression of
PKAinh in the c747 cells was truly inert,
simultaneous expression of PKAinh under
the control of both c107 and c747 should result in resistance to
ethanol, the phenotype displayed by
c107+UAS-PKAinh flies. However, expression
of PKAinh under the control of both c107
and c747 resulted in a MET similar to that of
c747+UAS-PKAinh flies; i.e., the lack of
effect of line c747 was epistatic to the ethanol resistance caused by
line c107 (Fig. 1D). One possible explanation for
this result is that the normal behavior displayed by
c747+UAS-PKAinh represents the balanced
sum of increased and decreased sensitivity caused by
PKAinh expression in different sets of
cells. Adding expression in c107 cells would not tip the balance toward
resistance if the GAL4-expressing cells in c107 are already present in
c747. That is, c107 may express GAL4 in resistance-causing cells that
are also present in c747; in c107, however, these cells would not be
counterbalanced by sensitivity-causing cells. It is also possible that
the expression of PKAinh in certain c747
cells clamps ethanol sensitivity at a relatively normal level, and that
these cells are epistatic to the resistance-causing cells of line c107;
these cells may also mask resistance-causing cells in c747. Regardless
of the exact mechanism, these data clearly show that broad
PKAinh expression under the control of
line c747 is not behaviorally inert but, rather, the result of complex
interactions between different populations of cells. It is therefore
important to exercise caution when drawing conclusions about neurons
(or brain regions) not involved in a behavior of interest based on a
lack of effect of a particular manipulation.
Decreased sensitivity to ethanol is not attributable to
altered pharmacokinetics
Because reduced ethanol sensitivity could be caused by impaired
ethanol absorption (Singh and Heberlein, 2000), we measured the ethanol
content in P[GAL4] flies in the absence and presence of
UAS-PKAinh after a 30 min exposure to
ethanol vapor (Table 2). Expression of
PKAinh did not affect the levels of
absorbed ethanol in these flies. Thus, the altered behavior is
attributable to functional differences in ethanol responsiveness rather
than changed pharmacokinetics.
Expression patterns of P[GAL4] lines
Expression of GAL4 was determined using a reporter
transgene, UAS-lacZ, encoding -galactosidase. The expression pattern
of the UAS-lacZ reporter was found to be indistinguishable in P[GAL4] and P[GAL4]+UAS-PKAinh flies on analysis
at the light microscope level (data not shown). Thus,
PKAinh expression does not alter the gross
morphology of the structures in which it is expressed, although it
could have effects at the ultrastructural level.
Of the three P[GAL4] lines that caused a specific change in ethanol
sensitivity in the presence of UAS-PKAinh,
two lines, c107 and c522, showed restricted expression in the CNS,
whereas 201Y expression was even more limited (Fig. 2). The most
prominent expression in c107 is in the optic lobes, small-field neurons
of the central complex projecting to the EB and FSB (Renn et al.,
1999), and the dorsal giant interneurons (DGIs), a bilateral pair of
neurons with characteristic dorsally looping projections (Ito et al.,
1997) (Fig. 2A). Line c522 is expressed in the
antennal lobes, the antenno-mechanosensory center, subesophageal
ganglion (SEG), central complex, and lateral protocerebrum (Zars
et al., 2000b) (Fig. 2A). Central complex expression
in c522 includes intrinsic neurons of the FSB and a small number of EB
intrinsic neurons. Lines c107 and c522 are also expressed in the
ventral nerve cord (VNC) (Fig. 2B). Line 201Y, used
extensively in previous behavioral (O'Dell et al., 1995; Connolly et
al., 1996; Martin et al., 1998; Joiner and Griffith, 1999; Zars et al.,
2000a; McGuire et al., 2001) and neuroanatomical (Yang et al., 1995;
Tettamanti et al., 1997; Armstrong et al., 1998; Kraft et al., 1998;
Lee et al., 2000) studies, is expressed in a subset of MB neurons that
project to the cores of the and lobes and broadly in 
lobe
neurons (Fig. 2A). In addition, 201Y is expressed in
the DGIs (K. Ito, personal communication), in ~13 neurons of
the PI projecting through the median bundle, and in 6 neurons located in the ventral SEG (see below). There is also very restricted and low
expression in the VNC (Fig. 2B).
