Transparent Peer Review - Nature Template 2022 (9) - Copy - Copy

Peer

Review

File

Manuscript

Title:

TRANSFORMATIONS

AND

FUNCTIONS

NEURAL

REPRESENTATIONS

SOCIAL

BEHAVIOR

NETWORK

Editorial

Notes:

Redactions

–

unpublished

data

Parts

this

Peer

Review

File

have

been

redacted

indicated

maintain

the

confidentiality

of unpublished

data.

Reviewer

Comments

Author

Rebuttals

Reviewer

Reports

the

Initial

Version:

Referees'

comments:

Referee

(Remarks

the

Author):

“BNST

promotes

regional

male

bias

within

female

biased

circuit

controlling

social

behavior

male mice” Yang and Anderson start with knowledge from a previous Shah lab study of bulk activity

of the

AB+

BNSTpr

neurons

which

concluded

that

they

encode

representations

of gender in

the

male

brain in an intensity

-dependent manner. The present study seeks to dissect the role of BNST-

ESR

neurons (a subset of AB+ neurons) that project to either the MPOA or the VMHvl using single cell

imaging. The approach is well used and the depth of the analysis performed is complete and

convincing, leading to technically rigorous

experiments. However, the difference between what was

previously shown and what is now understood is subtle and less convincing. Moreover, neither the

previous or the current study convince me that these neurons are actually encoding sex

representations, largely because in the previous study they were not found to encode gender in the

female brain (and it was not studied here). Is it likely that the female brain has evolved an entirely

different mechanism to encode such a key percept? The analysis in this study nicely supports that

they are responding to an analog comparison but it could be another function, such as motivation,

that drives their activity. Though it may be intuitive to think that they are encoding sex and is

supported by the PC and in silico analysis, it is not empirically shown by experimental means. It is

correlative.

The

author’s statement

that it

is “paradoxical that silencing

of BNSTpreEsr1

neurons

had

no effect on our ability to decode intruder sex from population activity in either the MPOA or

VMHvl” indicates that the data, model, and hypothesis are not yet fully aligned. The other major

finding from this study is the flexibility of the neural code downstream of the BNST. This part of the

conclusion echos back to earlier work from the Anderson lab finding scalable control of mounting

and attack in the VMH, and work from the Lin lab has shown heterogeneity in the VMH

-Esr

pulation

that

correlates

with

promoting

different

social

behaviors

(Hashikawa,

2017).

This

extends

some of that previous work by focusing on the function of the BNST to gate differential activity, but

there is no mechanistic understanding of how these neur

ons are dynamically recruited or how their

addition alters natural social behavior. Though I understand the meaning of the title, I do not agree

that the statistical difference of sampling of several 100 neurons in one area of the brain should be

interpret

as functional

male

or female

bias.

Though

the

experiments

in this

study are

elegant,

they

do not serve to clarify the significance of the BNST in the social behavior circuit and the

representation of sex in the brain.

Additional

concerns:

1) From fig 3 onwards, where the chemoinhibition

of BNST Esr1 neurons is being used to draw

conclusions about the information content in the VMHvl Esr1 and the MPOA Esr1 neurons, the

stated hypothesis is "that inhibiting BNSTprEsr1 neurons should alter sex representations in

MPOAand VMHvl."

To establish

this, it would be clearer to inhibit BNST Esr1 neurons, and then

image

neurons

in the

VMHvl

or MPOA

that

express

Esr1

AND

are

downstream

of the

BNST

Esr1

(or

BNST) neurons. In the absence of this kind of experiment, it is difficult to formally state that the

BNST

- VMHvl

or BNST

-MPOA

projection

has

anything

to do

with

the

distortions

in representations

at the

VMHvl

MPOA.

The

above

concern

also

applied

behavior.

When

the

BNST

Esr

VMH

Esr

MPOA

Esr

specifically

silenced,

what

the

effect

behavior?

The

rationale

focus

the

Esr

population

not

clear.

Are

the

ESR

negative

subset

neurons

not

responding

to male

and

female

cues

or projecting

to the

MPOA

and

VMH?

If not,

what

is their role in the model?

Many

the

BNST

Esr+

neurons

are

not

active

the

presence

either

male

female

(Figure

2). Are these neurons also projecting to the MPOA or VMH? The use of mating and aggression as a

behavioral proxy for sex identification is not very granular. Maybe the BNST contributes to other

aspects of social motivation that result in the phenotypes in figure 1. Do the authors have another

measure

for

sex

recognition

that

could

support

the

functional

conclusions

of the

Esr

neurons

driving

sex recognition?

Are

all

the

BNST

Esr

neurons

that

project

the

two

studied

targets

inhibitory?

not,

this

could

confound

the

model.

possible

manipulate

the

sensory

signals

that

BNST

and

VMH

activity

can

monitored

they

mis

-identify

the

sex

of the

partner?

A gender

illusion?

This

would

control

for

all

other

aspects

behavior and neural activity.

