Watanabe
et al
. eLife 2023;12:RP92380. DOI: https://doi.org/10.7554/eLife.92380
1 of 20
Whole-
brain in situ mapping of neuronal
activation in
Drosophila
during social
behaviors and optogenetic stimulation
Kiichi Watanabe
1†, ‡
, Hui Chiu
1§
, David J Anderson
1,2
*
1
Division of Biology and Biological Engineering, Tianqiao and Chrissy Chen Institute
for Neuroscience, California Institute of Technology, Pasadena, United States;
2
Howard Hughes Medical Institute, Chevy Chase, United States
eLife Assessment
This work reports an
important
new method for activity-
dependent neuronal labeling in
Drosophila
using in situ hybridization, with the potential to establish a new standard in the field. The authors
demonstrate the method's applicability by generating
compelling
evidence of the function of male-
specific neurons in both aggression and courtship behaviors. These results and the new method will
be of great interest to the neuroscience community.
Abstract
Monitoring neuronal activity at single-
cell resolution in freely moving
Drosophila
engaged in social behaviors is challenging because of their small size and lack of transparency.
Extant methods, such as Flyception, are highly invasive. Whole-
brain calcium imaging in head-
fixed, walking flies is feasible but the animals cannot perform the consummatory phases of social
behaviors like aggression or mating under these conditions. This has left open the fundamental
question of whether neurons identified as functionally important for such behaviors using loss- or
gain-
of-
function screens are actually active during the natural performance of such behaviors, and
if so during which phase(s). Here, we perform brain-
wide mapping of active cells expressing the
Immediate Early Gene
hr38
using a high-
sensitivity/low background fluorescence in situ hybridiza-
tion (FISH) amplification method called HCR-
3.0. Using double-
labeling for hr38 mRNA and for GFP,
we describe the activity of several classes of aggression-
promoting neurons during courtship and
aggression, including P1
a
cells, an intensively studied population of male-
specific interneurons. Using
HI-
FISH in combination with optogenetic activation of aggression-
promoting neurons (opto-
HI-
FISH), we identify candidate downstream functional targets of these cells in a brain-
wide, unbiased
manner. Finally, we compare the activity of P1
a
neurons during sequential performance of courtship
and aggression, using intronic vs. exonic
hr38
probes to differentiate newly synthesized nuclear
transcripts from cytoplasmic transcripts synthesized at an earlier time. These data provide evidence
suggesting that different subsets of P1
a
neurons may be active during courtship vs. aggression.
HI-
FISH and associated methods may help to fill an important lacuna in the armamentarium of tools
for neural circuit analysis in
Drosophila
.
Introduction
Monitoring neuronal activity during specific actions or activities is critical to understanding how the
brain controls behavior. Measurements within a local brain region of neural activity in non-
transparent
awake, behaving animals such as mice or fruit flies can be achieved with electrophysiological and
calcium imaging techniques (
Buzsáki, 2004
;
Grewe and Helmchen, 2009
). In
Drosophila
, two- photon
TOOLS AND RESOURCES
*For correspondence:
wuwei@caltech.edu
Present address:
†
International
Center for Cell and Gene
Therapy, Fujita Health University,
Toyoake, Japan;
‡
Department of
Medical Research for Intractable
Disease, Fujita Health University,
Toyoake, Japan;
§
Department of
Immunobiology, Yale University
School of Medicine, New Haven,
United States
Competing interest:
The authors
declare that no competing
interests exist.
Funding:
See page 15
Sent for Review
16 September 2023
Preprint posted
29 September 2023
Reviewed preprint posted
11 December 2023
Reviewed preprint revised
16 October 2024
Version of Record published
28 November 2024
Reviewing Editor:
Esteban J
Beckwith, Universidad de Buenos
Aires - CONICET, Argentina
Copyright Watanabe
et al
.
This article is distributed under
the terms of the Creative
Commons Attribution License,
which permits unrestricted use
and redistribution provided that
the original author and source
are credited.
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Neuroscience
Watanabe
et al
. eLife 2023;12:RP92380. DOI: https://doi.org/10.7554/eLife.92380
2 of 20
calcium imaging (TPI) has been widely used (
Seelig et al., 2010
) because single-
unit electrical recording
in the fly central brain remains exceedingly difficult (
Turner et al., 2008
;
Wilson et al., 2004
). But TPI
in
Drosophila
requires head-
fixation, which limits the repertoire of behaviors that flies can perform
under these conditions. For example, it is difficult to use TPI to image neuronal activity during the
consummatory phases of complex social behaviors, such as male–male aggression or male–female
courtship (but see
Clowney et al., 2015
). Recently, a new method called Flyception2 was introduced
for brain imaging in freely walking flies (
Grover et al., 2020
;
Grover et al., 2016
). However, the
spatial resolution of this method is limited, it is invasive, and monitoring the activity of single neurons
in freely moving animals is still challenging.