It is unlikely that PKAinh expression in
the optic lobes of c107 affects ethanol sensitivity, because flies with
severely abnormal optic lobes attributable to the small optic
lobes mutation (sol1) are normal
(Scholz et al., 2000). As mentioned above, small-field neurons of c107
project onto the EB and FSB, whereas expression of c522 includes
intrinsic neurons of both of these structures, providing a possible
functional link between the two P[GAL4] lines. As described above,
however, PKAinh expression in 25 P[GAL4]
lines with EB expression and 11 lines with FSB expression did not
result in a specific effect of the inhibitor on behavior (Table 1).
Therefore, it is unclear whether PKA signaling in these structures
plays a role in modulating ethanol sensitivity. Because of the more
limited expression of 201Y, we focused most subsequent experiments on
this line.
Effect of PKAinh is suppressed by overexpression
of catalytic subunit
As discussed above, the effect of
PKAinh expression on ethanol sensitivity
appears to require the binding of PKAinh
to catalytic subunits, because PKAm-inh,
which lacks this function, is inert. If this is correct, overexpression of the PKA catalytic subunit (PKAc) should counteract
PKAinh and therefore suppress its effect
on ethanol sensitivity. Indeed, PKAinh-induced defects in wing development
are suppressed by coexpression of PKAc (Kiger et al., 1999). We
therefore expressed both UAS-PKAinh and
UAS-PKAc simultaneously under the control of line 201Y. As shown in
Figure 3, coexpression of PKAc did
suppress the PKAinh-induced reduction in
ethanol sensitivity. This effect was not caused by the presence of two
UAS transgenes, which may reveal limiting amounts of GAL4, because
coexpression of an inert transgene, UAS-lacZ, with
UAS-PKAinh did not cause phenotypic
suppression (Fig. 3). Overexpression of PKAc alone with the 201Y driver
did not alter behavior, suggesting that ethanol sensitivity was not
affected by enhanced PKA function in the 201Y-expressing cells.
Expression of any combination of transgenes with the control line c290
had no effect on ethanol sensitivity. These data strongly suggest that
inhibition of PKA function in the 201Y-expressing cells decreases
ethanol sensitivity, and that PKAinh acts
by sequestering endogenous PKA catalytic subunits.
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Figure 3.
PKAc coexpression suppresses
PKAinh-induced ethanol resistance. Flies of the
indicated genotypes were tested in the inebriometer; METs are shown.
Simultaneous expression of PKAinh and lacZ under the
control of 201Y resulted in an increase in MET. This increase in MET
was suppressed by coexpression of PKAc with PKAinh.
Expression of PKAc alone did not alter MET, nor did the expression of
any of the transgenes under the control of c290. The values for
P[GAL4] were obtained from the experiments shown in Figure 1 and are
displayed for comparison. One-way ANOVA revealed no significant effect
of genotype for c290 (F(2,12) = 0.45;
p = 0.647). A significant effect of genotype was
observed for 201Y (F(2,12) = 45.5;
p < 0.0001). Planned pair-wise comparisons with
the critical p value adjusted to = 0.0167 revealed a significant difference between
201Y+UAS-lacZ+UAS-PKAinh and both
201Y+UAS-PKAc+UAS-PKAinh and 201Y+UAS-PKAc
(p < 0.0001 for both comparisons) but not
between 201Y+UAS-PKAc+UAS-PKAinh and 201Y+UAS-PKAc
(p = 0.019) (n = 4 for
each UAS genotype; n = 5 for all others).
Asterisks denote significant differences.
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Mushroom bodies are not involved in the regulation of
ethanol sensitivity
As has been documented previously (O'Dell et al., 1995; Yang et
al., 1995; Connolly et al., 1996; Tettamanti et al., 1997; Armstrong et
al., 1998; Kraft et al., 1998; Martin et al., 1998; Joiner and
Griffith, 1999; Lee et al., 2000; Zars et al., 2000a,b; McGuire et al.,
2001), 201Y is expressed in the mushroom bodies (Fig.
2A), structures in the fly's brain that play an
important role in olfactory classical conditioning. Interestingly,
several olfactory learning and memory mutants, such as
amnesiac (amn), rutabaga
(rut), and the cell adhesion molecule fasciclin
II (fasII), show altered ethanol
sensitivity (Moore et al., 1998; Cheng et al., 2001). rut
and fasII are preferentially expressed in the MBs, along
with the catalytic and regulatory subunits of PKA (for review, see
Roman and Davis, 2001); amn is expressed in two prominent neurons that project onto the MB axons (Waddell et al., 2000). We were
therefore interested in determining whether the MBs are required for
normal ethanol sensitivity and whether the expression of
PKAinh in the MBs is responsible for the
altered sensitivity seen in 201Y+UAS-PKAinh flies.