The

model

(EDF8)

suggests

that

absolute

incoming

sensory

activity

weighed,

female

activity

= mating,

male

activity

= aggression.

Can

you

manipulate

this

allowing

your

subjects

to interact with multiple females and a single male simultaneously? It looks like the BNST activity

persists as long as a female is present (fig 2j). Is this correct? If the subject first interacts with

females

and

then

a male

is also

added

subsequently,

does

the

simultaneous

presence

of a male

alter

the balance of activity in the VMH? (In both cases, I expect the presence of a male would promote

aggression, but the representation of male and female shoul

d not change.)

Figure

4a,

appears

that

with

CNO

(BNST

)

the

female

responding

cells

are

largely

spatially

segregated

from

the

male

responding

cells

and

about

half

of the

male

responding

cells

remain

male

responding. Are these two populations, those that switch vs retain sex tuning without BNST input

different molecular subsets of the VMHvl-

ESR population? On repeated trials, is the same neuron

able to switch sometimes and remain stable other times, or

are they set to be either flexible or

fixed?

Minor

Concerns:

I cannot

find

CNO

only

controls

neural

activity

and

behavior,

and

some

quantification/analysis

for the silencing of BNST

-Esr expressing hm4di neurons.

The

term

chemoscope

doesn't

seem

to add

much,

it is fundamentally

chemogenetics

and

regular

miniscope imaging combined.

Fig

Representative

images

of GCaMP

infections

in BNSTEsr1

neurons

will

useful.

Fig

Representative

images

of DREADDs

infections

in BNSTEsr1

neurons

+ GCaMP

MPOA/VMHvl Esr1 neurons will be useful.

Ext

Fig

Why

are

error

bars

missing

Referee

(Remarks

the

Author):

Neural

circuits

that

mediate

social

behaviors

mating

and

aggression

are

not

well understood.

This

paper is a technical tour

-de

-force, using in vivo calcium imaging in several limbic system nuclei of

awake

mice

to provide

single

neuron

representations

during

male

and

female

social

encounters.

This

study significantly advances our understanding of sensory coding in

the BNST (a limbic system

structure that receives chemosensory inputs), revealing that different neurons are tuned to male

and female sensory cues,

with smaller groups

of neurons activated during behavioral displays rather

than by sensory cues per se. Pri

or studies involving fiber photometry in this region were important,

claiming a female

-bias in the overall response, but as they lacked single neuron resolution, they

missed the presence of male-

selective neurons, which are sparser, as well as the distribu

tion of

neurons tuned to sensory vs motor actions. Moreover, Yang and Anderson used chemogenetics to

show that BNST inputs re

-shape sex representations in a downstream hypothalamic nucleus (the

VMH). I am enthusiastic about this study, but also note that s

ome additional controls are needed to

validate

claims and

that

some questions remain

to the

mechanism underlying the interesting

BNST

-VMH transformation.

interesting

that

the

VMH

and

MPOA

still

display

sex

biased

responses

after

chemogenetic

inhibition

of the

BNST.

This

could

due

to factors

discussed,

such

as a role

for

another

brain

region,

but also

could

due to

incomplete

BNST inhibition

in chemogenetic

experiments, related to

either

(1)

the

extent

of coverage

of the

large

BNST

area

AAV

injections,

and

2) the

Cre

lines

used.

For

(1),

what

% of Cre

-expressing

BNST

cells

are

labeled

and

silenced

AAV

injections,

and

for

(2)

are

there

sex

-selective BNST neurons that do not express aromatase and/or ESR and could

contributing to

downstream representations?

The

authors

should

ensure

that

AAV

injection

the

BNST

does

not

label

neurons

the

VMH

MPOA

(for

example

retroactive

labeling

of neurons

providing

feedback

control),

which

could

confound

interpretations.

The

authors

use

nicely

comprehensive

set

stimuli

Figure

2j,

and

showed

that

BNST

response variance due to intruder sex was larger than the associated behavior. It would be worth

discussing

these

observations

in the

context

of the

lab's

work

VMH/MPOA.

Is there

increased response variance due to behavior in the VMH and MPOA, suggesting further input

transformation as information moves to the hypothalamus?

The

authors

focus

here

neuronal

epresentations

male

mice;

similar

sex

biases

BNST

and

VMH

exist

female

mice?

Both

the

title

and

last

sentence

the

abstract

should

edited

for

clarity

make

the

manuscript

accessible

to a general

audience.

(I would

with

something

'Transformations

of sex

representations in the ascending limbic system' but of course this is just a suggestion!)

For

4b,

seems

activity

synchronized

particular

events

the

time

series

true,

would

helpful

to provide

annotation

of whether

such

synchronized

events

correspond

sniffing

or other social episodes.

Referee

(Remarks

the

Author):

Yang

Anderson

demonstrate

that

chemogenetic

silencing

BNSTprEsr1

alters

sexual

behavior

and

aggression. Optogenetic silencing of this same neuronal population showed that activity of

BNSTprEsr1

neurons

is required

for

the

transition

from

appetitive

to consummatory

social

behaviors

towards both sexes (i.e. initiation and duration of aggression and

sexual behavior).