In mice, immediate early genes (IEGs) such as c-
fos, Arc, or Egr1 have been used to map the activity
of neurons by visualizing their expression histologically using immunohistochemistry (IHC) or in situ
hybridization (ISH) (
Morgan and Curran, 1991
). More recently, this method has been combined with
tissue-
clearing and light-
sheet microscopy to perform systematic and unbiased whole-
brain mapping
of neurons activated during different behaviors (
Kim et al., 2015
;
Renier et al., 2016
). An extension
of this approach, called cellular compartment analysis of temporal activity by fluorescence
in
situ
hybridization (catFISH), enables within-
animal comparisons of neuronal populations activated during
two different sequential behaviors (
Guzowski et al., 1999
;
Lin et al., 2011
).
In
Drosophila
, by contrast, the application of IEGs to map whole-
brain neuronal activation patterns
has been limited. In part, this is because c-
fos
is not a particularly good activity marker in flies (
Chen
et al., 2016
). More recently, several studies using fly IEGs such as
Hr38
and
stripe/Egr1
have been
reported (
Chen et al., 2016
;
Fujita et al., 2013
;
Takayanagi-
Kiya et al., 2023
;
Takayanagi- Kiya and
Kiya, 2019
). Nevertheless, this technique has been relatively under-
utilitized in flies. This may be the
case, at least in part, because application of FISH to the adult
Drosophila
brain has been technically
challenging, due to limited probe penetration and high background caused by traditional amplifica-
tion methods such as Tyramide (
Raap et al., 1995
).
To circumvent these problems, we have applied in this study an ISH amplification technique called
the hybridization chain reaction (HCR), v.3.0 (
Choi et al., 2018
) to visualize the expression of
Hr38
, an
IEG expressed by active neurons in the adult male fly brain (
Fujita et al., 2013
). The design of HCR,
which requires conjoint hybridization to target sequences of adjacent pairs of ‘half-
probes’ to achieve
amplification, affords both greater sensitivity and specificity, allowing detection of low-
abundance
transcripts with minimal background noise and off-
target hybridization. We call this approach HCR3.0-
amplified
IEG
Fluorescent
In
Situ
Hybridization, or ‘HI-
FISH’ (
Figure 1A
). Although we use
Hr38
as
proof-
of-
concept, this approach is in principle applicable to any IEG.
Here, we demonstrate three different applications of HI-
FISH relevant to understanding the neural
circuitry underlying social behaviors in flies. First, we have asked whether specific neuronal popu-
lations discovered in functional screens for aggression-
promoting neurons (
Asahina et al., 2014
;
Hoopfer et al., 2015
;
Watanabe et al., 2017
) are indeed active during natural aggressive behavior,
courtship, or both. We have further investigated whether such activity is only observed during the
contact-
mediated consummatory phase of these behaviors, or can also be detected in freely moving
flies in the absence of fighting or mating. Second, we have performed unbiased, whole-
brain func-
tional mapping of the downstream targets activated by optogenetic stimulation of a neuronal popula-
tion of interest, a variant method we call ‘opto-
HI-
FISH’. We have applied this method to P1
a
neurons
(
Anderson, 2016
;
Clowney et al., 2015
;
Hoopfer et al., 2015
;
Inagaki et al., 2014
) a subset of
the male-
specific P1 class of interneurons originally identified based on their ability to trigger male
courtship behavior when stimulated (
von Philipsborn et al., 2011
). Finally, we have developed a
variant of the catFISH technique (
Guzowski et al., 1999
), using dual-
color FISH combining intronic
and exonic
Hr38
probes, to compare neuronal populations activated during two different sequential
behaviors, a method we call ‘HI-
catFISH’. Since experimental activation of P1
a
neurons can promote
both aggression and courtship in pairs of male flies (
Hoopfer et al., 2015
), we have used HI-
catFISH
to investigate whether separate or overlapping subpopulations of P1
a
neurons are active during
natural occurrences of these two social behaviors. Together, the results of these studies illustrate the
utility of HI-
FISH, opto-
HI-
FISH, and HI-
catFISH to detect the activation of both known and previously
unidentified neurons during naturalistic social behaviors that are not amenable to head-
fixation. The
results have also yielded new biological insights into the neural circuit-
level control of aggression
and courtship.