During the first few hours of larval life, the only proliferating
neuroblasts are four that give rise to the MBs and one that contributes
to the antennal lobes (Truman and Bate, 1987; Prokop and Technau, 1991,
1994; Ito and Hotta, 1992). The MBs can thus be selectively ablated by
feeding hydroxyurea to newly hatched larvae, a manipulation that has
been shown to impair olfactory and courtship conditioning (de Belle and
Heisenberg, 1994; McBride et al., 1999). To determine whether MB
ablation alters ethanol sensitivity, we fed 201Y+UAS-lacZ larvae yeast
paste with or without hydroxyurea and tested the resulting adults in
the inebriometer; MB ablation was assessed by examining
-galactosidase expression (Fig. 4; see
Materials and Methods). As reported previously (Armstrong et al.,
1998), hydroxyurea treatment eliminated most MB neurons in which 201Y
is expressed (Fig. 4C); a few lobe neurons remain, probably those arising during embryogenesis. Also consistent with previous results (Armstrong et al., 1998), hydroxyurea treatment spared
neurons outside of the mushroom bodies, such as the DGIs (Fig.
4C). Flies lacking the MBs showed normal sensitivity in the
inebriometer (Fig. 4A). Thus, unlike olfactory
learning, in which the MBs play a central role, ethanol sensitivity
does not appear to be regulated by these structures.
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Figure 4.
Ablation of mushroom bodies does not alter ethanol
sensitivity. A, The MET in the inebriometer of
hydroxyurea-treated (+HU) and mock-treated
( HU) flies is shown. METs were subjected to
two-way ANOVA revealing a significant main effect of genotype
(F(1,12) = 148.7; p < 0.001) but no effect of hydroxyurea treatment
(F(1,12) = 3.3; p = 0.093) and no significant interaction
(F(1,12) = 0.62; p = 0.446). These data indicate that ablation of the mushroom bodies had
no effect in either genotype. (n = 4 for all
groups). Asterisks denote significant differences.
B, C, Males eluting from each
inebriometer run were collected, and their brains were dissected and
stained with X-gal. Examples of an unablated brain
(HU) and ablated brain
(+HU) are shown. HU treatment ablated all but a
few gamma lobe neurons (L) but spared other
neurons such as the DGIs. D-F, Close-up view of the
expression of P[GAL4] outside the MBs in 201Y. D,
Expression in the PI neurons, which extend axons through the median
bundle (MedB). E, Expression in the vSEG
neurons. Note axons extending from the cell bodies toward the esophagus
(E). F, Expression in the DGI
neurons. G, Expression of the microtubule-binding
protein tau under the control of 201Y in third instar larvae.
Expression is observed in MB lobes (L) and the
cell bodies and axons (arrowheads) of PI and vSEG
neurons. These axons project to the base of the ring gland
(RG, the outline of the unstained gland, based on a
phase-contrast image, gray lines).
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To determine whether expression of PKAinh
in MBs contributes to the reduced sensitivity of
201Y+UAS-PKAinh flies, we treated larvae
of this genotype with hydroxyurea as described above. These flies also
carried UAS-lacZ to monitor MB ablation. Decreased ethanol sensitivity
of 201Y+UAS-PKAinh flies remained even
after mushroom body ablation (Fig. 4A). We conclude
from these results that PKA inhibition in neurons other than those
forming the MBs is responsible for the decreased ethanol sensitivity of
201Y+UAS-PKAinh flies. This is consistent
with our finding that expression of PKAinh
in MB neurons under the control of c747 (and five additional lines) did
not alter MET (Fig. 1B, Table 1).
Because few neurons outside of the MBs express GAL4 in 201Y, this
limits the number of neurons that cause the altered inebriometer phenotype. These include the PI neurons (Fig. 4D),
the ventral SEG neurons (Fig. 4E), the DGIs (Fig.
4F), and a very small number of other neurons in the
protocerebrum and abdominal ganglion. Diffuse staining near the
esophagus (Fig. 4E) is attributable to expression in
the axons and nerve endings of PI and SEG neurosecretory cells known to
project to this area (Rajashekhar and Singh, 1994; Shiga et al., 2000).