These findings (reported already in previous studies, as noted by the authors) set the foundation to

determine

how

sex

is presented

in this

neuronal

population.

The

authors

performed

calcium

imaging

of the BNSTprEsr1 neuronal population in sexually experienced male mice during social interaction

with an intruder male or female, using a miniature head

-mounted microscope, and specifically

monitored the decoding of the intruder's sex, at a single cell level. They identified subpopulations

that were female preferring or male preferring, with or without physical interaction with the

intruders, suggesting that sex is represented by population coding.

Next,

they

combined

chemogenetic

silencing

of BNSTprEsr1

neurons,

with

microendoscopic

imaging

of VMHvlEsr1 or MPOAEsr1 neurons expressing. They revealed that such manipulation led to a

decrease in the neuronal response to male intruders in both MPOA and VMHvl neurons, and an

increase in the response to female intruders in VMHvl. Moreover, they revealed that silencing

BNSTprEsr1 neurons inverted the 2:1 ratio of male

- to female

-preferring units in VMHvl, to the

female

-dominant ratio seen in MPOA.

The

main

novel

findings

and

conclusion

of the

authors

is that

"the

activity

of BNSTprEsr1

neurons

not

required for the coding of intruder sex identity by MPOAEsr1

and

VMHvlEsr1

neurons.

Rather,

it is required

to invert,

in VMHvl,

the

female

bias

in population

representations of intruder sex seen in BNSTpr, MPOA and MeApd, to a male bias".

The

set

methodologies

used

impressive,

contain

appropriate

controls

and

the

data

analyzed

well.

doubt

will

provide

great

dataset

for

better

understanding

how

sex

specific

stimuli

are

encoded

in neuronal

networks,

at single

cell

resolutions.

However, the neuroimaging-

chemogenetic data set (the main part which provide novel findings),

although interesting, is purely descriptive and missing any mechanistic explanation (beyond the

proposed hypothesis). Namely, how does sex

-biased neu

ronal coding in the MPOA/VMH/BNST (or

the altered sex-

bias in the VMH following chemogenetic silencing) encode sex

-typical stimuli and

control different reproductive behaviors (mating and aggression) towards males and females? As

stated by the authors them

selves in the discussion, the manuscript does not provide any

experimental data to answer whether and how the changes in the ratio of female-

male responsive

neurons induced by silencing of BNSTprEsr1 are required for sex discrimination, sex

-typical

sexual/

aggressive

behavior,

or any

other

phenotype.

Moreover,

it is essential

to confirm

the

findings

with additional complementary manipulations such as chemogenetic or optogenetic activation of

the same neuronal population.

Additional

concerns:

1. The use

of restrained

individuals as social

stimui (first set of

experiment) is

very problematic, as

may

trigger

a massive

stress

response

in both

mice.

If the

authors

wish

to examine

the

response

conspecific mouse without enabling attack or mount responses,

they can present the conspecific

separated by a perforated barrier, or use a stimuli such as urine or soiled bedding.

The

link

between

the

behavioral

and

neural

effects

the

chemogenetic

silencing

poorly

explained,

thus

not

clear

what

the

biological/functional

significance

the

neural

findings.

continuous

the

prior

comment,

did

the

authors

notice

any

sex

reversed

behaviors

during

the

silencing

period

(i.e.

attack

of subject

females

or sexual

behaviors

towards

males)?

These

behavior

should be quantified for pre

CNO and CNO segments.

Also,

the

silencing

effect

reversible?

What

would

happen

optogenetic

chemogenetic

activation

these

neurons?

order

support

their

sex

discrimination

claim,

the

authors

need

conduct

separate

discrimination

assay,

during

BNSTprEsr1

silencing.

the

response

characteristics

specific

neurons

remain

stable

for

days/weeks?

Author

Rebuttals

Initial

Comments:

Nature

2021

04882

Yang

al.

Point

point

response

Reviewer

We thank this reviewer for their incisive and helpful questions and comments. While it was

not

possible

to perform

every

experiment

requested,

both

for

technical

reasons

and

because

of the contraction of our mouse

colonies necessitated by the COVID pandemic, we have

done

our

best

provide

new

data

address

them.

Comment

“neither

the

current

study

convince

that

these

neurons

are

actually

encoding sex

representations,

largely

because

in the

study

they

were

not

found to encode gender in the female brain (and it was not studied here).”

Response

: We appreciate the reviewer’s comment, but find it puzzling. We do not

understand why evaluating the function of a neuronal population i

n males is

dependent on knowing what a similar population does in females (or vice-

versa),

especially when the circuit we are studying is well

-known for sexual dimorphisms in

structure and function. Perhaps the reviewer thinks that by

“sex representations”

mean a representation of the animal’s OWN sex?

If so, that is a misunderstanding:

we meant the representation of the sex of a conspecific intruder, either male or

female (see Remedios et al. (2017)

Nature

550

:388

-392). In any case, as detailed

below our new data indicate that while BNSTpr may contain a neural representation

of intruder

sex

(or

a sex

-specific

internal

state),

it is not

required

functionally

males

to identify

and

distinguish

male

from

female

conspecifics.