We do not believe that PKAinh expression
in the DGIs is responsible for the altered behavior, because three
lines with DGI expression did not cause altered ethanol sensitivity
when driving PKAinh (Table 1; data not
shown). We also tested P[GAL4] lines with PI and vSEG expression: 8 lines with adult expression in PI neurons and 10 lines with expression
in larval PI and SEG neurosecretory cells projecting to the ring gland
did not result in altered ethanol sensitivity (Table 1). However, PI
and SEG neurons are heterogeneous; there are at least three different
subtypes of larval PI neurons and two subtypes of SEG neurons based on
their projection patterns (Siegmund and Korge, 2001). In addition,
different PI neurons in the blowfly Calliphora express
different neuropeptides (Duve and Thorpe, 1980, 1981, 1983; Duve et
al., 1983). This leaves open the possibility that a specific subset of
these neurosecretory cells expresses GAL4 in 201Y but not in the other
GAL4 lines tested.
Acute PKA inhibition in the adult fly increases
ethanol sensitivity
Previous experiments had shown that global reduction of PKA
activity throughout development (by reducing the dose of the PKA catalytic subunit) or acute pharmacological inhibition of PKA in the
adult led to increased ethanol sensitivity (Moore et al., 1998). Yet we
were unable to find a GAL4 line that mimicked that effect when driving
PKAinh. To test the effect of expression
of PKAinh throughout the fly, we used a
heat shock-inducible GAL4 transgene, hsGAL4, in which GAL4 is under the
control of the hsp70 promoter. Flies carrying the hsGAL4 transgene and
either UAS-PKAinh or
UAS-PKAm-inh were heat-shocked for 1 hr
and tested in the inebriometer the following day. Under these
conditions, heat shock alone did not affect ethanol sensitivity,
because the MET of UAS-PKAinh flies was
unchanged by heat shock (Fig.
5A). Heat shock-induced expression of GAL4 (in hsGAL4 flies) or GAL4 and
PKAm-inh (in
hsGAL4+UAS-PKAm-inh flies) resulted in a
significant increase in ethanol resistance (Fig. 5A),
probably a nonspecific effect of protein overexpression. However, heat
shock-induced expression of PKAinh (in
hsGAL4+UAS-PKAinh flies) resulted in
increased sensitivity to ethanol (Fig. 5A). Thus, in
contrast to restricted expression of
PKAinh, short-term ubiquitous expression
of the inhibitor in adult flies led to increased ethanol sensitivity,
an observation that is consistent with previous pharmacological
experiments (Moore et al., 1998).
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Figure 5.
Heat shock-induced expression of
PKAinh results in increased ethanol sensitivity.
Male flies of the indicated genotypes were grown at 25°C. Flies were
either heat-shocked for 1 hr at 37°C or kept at 25°C. Twenty-four
hr after the heat shock, flies were tested in the inebriometer, or
extracts were prepared for kinase activity assays. A,
PKAinh-expressing flies are more sensitive to
ethanol after heat shock. Two-way ANOVA revealed a significant effect
of genotype (F(3,24) = 94.5;
p < 0.0001), a significant effect of heat shock
treatment (F(1,24) = 4.6;
p < 0.05), and a significant interaction between
genotype and treatment (F(3,24) = 53.5;
p < 0.0001). Newman-Keuls post hoc
testing revealed no significant difference between
UAS-PKAinh with or without heat shock
(p = 0.6) but did reveal significant
differences for hsGAL4, hsGAL4+UAS-PKAm-inh, and
hsGAL4+UAS-PKAinh with and without heat shock
(p < 0.001 in all cases). Newman-Keuls
tests also revealed significant differences between heat-shocked
hsGAL4+UAS-PKAinh and heat-shocked
UAS-PKAinh (p = 0.02),
hsGAL4 (p = 0.0001), and
hsGAL4+UAS-PKAm-inh (p = 0.0001). B, PKAinh expression results
in reduced PKA activity. Two-way ANOVA revealed a significant effect of
genotype (F(3.16) = 55.1;
p < 0.0001) but no effect of heat shock
(F(1,16) = 0.5; p = 0.5) and no interaction between genotype and heat shock
(F(3,16) = 1.9; p = 0.2). Newman-Keuls post hoc testing revealed
significant differences between hsGAL4+UAS-PKAinh
(with or without heat shock) and all other genotypes
(p < 0.001 in all cases). The only other
significant difference was between
hsGAL+UAS-PKAm-inh (without heat shock) and hsGAL4
(with heat shock) (p = 0.046;
n = 3 for each genotype and condition).
Asterisks denote significant differences.
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To determine whether expression of PKAinh
inhibited PKA activity as expected, we measured cAMP-stimulated PKA
activity in extracts of flies expressing either
PKAinh or
PKAm-inh under the control of hsGAL4. PKA
activity was strongly reduced by expression of
PKAinh but not
PKAm-inh (Fig. 5B). However,
the degree of PKA inhibition was the same in the presence or absence of
heat shock. These data suggest that there is considerable "leaky"
expression of GAL4 even in the absence of heat shock, an observation
that has been made before with transgenes expressed under the control
of heat-inducible promoters (for example, see Grotewiel et al., 1998;
Cheng et al., 2001). However, as shown above, this leaky expression of
PKAinh did not result in altered ethanol
sensitivity, whereas heat-induced expression of the inhibitor led to
strongly increased ethanol sensitivity (Fig. 5A). There are
several possible explanations for this observation. For example, the
assay used to detect kinase activity may not be sensitive enough to
detect subtle differences in activity between heat-shocked and
non-heat-shocked flies. Alternatively, heat induction of
PKAinh above baseline levels may occur in
only a few cells, an effect that would not be detected when assaying
kinase activity in extracts of whole flies. Although it has not been
reported, it is possible that PKA inhibition may alter the flies'
ability to cope with the heat shock itself, subsequently altering
the flies' response to ethanol. Finally, PKA activity may be reduced
transiently between heat shock and testing, altering nervous system
susceptibility to ethanol. Extracts prepared 4 hr after the heat shock
did not reveal such a difference (data not shown), but this does not
preclude an effect at other time points.
In summary, we have shown that PKAinh and
PKAm-inh display the expected effects on
cAMP-stimulated kinase activity, and that ubiquitous expression of
PKAinh resulted in increased ethanol
sensitivity, similar to that observed with genetic and pharmacological
manipulations that globally reduce cAMP signaling (Moore et al.,
1998).
PKA inhibition in different regions has distinct effects on
ethanol-induced locomotor activity
To further analyze the ways in which
PKAinh expression affects ethanol
sensitivity, we examined the flies' response to ethanol in a different
behavioral assay. The locomotor video-tracking system allows the
monitoring of horizontal walking activity of groups of flies as they
are exposed continuously to ethanol vapor of defined concentrations
(Scholz et al., 2000). Immediately after the start of ethanol exposure,
flies showed a transient increase in locomotor velocity (Fig.
6); this is probably a startle response to a novel odor (F. Wolf and U. Heberlein, unpublished observations). All P[GAL4]+UAS-PKAinh flies tested had
robust startle responses, indicating that locomotor function in these
flies is unaffected by PKAinh expression
(Fig. 6). With continued exposure to the relatively high ethanol
concentration used in these experiments, locomotor activity decreased
gradually until the flies became completely sedated, at which point
they became resistant to arousal by mechanical stimulation. When
compared with 201Y and UAS-PKAinh
controls, 201Y+UAS-PKAinh flies were
resistant to the locomotor-depressant effects of ethanol (Fig.
6A). This effect can be seen clearly in examples of
locomotor traces obtained 12.5 min after the start of ethanol exposure
(Fig. 6E); whereas control flies were nearly
completely immobile, most 201Y+UAS-PKAinh
flies remained active. Because the kinetics of ethanol absorption are
indistinguishable in control and experimental flies (Table 1), these
data show that higher levels of ethanol are needed to sedate flies with
targeted inactivation of PKA in 201Y-expressing cells. We note that
baseline locomotion levels (before the start of ethanol exposure) are
somewhat variable, because flies are still in the process of adapting
to their new environment (Fig. 6). These differences in spontaneous
locomotion, however, do not affect the extent of the response to
ethanol (F. W. Wolf and U. Heberlein, unpublished
observations).
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Figure 6.
PKA inhibition differentially affects the
ethanol-induced locomotor activity pattern. The average velocity of a
population of 20 flies is shown as a function of time. Ethanol exposure
started at time 0. A, 201Y and
UAS-PKAinh flies were relatively calm in air (2-0
min), startled on exposure to alcohol (0-2 min), remained relatively
active until ~6 min, but were strongly sedated by ~8.5 min of
ethanol exposure. 201Y+UAS-PKAinh flies behaved
similarly at early time points but remained active during the later
time points. One-way ANOVA across each time point, with the critical
p value adjusted to = 0.003, revealed a
significant effect of genotype at 10 min
(F(2,9) = 34.5; p < 0.0001), 12.5 min (F(2,9) = 130.8;
p < 0.0001), 15 min
(F(2,9) = 74.3; p < 0.0001), and 17.5 min (F(2,9) = 27.7; p < 0.0005). Post hoc
Newman-Keuls testing showed that at each starred
time-point, P[GAL4]+UAS-PKAinh flies were
different from both P[GAL4] and UAS-PKAinh
controls (p < 0.001 for all comparisons).