This

in turn

argues

that

the

requirement for BNSTpr in mounting and attack behavior is not an indirect

consequence of deficient sex identification. These new findings have substantially

changed our view of BNSTpr function, as explained in our responses below and in

the

revised

man

uscript.

Comment 2.

“The analysis in this study nicely supports that they are responding to an

analog comparison, but it could be another function, such as motivation, that drives their

activity. Though

it may

be intuitive

to think

that

they

are

encodin

g sex

and

is supported

the PC and in silico analysis, it is not empirically shown by experimental means. It is

correlative.”

Response

: The reviewer is correct that we cannot distinguish a function in encoding

sex from encoding a

motivational state that is closely associated with the intruder’s

sex.

Our

experiments

using

functional

silencing

demonstrate

that

BNSTpr

Esr1

neurons

are necessary for the transition from sniffing to consummatory behavior (mating or

fighting).

have

now

revised

our

manuscript

to indicate

that

BNSTpr

could

encode

either

intruder

sex,

or a motivational

state

that

is strongly

correlated

with

intruder

sex,

pg. 10

and

12.

Comment 3.

“From fig 3 onwards, where the chemo

inhibition of BNST Esr1 neurons is

being used to draw conclusions about the information content in the VMHvl Esr1 and the

MPOA Esr1 neurons, the stated hypothesis is "that

inhibiting BNSTprEsr1 neurons should

alter sex

representations in MPOA and VMHvl

." To establish this, it would be clearer to

inhibit

BNST Esr1

neurons,

and

then

image

neurons

in the

VMHvl

or MPOA

that

express

Esr1

AND are downstream of

the BNST

Esr1 (or BNST) neurons

. In

the

absence of

this

kind of experiment, it is difficult to formally state that the BNST

-VMHvl or BNST

-MPOA

projection

has

anything

with

the

distortions

representations

the

VMHvl

MPOA”.

Response

: The experiment the reviewer suggests (

bolded

above) is not technically

feasible

at present.

our

knowledge,

there

is no method

that

will

efficiently

transfer

GCaMP anterogradely from a pre

-synaptic neuron to its post

-synaptic target while

maintaining

the

viability

of the

latter

cells

for

imaging.

However, we

now

present

new

data

Fig.

showing

that

silencing

of BNSTpr

Esr1

terminals

in VMHvl

and

MPOA

blocks attack and mating, respectively (but not vice-

versa). We also present new

data showing that the female-

biased representations of intruder sex are present in

BNSTpr

neurons

that

project

to MPOA

and

VMHvl

(

Extended

Data

Fig.

Together

these data make it highly likely that the BNSTpr



VMHvl and BNSTpr



MPOA

projections control sex representations in these two hypothalamic nuclei.

Nevertheless,

have

included

a caveat

to state

that

cannot

formally

exclude

indirect

influences

of BNSTpr

silencing

on the

altered

representations

in VMHvl

MPOA (

pg. 11)

Comment

“The

above

concern

also

applied

behavior.

When

the

BNST

Esr

VMH

Esr

MPOA

Esr

specifically

silenced,

what

the

effect

behavior?”

Response

have

now

performed

these

experiments,

suggested

the

reviewer.

have

silenced

BNSTpr

Esr1

terminals

VMHvl

and

MPOA

using

Halorhodopsin,

during

resident

intruder

assays.

Silencing

the

BNSTpr

Esr1



VMHvl

projections strongly inhibited ongoing aggression towards males, while causing a

modest reduction in the transition from approach/sniff to mounting towards females;

Silencing

of the

BNSTpr

Esr1



MPOA

projections

strongly

inhibited

the

transition

from

approach/sniff to mounting towards females, while having little effect on male-

male

aggression. These results phenocopy the effects of silencing BNSTpr cell bodies

(

Fig. 1

) and therefore cannot be ascribed to

paradoxical effects of eNpHR3.0 at

nerve

terminals.

have

now

included

these

results in

the

revised

manuscript

pg. 3

and

Figure

Comment

“The

rationale

focus

the

Esr

population

not

clear.

Are

the

ESR

negative

subset

of AB

neurons

not

responding

to male

and

female

cues

or projecting

the

MPOA and VMH? If not, what is their role in the model?”

Response

focused

the

Esr1

populations

all

structures

because

these

neurons have been functionally implicated in sniffing,

mounting and attack by

perturbation experiments in VMHvl and MPOA; 2) it allowed us to silence Esr1

neurons in BNSTpr while simultaneously imaging Esr1

neurons in VMHvl and

MPOA, using the same Esr1

-Cre driver, simplifying the genetics; Aromatase-

Cre is

not

useful

for

that

purpose.

In response to the reviewer’s question, AB neurons constitute a relatively minor

population in BNSTpr. Aromatase

marks

a subset of Esr1

neurons, and

the Esr1

negative

subset

of AB

neurons

is even

smaller.