A', 201Y, 201Y+UAS-PKAinh and
201Y+UAS-PKAm-inh flies were exposed to air,
followed by an ethanol/air dose of 40/25 U beginning at time 0 for 1 min. Consecutive 10 sec clips were analyzed beginning 20 sec
before the onset of ethanol and ending 20 sec after ethanol exposure
was terminated; an additional 10 sec was analyzed 1 min after the end
of exposure. The startle response was similar in all three genotypes
(n = 3 for each genotype). B,
c107+UAS-PKAinh flies are also resistant to the
locomotor-depressant effects of ethanol compared with controls. A
significant effect of genotype was seen at 7.5 min
(F(2,9) = 18.1; p < 0.001), 10 min (F(2,9) = 30.9;
p < 0.0001), 12.5 min
(F(2,9) = 47.0; p < 0.0001), 15 min (F(2,9) = 65.3;
p < 0.0001), and 17.5 min
(F(2,9) = 37.7; p < 0.0001). Newman-Keuls post hoc tests showed that
c107+UAS-PKAinh flies were significantly different
from both c107 and UAS-PKAinh controls at 10, 12.5, 15, and 17.5 min (p < 0.01 for all
comparisons). C, c522+UAS-PKAinh
flies have normal sensitivity to the locomotor depressant effects of
ethanol. A significant effect of genotype was observed only at 15 min
(F(2,9) = 12.1; p = 0.0028). In Newman-Keuls post hoc tests,
c522+UAS-PKAinh flies were significantly different
from both c522 and UAS-PKAinh controls
(p < 0.01) at this time point.
D, c290+UAS-PKAinh flies were also
similar to controls. In A-D, n = 4 for all genotypes. E, Examples of locomotor traces of 20 flies corresponding to a 10 sec period at 12.5 min of ethanol exposure.
Although almost all 201Y and UAS-PKAinh flies were
completely sedated at 12.5 min, 201Y+UAS-PKAinh
flies were still active at this time. Asterisks denote
significant differences.
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c107+UAS-PKAinh flies were also resistant
to ethanol-induced sedation, although to a lesser degree than
201Y+UAS-PKAinh flies (Fig.
6B). In contrast, both
c522+UAS-PKAinh and
c290+UAS-PKAinh flies did not differ
significantly from controls, despite their different inebriometer
phenotypes (Figs. 1, 6C,D). Thus, although PKAinh expression under the control of
201Y, c107, and c522 resulted in decreased ethanol sensitivity in the
inebriometer, only 201Y and c107 showed an altered response to the
locomotor-depressant effects of ethanol.
We suggest that distinct components of ethanol sensitivity are
differentially modulated by cAMP signaling in different neurons in the
flies' CNS. Signaling in the 201Y and c107 cells regulates sensitivity
to the effects of ethanol on both motor coordination (postural control)
and sedation, whereas signaling in the c522 cells appears to only
modulate sensitivity to the effect of ethanol on postural control.
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DISCUSSION |
A role for cAMP signaling in the modulation of acute sensitivity
to ethanol in Drosophila has been demonstrated previously (Moore et al., 1998). Here we show that several neuroanatomical loci
interact in complex ways to regulate normal responsiveness to acute
ethanol exposure, yet inhibition of PKA in just a few cells in the
brain, possibly neurosecretory cells, is sufficient to reduce the
flies' sensitivity to the effects of ethanol on postural control.
Using a different behavioral assay that measures the
locomotor-depressant effects of ethanol, we show that cAMP signaling in
different populations of neurons regulates distinct aspects of the
acute behavioral response to ethanol. In addition, we show that despite
the regulation of olfactory learning and ethanol sensitivity by a
common set of genes, these two complex Drosophila behaviors
are controlled by distinct neural circuits.