[REDACTED]

The

curiosity

driven

question

of determining

the

role

of the

Esr1

-negative

subset of AB

neurons

would

require

complex,

expensive

and

time

-consuming

genetic

intersectional

strategies,

and

tangential

the

central

point

this

paper.

Comment 6.

“Many of

the BNST

Esr+ neurons are not active in the presence of either a

male

or female

(Figure

2).

Are

these

neurons

also

projecting

to the

MPOA

or VMH?

The

use

of mating and aggression as a behavioral proxy for sex identification is not very granular.

Maybe

the

BNST

contributes

other

aspects

social

motivation

that

result

the

phenotypes

figure

the

authors

have

another

measure

for

sex

recognition

that

could

support

the

functional

conclusions

the

Esr

neurons

driving

sex

recognition

?”

Response

: In response to the reviewer’s initial question, we believe that most of the

BNSTpr

Esr1

neurons

that

are

not

active

in the

presence

of either

a male

or female

are

either interneurons, or cells that do not project to VMHvl

or MPOA. To confirm this,

we have performed a new

imaging analysis of BNSTpr

Esr1



VMHvl and BNSTpr

Esr1



MPOA

projection

neurons

(labeled

by retrograde

delivery

of GCaMP),

and

show

that

most

of the

back

-labeled

BNSTpr

Esr1

neurons

respond

to either

male

or female

cues (

ED Figure 9g

To address the reviewer’s second point (italics), we have performed optogenetic

silencing of

BNSTpr

Esr1

neurons

during

male

vs.

female

urine

preference

assays

and

(pencil cup-

enclosed) male vs. female preference tests.

Our results indicate that the

preferences

for

female

vs.

male

urine,

as well

as the

preference

for

interacting

with

female vs. a male restrained in a pencil cup, are lost upon BNSTpr

Es1

silencing,

consistent with results reported in Bayless et al. (2019)

. Surprisingly, however, we

found that despite the loss of

preference

for female cues when BNSTpr is silenced,

ultrasonic vocalizations (USVs) towards females or female urine but not males or

male

urine;

Karigo

al.

2021

Nature

)

remained

intact

(

Figure

)

This unexpected new finding has caused us to revise our original conclusion. It

suggests that in sexually experienced males, activity in BNSTpr is not required to

identify or recognize intruder sex, but rather to exhibit a

preference

for

female cues

over male cues. (By analogy, a person who once preferred apples to oranges may

lose

that

preference,

while

still

being

able

to identify

apples

vs.

oranges

apart

their

smell.) The observation that sex recognition is still intact following BNS

Tpr silencing

fits with our original observation that intruder sex

can still be efficiently decoded from

neuronal activity in either MPOA

Esr1

or VMHvl

Esr1

neurons following such silencing

(Fig. 3l, 3m). These data suggest that the deficits in mounting and attack caused by

silencing BNSTpr neurons cannot be explained by a failure of sex identification,

[REDACTED]. Rather, when taken together with our new results from silencing

BNSTpr



MPOa/VMHvl terminals during social interactions (see

ED Figure 2

and

response

to Comment

4, above),

the

data

suggest

that

BNSTpr

Esr1

silencing

prevents

mounting and attack as a consequence of its effects on neural activity and sex

representations

VMHvl

and

MPOA.

have

now

included

these

results

the

revised

manuscript

pg.

and

illustrated

them

with

new

diagram

Fig.

summary,

our

new

data

argue

for a

view

of BNSTpr

function

that

substantially

different

from

that

suggested

[REDACTED].

thank

the

reviewer

for

suggesting

the

key

experiments

that

brought

this

important

revision

light.

Comment

“Are

all

the

BNST

Esr

neurons

that

project

the

two

studied

targets

inhibitory?

not,

this

could

confound

the

model.”

Response

: We thank the reviewer for raising this important question. Recently

published BNSTpr single

-cell RNA sequencing data (Welch et al., 2019, Cell, 177,

1873–

1887) indicate that 95% of the BNSTpr

Esr1

neurons are inhibitory. Thus, it’s

reasonable to assume th

at the majority of the BNSTpr

Esr1

projection neurons are

inhibitory as well. To confirm this, we have performed new experiments using cre-

dependent

retrograde

AAVs

expressing

mNeongreen

or mScarlet

in MPOA

VMHvl

of VGAT

-Cre mice. The results (

ED Fig. 9

a-f

) indicate that most of the back labeled

VGAT+ neurons are Esr1

. We have now included both the citations and our

retrograde

tracing

results

the

revised

manuscript

pg.

Comment

“Is

possible

manipulate

the

sensory

signals

that

BNST

and

VMH

activity

can

monitored

as they

mis

-identify

the

sex

of the

partner?

A gender

illusion?

This

would

control for all other aspects of behavior and neural activity.”

Response

appreciate

the

utility

the

experiment

suggested

the

reviewer,

principle.