Neuroanatomy of ethanol sensitivity
Of the neurons we have identified as candidates for mediating the
resistance to ethanol intoxication, the PI and ventral SEG (vSEG)
neurons are the most interesting. These resemble known neuropeptide-expressing neurosecretory cells that extend axons toward
the esophagus, where they intermingle with the projections from the
neurosecretory PI neurons (Rajashekhar and Singh, 1994; Shiga et al.,
2000). Both PI and vSEG neurons also send projections to the
hypocerebral ganglion-corpus cardiacum complex, a neuroendocrine organ
that is an adult derivative of the larval ring gland (Thomsen, 1969;
Nassel, 1993; Shiga et al., 2000). Interestingly, an enhancer-trap insertion in the neuropeptide-encoding amnesiac gene,
amnchpd, shows strong galactosidase
expression in the ring gland (data not shown), suggesting a possible
association between the PI and vSEG neurons and neuropeptides
modulating ethanol sensitivity.
Ethanol sensitivity and the cAMP pathway
Several conditions known or expected to decrease the function of
the cAMP pathway in the whole fly were shown previously to increase the
flies' ethanol sensitivity in the inebriometer. These include
mutations in amn, rut, and the catalytic subunit
of PKA, as well as acute pharmacological inhibition of PKA (Moore et
al., 1998). Similarly, ubiquitous expression of
PKAinh in the adult fly under the control
of hsGAL4, as shown here, resulted in increased ethanol sensitivity. In
contrast, restricted inhibition of PKA under the control of specific
GAL4 lines caused reduced ethanol sensitivity. Several mechanisms may
contribute to this apparent paradox. First, the different phenotypes
may be the consequence of cAMP pathway requirements in different
populations of CNS cells. Although we identified cells that confer
reduced sensitivity to ethanol, we failed to find those that confer
increased sensitivity. The existence of the latter cells is, however,
inferred from the fact that ubiquitous expression of
PKAinh results in increased ethanol
sensitivity. If so, cells conferring increased sensitivity likely act
downstream in the neural circuits regulating the effects of ethanol,
because they mask the effect of cells that confer increased resistance.
This is consistent with the result obtained with coexpression of the
PKAinh with both c107 and c747, in which
the resistant phenotype obtained with c107 was masked by simultaneously
expressing PKAinh in the c747 neurons.
It is also possible that the specific effect of PKA inhibition on
ethanol sensitivity depends on the timing of such inhibition. Perhaps
inhibition in the adult fly, such as that expected to be caused by
feeding flies a PKA inhibitor and that caused by heat shock
induction of PKAinh, results in increased
sensitivity, whereas expression under the control of c107, c522, and
201Y during development results in reduced sensitivity. These three
lines all exhibit GAL4 expression in the CNS in third instar
larvae (Fig. 4G; data not shown).
Finally, it is possible that quantitative differences between the
degree of PKA inhibition in the three GAL4 lines that cause ethanol
resistance, c107, c522, and 201Y, and the experimental conditions that
result in increased ethanol sensitivity are responsible for the
dichotomy. Strong and global reductions in PKA activity, for example in
pka-C1 null mutants or by expressing
PKAinh pan-neurally, result in lethality
attributable to developmental defects (Lane and Kalderon, 1993; data
not shown), precluding behavioral analyses. It is therefore possible
that the degree of PKA inhibition achieved in the c107, c522, or 201Y
cells is greater than that obtained with other GAL4 lines, but flies
survive because expression is restricted in these lines.
The regulation of ethanol sensitivity by cAMP signaling is also complex
in mammals. As in flies with a mutation in PKA-RII, cAMP-stimulated PKA activity is greatly reduced in mice with a targeted
disruption in the PKA-RII gene (Park et al., 2000; Thiele et al., 2000); like the PKA-RII mutant flies, these mice
show decreased sensitivity to the sedating effects of ethanol.
Interestingly, mice expressing a transgene similar to
PKAinh in the forebrain, which therefore
have decreased PKA activity in these brain regions, display increased
ethanol sensitivity (Wand et al., 2001). Mice heterozygous for a
deletion of a Gsa gene are also more
sensitive to ethanol. Thus, as with flies, different genetic
manipulations that decrease cAMP signaling result in opposite effects
on ethanol sensitivity.
Regulation of different ethanol-induced behaviors
Exposure of both mammals and flies to varying concentrations of
ethanol has distinct behavioral consequences. In Drosophila, these can be separated using different assays, such as the inebriometer and the locomotor tracking system. Using these assays, we have shown
that expression of PKAinh under the
control of c107, c522, and 201Y, which in all three cases resulted in
similar resistance to the incoordinating effects of ethanol in the
inebriometer, altered sensitivity to the locomotor-depressant effects
of ethanol to varying degrees. Thus, PKA inhibition in different sets
of neurons affects distinct aspects of the acute behavioral effects of
ethanol. The neural circuitry controlling different aspects of ethanol
sensitivity has not been studied in mammals, but different inbred mouse
strains, as well as mice with targeted disruption of the
5-HT1B receptor, show different relative
sensitivities to acute ethanol administration in different behavioral
assays, such as the stationary dowel, rotarod, and loss of righting
reflex tests (Crabbe et al., 1994; Boehm et al., 2000; Browman and
Crabbe, 2000). Together, the data from flies and mice suggest that
different aspects of ethanol sensitivity are both genetically and
neuroanatomically separable.
Ethanol sensitivity and olfactory conditioning
Flies learn to avoid an odor that has been paired previously with
an electric shock, a fact that they can remember for hours to days,
depending on the training paradigm (for review, see Dubnau and Tully,
1998). cAMP signaling plays a central role in this classical
conditioning paradigm (for review, see Davis, 1996; Dubnau and Tully,
1998). Interestingly, the genetic overlap between olfactory
conditioning and ethanol sensitivity includes not only rut,
amn, and pka-C but also the cell adhesion
molecule fasciclin II (Cheng et al., 2001).
Because the mechanisms that regulate olfactory learning and ethanol
sensitivity appear to share molecular components, we investigated whether some of the same neuroanatomical structures might be involved in both behaviors. Multiple different lines of experimentation, including hydroxyurea-induced MB ablation (de Belle and Heisenberg, 1994), have shown that these structures play a central role in olfactory learning and memory (for review, see Roman and Davis, 2001).
In contrast, our experiments show that MBs are not necessary for proper
regulation of ethanol sensitivity measured in the inebriometer.
amn, although not expressed in the MBs, is expressed in the
dorsal paired medial (DPM) interneurons, which project to the neuropil
containing MB axons; preventing neurotransmission of these neurons
impairs olfactory learning (Waddell et al., 2000). It seems unlikely
that amn expressed in the DPM neurons plays a role in
regulating ethanol sensitivity, because ablation of the presumed DPM
targets, the MBs, has no effect. Thus, amn expressed elsewhere in the nervous system (Waddell et al., 2000) must modulate ethanol sensitivity. As discussed above, an interesting possibility is
the neuroendocrine hypocerebral ganglion-corpus cardiacum complex. Consistent with distinct roles for amn in ethanol
sensitivity and olfactory learning is the finding that the ethanol
sensitivity defect of amn can be rescued by ubiquitous
expression of an amn transgene in the adult fly, a protocol
that fails to restore normal learning (Moore et al., 1998; DeZazzo et
al., 1999).
These observations show that despite the overlap among the genes
involved in regulating ethanol sensitivity and olfactory conditioning,
the neural circuitry underlying these behaviors is separable. It is
therefore important to determine not only the identity of genes
involved in the behavior of interest but also the neuroanatomical
circuits that regulate its manifestations. The ability to manipulate
subsets of neurons in the fly's nervous system allows dissection of
neuroanatomical loci regulating specific behaviors, in this case
different aspects of ethanol sensitivity, allowing exploration of both
the molecular and neural circuitry underlying behavior.
| |
FOOTNOTES |
Received April 9, 2002; revised July 15, 2002; accepted Aug. 9, 2002.
This work was supported by an award from the McKnight Foundation for
Neuroscience (U.H.), National Institutes of Health Grant AA10035
(U.H.), and the Medical Scientist Training Program (A.R.R.). We are
grateful to Kim Kaiser, Dan Kalderon, Grae Davis, Leslie Griffith,
Thomas Siegmund, Gunter Korge, and Cahir O'Kane for providing fly
lines used in this study. Kei Ito generously shared unpublished lines
and knowledge of fly neuroanatomy. This manuscript benefited from the
comments of Linus Tsai, Adrian Rothenfluh, Doug Guarnieri, Henrike
Scholz, Fred Wolf, and Ammon Corl. We thank Linus Tsai for outcrossing
many of the lines used as well as for thoughtful suggestions throughout
the project.
Correspondence should be addressed to Ulrike Heberlein, 513 Parnassus
Avenue, S-1332, San Francisco, CA 94143-0452. E-mail: ulrike{at}itsa.ucsf.edu.
| |
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ABSTRACT
INTRODUCTION