Unfortunately, there is no validated procedure for creating “gender

illusions” in mice. However, we present dual

-site (VMHvl and MPOA) fiber

photometry data as a reviewer figure (Reviewer Figure 1) from triadic interactions

with bot

h a male and a female intruder, in which paradoxical male-

directed sexual

behavior (which could reflect a “gender illusion” of the sort referred to by the

reviewer) was observed. These results are described in detail in our response to

comment

below.

Comment 9.

“The model (EDF8) suggests that absolute incoming sensory activity is

weighed, more female activity = mating, more male activity = aggression. Can you

manipulate

this

by allowing

your

subjects

to interact

with

multiple

females

and

a single

male

simultaneously?

looks

the

BNST

activity

persists

long

female

present

(fig

2j).

Is this correct?

If the subject first interacts with females and then a male is also added

subsequently,

does

the

simultaneous

presence

of a male

alter

the

balance

of activity

the

VMH

? (In both cases, I expect the presence of a male would promote aggression, but the

representation

male

and

female

should

not

change.)

Response

: The exact experiment the reviewer requested is extremely

difficult to

perform, for reasons that will become apparent in the following description. As an

approximation

to this

experiment,

have

now

included

bulk

calcium

measurements

acquired by dual fiber

-photometry performed simultaneously in VMHvl and MPOA

r1+

neurons

in the

same animal

(described and validated in in Karigo

et al., 2021,

Nature

), recorded during

triadic interactions

between a resident male, an intruder

female and an intruder male present in the same cage

(Reviewer Fig. 1

), as the

reviewer

suggested.

provide

examples

from

two

such

interactions.

In both

cases,

the male resident first interacts with the female intruder and then interacts with the

male

intruder.

In the first, characteristic example (

Reviewer Fig. 1

upper recording)

when

the

recorded

male

is sniffing

or mounting

the

female

(purple

outlined

box;

red

and

green

rasters, respectively), activity in MPOA (purple trace) is higher than in VMHvl (gray

trace). The converse (VMHvl>MPOA activity) is observed when the resident is

sniff

ing the male intruder (gray box; blue raster). Towards the end of the recording

session, as the animal switches from mounting a female to sniffing a male, MPOA

activity

rapidly

declines

and

VMHvl

activity

increases

(red

box).

These results from a triadic interaction confirm observations made on dyadic pairs

published in Karigo et al. (2021), which showed that in the presence of a female

intruder, MPOA>>VMHvl activity, while in the presence of a male intruder,

VMHvl>MPOA

activit

y. These

opposite

ratios

of MPOA:VMHvl

activity

in bulk

calcium

measurements reflect the fact that in MPOA (as in BNSTpr), female-

tuned neurons

outnumber male tuned neurons by ~2:1, whereas the converse is true in VMHvl

(Remedios

al.,

2017;

Karigo

2021).

The

lower recording in

Reviewer Fig. 1

shows a

rare case

of what appears to

be a

“gender illusion,” as mentioned by the reviewer in comment 8.

In this [different]

triadic assay, following a sniffing/mounting interaction with the f

emale (purple box,

red and green rasters), the male exhibits paradoxical

sexual

mounting (marked by

ultrasonic vocalizations, USVs; see Karigo et al. 2021) towards the intruder male

(yellow raster). During this USV

male mounting behavior, MPOA activity initially

increases

relative

to VMHvl

activity

(red

box

1, dashed

vertical

line),

the

opposite

what is typically observed (see above description of upper recording).

The

fact that

the

resident

male

sings

when

mounting

the

male

intruder

confirms

that

perceives

the intruder as a female (Karigo et al., 2021). Following this male-

directed USV

mounting

bout,

the

animal

resumes

sniffing

the

male.

During

that

period,

MPOA

[REDACTED]

activity

slowly

falls

while

VMHvl

activity

increases

(lower

recording,

red

box

traces

to right of dashed vertical line). This suggests the resident male now perceives this

intruder as a male. Eventually, following another interaction with the female, during

sniffing of the male (gray box), the relative level of

MPOA vs. VMHvl activity flips,

reflecting the typical pattern (upper panel): MPOA shows a characteristic decline in

activity, while VMHvl activity rises.

The

data

in the

second

(lower)

recording

suggest

that

the

initial

attempted

sexual

mounting

the

mal

(red

box

may

reflect

spontaneous

“gender

illusion,”

which

the

resident’s

brain

mistakenly

identified

the

male

intruder as

female

(MPOA

activity>VMHvl

activity).

They

reinforce

our

conclusion

that

activity

MPOA

and VMHvl does not simply reflect sensory responses to male-

or female

-specific

cues, but rather a percept of intruder sex (Karigo et al., 2021). These data further

support the

reviewer’s inference

that more

“female activity” (MPOA>VMHvl) leads

mat

ing

and

“male

activity”

(VMHvl>MPOA)

leads

to aggression,

consistent

with

the female-

vs. male

- tuning bias in MPOA vs. VMHvl, respectively. Unfortunately,

the low frequency of these spontaneous “gender illusion” events (1 in ~20 mice

tested) makes it

impractical to perform single-

cell calcium imaging during such

events,

due

to the

very

large

number

of animals

that

would

have

to be

implanted

with

miniscopes

image

activity

during

even

single

such

behavioral

event.

Comment

10.

“Figure

4a,

appears

that

with

CNO

(BNST

)

the

female

responding

cells

are

largely spatially segregated from the male responding cells and about half of the male

responding cells remain male responding.

Are these two populations, those that switch vs

retain sex tuning without BNST input

different molecular subsets

of the VMHvl

-ESR

population

? On repeated trials, is the same neuron able to switch sometimes and remain

stable

other

times,

are

they

set

either

flexible

fixed?

Response

: The apparent spatial

segregation of female

vs. male

responding cells to

which the reviewer refers is not a consistent observation across animals. To our

knowledge, there is no method that would allow us to track the neurons that switch

vs. retain sex tuning following BNST sil

encing via microendoscopic calcium imaging

in freely

moving animals, and

then determine

the

molecular profile of these neurons,

as the reviewer suggested. (Such an experiment might be possible in head-

fixed

animals,

but

they

will

not

perform

the

social

behaviors

studied

here.)

look

forward

to the time when such technology has been developed. In answer to the last

question, we did not perform repeated CNO trials in the same animals, in order to

prevent experience-

dependent changes in the animals that coul

d confound the

results.

Comment

11.

“I

cannot

find

CNO

only

controls

neural

activity

and

behavior,

and

some

quantification/analysis

for

the

silencing

BNST

Esr

expressing

hm4di

neurons.”

Response

have

now

reported

the

percentage

BNSTpr

Esr1

neurons

that

express

hM4Di,

and

included

CNO

only

controls,

Figure

1c,

Comment

12.

“Fig

Representative

images

GCaMP

infections

BNST

Esr1

neurons

will

be useful.

Response:

have

now

provided

a representative

image

of GCaMP

infections

BNST

Esr1

neurons

Figure

3i.

Comment

13.

“Fig

Representative

images

DREADDs

infections

BNSTEsr1

neurons

GCaMP

MPOA/VMHvl

Esr1

neurons

will

useful.”

Response

have

added

the

requested

images

Figure

well

schematic

diagrams

facilitate

their

interpretation

the

reader

(

Fig.

5a,

Comment

14.

“Ext

[Data]

Fig

“Why

are

error

bars

missing

k?”

Response

only

imaged

single

animal

(the

only

one

of 2

implanted

animals

that

exhibited adequate GCaMP expression), to confirm that separate populations of

female-

and male-

tuned neurons exist in the BNST aromatase+ (AB) population.

Therefore

error

bars

were

shown.

This

experiment

is simply

existence

proof

demonstrate

that

the

difference

between

our

conclusions

and

those

of Bayless

et al.

(2019) is not due to the use of different Cre drivers.

again

thank

this

reviewer

for

their

insightful

and

thoughtful

questions,

and

hope

that

our

efforts

address

them

have

improved

the

paper.

Reviewer

thank

the

reviewer

for

their

helpful

questions

and

suggestions,

which

respond

below.

Comment 1.

“It is interesting that the VMH and MPOA still display sex

biased responses

after

chemogenetic

inhibition

of the

BNST.

This

could

due

to factors

discussed,

such

role for another brain region, but also could be due to incomplete BNST inhibition in

chemogenetic

experiments, related to either (1) the extent of coverage of the large BNST

area by AAV injections, and 2) the Cre lines used. For (1), what % of Cre

-expressing BNST

cells are labeled and silenced by AAV injections, and for (2) are there sex

-selective BNS

neurons that do not express aromatase and/or ESR and could be contributing to

downstream

representations?”

Response

: These are reasonable questions, for which we thank the reviewer. To

address them, we have now included a panel showing

that over 90% of the Esr1

BNSTpr neurons are labeled by AAV expressing hM4D

-mCherry, as the reviewer

requested (

ED figure 1c

). Single cell RNA

-seq data (Welch et al., 2019, Cell, 177,

1873–

1887) indicate that 25% of the inhibitory population in BNSTpr is

Esr1

. We

cannot exclude that there would be a stronger behavioral or circuit

-level perturbation

if additional, non

-Esr1

BNSTpr neurons were silenced. That said, if silencing the

Esr1

subset

of BNSTpr

neurons

was

inadequate

to strongly

perturb

the

syste

then

we would have expected to see NO behavioral phenotype.

To the contrary, we

observed very robust and highly penetrant behavioral phenotypes, including the loss

of preference for female over male cues, and the inhibition of both mounting and

attack

(Fig.

1f,

and

Fig.

1b).

Comment

“The

authors

should

ensure

that

AAV

injection

the

BNST

does

not

label

neurons

in the

VMH

or MPOA

(for

example

by retroactive

labeling

of neurons

providing

feedback control), which could confound

interpretations.”

Response

The

reviewer

correct

bring

this

important

concern.

have

added

a figure

demonstrating

the

lack

of cell

body

labeling

in VMHvl

and

MPOA

following AAV injections into BNST in

ED Figure 5a

Comment 3

. “The

authors use a nicely comprehensive set of stimuli in Figure 2j, and

showed that BNST response variance due to intruder sex was larger than the associated

behavior.

It would

worth

discussing

these

observations

in the

context

of the

lab's

work on

VMH/MPOA. Is there an increased response variance due to behavior in the VMH

and MPOA, suggesting further input transformation as information moves to the

hypothalamus?”

Response

: The reviewer brings up a great point.

There is indeed an increase

fraction of variance explained by behavior in MPOA, relative to BNST, and we have

now mentioned those data in the

revised manuscript pg. 9 and Figure 5g

. In

VMHvl,

the

fraction

of variance

due

to intruder

sex

is larger

than

that

due

behavior,

as descr

ibed in Karigo et al. (2021). These results are now summarized in a new

diagram

Fig.

5m.

Comment

The

authors

focus

here

neuronal

representations

male

mice;

similar

sex

biases

BNST

and

VMH

exist

female

mice?

Response

: This is an important question that we appreciate, but it would require

repeating all of the imaging experiments in female mice, with animals analyzed at

different

stages

during

the

estrus

cycle.

This

represents

a large

amount

additional

work

that

likely

would

raise

questions

than

it would

answer.

therefore

feel

appropriate

for

separate,

study.

Comment 5

. “Both the title and last sentence of the abstract should be edited for clarity to

make

the

manuscript

accessible

to a general

audience.

(I would

go with

something

'Transformations

of sex

representations in

the ascending limbic

system' but of course

this is

just

suggestion!)”

Response

appreciate

the

suggestion

and

have

modified

the

title

along

the

lines

the

reviewer’s

suggestion.

Comment

“For

4b,

seems

activity

synchronized

particular

events

the

time

series

- if true,

it would

helpful

to provide

annotation

of whether

such

synchronized

events correspond to sniffing

or other social episodes.”

Response

thank

the

reviewer

for

this

suggestion,

and

have

now

added

such

behavioral

annotation

Figure

4b.

Reviewer

thank

this

reviewer

for

taking

the

time

read

the

manuscript

carefully,

and

for

their

thoughtful

comments.

Comment 1

. “The neuroimaging

chemogenetic data set (the main part which provide novel

findings),

although

interesting,

is purely

descriptive

and

missing

any

mechanistic

explanation

(beyond the proposed hypothesis). Namely, how does sex

-biased neuronal coding in the

MPOA/VMH/BNST (or the altered sex

-bias in the VMH following chemogenetic silencing)

encode sex

-typical stimuli and control different reproductive behaviors (mating and

aggression)

towards

males

and

females?”

Response

respectfully

disagree

with

the

reviewer’s

characterization

our

experiments as “purely descriptive.” We have performed a genetically based

perturbation

experiment

(silencing

BNSTpr)

and

have

characterized

the

phenotypes of that perturbation at both the behavioral and circuit levels, in two

different downstream targets of BNSTpr, a first

-in-class study.

Our observations

reveal

unexpected

transformation

performed

by BNS

Tpr

Esr1

neurons

(inverting

the

ratio

female:male

specific

neurons

VMHvl,

relative

MPOA).

We originally provided two complementary mechanistic hypotheses to explain how

the

observed

perturbations

of neural

activity

could

explain

the

observed

perturbations

in social behavior, following BNSTpr silencing. [REDACTED] In the interests of

distinguishing these hypotheses, and thereby gaining further mechanistic insight as

the reviewer requested, we have performed an extensive additional series of

Che

moScope experiments to monitor activity in MPOA and VMHvl before vs. after

silencing BNSTpr, during unrestrained male-

male and male-

female social

interactions.

(As

the

reviewer

may

recall,

our

original

ChemoScope

experiments

were

performed using dangled intruders, preventing free social interactions). In this

experiment, we performed the DREADD

-mediated silencing of BNSTpr

Esr1

neurons

unilaterally

(rather than bilaterally as in the case of behavioral assays, e.g., Fig. 1).

Because there are very few inter

-hemispheric connections in this circuit, the

unperturbed contralateral side of the brain is able to compensate for silencing on the

ipsilateral (imaged/silenced) side, and therefore social behavior is intact. This design

eliminates

the

confound

that

any

obs

erved

changes

in neural

activity

MPOA/VMHvl

are simply a reflection of changes in behavior.

It also allows us to temporally

correlate alterations in MPOA or VMHvl activity caused by silencing BNSTpr, with

specific behavioral states or transitions made by the animal. The results of these

experiments

are

now

presented

new

Figure

well

Figures

and

These

new

data

include

unexpected

results

that

strongly

favor

one

hypothesis

over

the

other,

and

provide

mechanistic

insights.

There

are

basic

findings

report:

1)[REDACTED] a requirement for BNSTpr in sex recognition was inferred

from the

observation

that

preference

for

female

over

male

cues

was

lost

when

Aromatase

neurons

BNSTpr

were

ablated

chemogenetically

silenced.