Low intensity repetitive transcranial magnetic stimulation modulates brain-wide functional connectivity to promote anti-correlated c-Fos expression

Abstract

Repetitive transcranial magnetic stimulation (rTMS) induces action potentials to induce plastic changes in the brain with increasing evidence for the therapeutic importance of brain-wide functional network effects of rTMS; however, the influence of sub-action potential threshold (low-intensity; LI-) rTMS on neuronal activity is largely unknown. We investigated whether LI-rTMS modulates neuronal activity and functional connectivity and also specifically assessed modulation of parvalbumin interneuron activity. We conducted a brain-wide analysis of c-Fos, a marker for neuronal activity, in mice that received LI-rTMS to visual cortex. Mice received single or multiple sessions of excitatory 10 Hz LI-rTMS with custom rodent coils or were sham controls. We assessed changes to c-Fos positive cell densities and c-Fos/parvalbumin co-expression. Peak c-Fos expression corresponded with activity during rTMS. We also assessed functional connectivity changes using brain-wide c-Fos-based network analysis. LI-rTMS modulated c-Fos expression in cortical and subcortical regions. c-Fos density changes were most prevalent with acute stimulation, however chronic stimulation decreased parvalbumin interneuron activity, most prominently in the amygdala and striatum. LI-rTMS also increased anti-correlated functional connectivity, with the most prominent effects also in the amygdala and striatum following chronic stimulation. LI-rTMS induces changes in c-Fos expression that suggest modulation of neuronal activity and functional connectivity throughout the brain. Our results suggest that LI-rTMS promotes anticorrelated functional connectivity, possibly due to decreased parvalbumin interneuron activation induced by chronic stimulation. These changes may underpin therapeutic rTMS effects, therefore modulation of subcortical activity supports rTMS for treatment of disorders involving subcortical dysregulation.

Full text

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Low intensity repetitive
transcranial magnetic stimulation
modulates brain‑wide functional
connectivity to promote
anti‑correlated c‑Fos expression
Jessica Moretti
1,2,5*, Dylan J. Terstege
3,5, Eugenia Z. Poh
1,2,4, Jonathan R. Epp
3 &
Jennifer Rodger
1,2*
Repetitive transcranial magnetic stimulation (rTMS) induces action potentials to induce plastic
changes in the brain with increasing evidence for the therapeutic importance of brain‑wide functional
network eects of rTMS; however, the inuence of sub‑action potential threshold (low‑intensity;
LI‑) rTMS on neuronal activity is largely unknown. We investigated whether LI‑rTMS modulates
neuronal activity and functional connectivity and also specically assessed modulation of parvalbumin
interneuron activity. We conducted a brain‑wide analysis of c‑Fos, a marker for neuronal activity, in
mice that received LI‑rTMS to visual cortex. Mice received single or multiple sessions of excitatory
10 Hz LI‑rTMS with custom rodent coils or were sham controls. We assessed changes to c‑Fos
positive cell densities and c‑Fos/parvalbumin co‑expression. Peak c‑Fos expression corresponded
with activity during rTMS. We also assessed functional connectivity changes using brain‑wide c‑Fos‑
based network analysis. LI‑rTMS modulated c‑Fos expression in cortical and subcortical regions.
c‑Fos density changes were most prevalent with acute stimulation, however chronic stimulation
decreased parvalbumin interneuron activity, most prominently in the amygdala and striatum. LI‑rTMS
also increased anti‑correlated functional connectivity, with the most prominent eects also in the
amygdala and striatum following chronic stimulation. LI‑rTMS induces changes in c‑Fos expression
that suggest modulation of neuronal activity and functional connectivity throughout the brain.
Our results suggest that LI‑rTMS promotes anticorrelated functional connectivity, possibly due to
decreased parvalbumin interneuron activation induced by chronic stimulation. These changes may
underpin therapeutic rTMS eects, therefore modulation of subcortical activity supports rTMS for
treatment of disorders involving subcortical dysregulation.
Use of repetitive transcranial magnetic stimulation (rTMS) is increasing for the treatment of a range of neurologi-
cal conditions, however there is still limited understanding of the eects of electromagnetic stimulation in the
brain. Conventional rTMS is generally linked to direct electromagnetic activation of cortical tissue underneath
the coil, where induced electric elds lead to plastic changes in the brain. However, rTMS-induced changes in
brain activity also occur outside of the initial stimulation site, which is thought to be due to indirect modulation
of connected brain structures1–4; these signicant changes to network connectivity may underpin the therapeu-
tic eects of rTMS5–11. As a result, there has been growing interest in understanding the brain-wide changes in
functional connectivity in response to rTMS. Since e-eld models of rTMS stimulation only show the initial
stimulation point, they cannot account for induced network eects. However, in humans, understanding the
relationship between initial stimulation and induced network eects is limited to EEG and fMRI techniques,
which have limitations in spatial and temporal resolution. Due to technical restrictions of ferromagnetic TMS
OPEN
1School of Biological Sciences, The University of Western Australia, Perth, WA, Australia. 2Perron Institute
for Neurological and Translational Science, Perth, WA, Australia. 3Department of Cell Biology and Anatomy,
Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Alberta, Canada. 4Present address:
Netherlands Institute for Neuroscience, Amsterdam, The Netherlands. 5These authors contributed equally: Jessica
Moretti and Dylan J. Terstege. *email: jmoretti.research@gmail.com; jennifer.rodger@uwa.edu.au
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coils, these techniques are also dicult to apply during stimulation. BOLD changes are also indirectly related to
neural activity and therefore are unable to separate the contribution of dierent neuronal subtypes12.
In order to explore rTMS-induced changes in neuronal activity at cellular resolution (without electrophysi-
ology), previous studies used the immediate-early gene c-Fos as an indirect marker of neuronal activity in the
brains of mice that had received rTMS13–15. However, those studies used a human rTMS coil, which was too large
to deliver focal stimulation to the small mouse brain, precluding the study of connectivity changes16. To better
emulate the spatial characteristics of human rTMS, and provide the opportunity to study activation of networks
downstream of dened brain regions, here we delivered stimulation to the mouse brain using a miniaturised
coil17. Despite the low intensity magnetic eld delivered by these miniaturised coils (low-intensity (LI-) rTMS),
they have been shown to induce a range of neurobiological changes in rodents, including changes in resting state
connectivity that are comparable to those observed in humans1,2,18. A further advantage of the miniaturised coils
is that they can be attached to the head of awake and freely moving animals, avoiding the confounds of restraint
and anaesthesia required by larger coils19,20.
To better understand how rTMS alters activity in, and connectivity between, dierent parts of the brain,
we conducted a brain-wide analysis of c-Fos positive (c-Fos+) cell density in mice that were euthanised 90min
aer LI-rTMS over the visual cortex to capture the peak of c-Fos expression corresponding with brain activity
during stimulation. We specically included analysis of c-Fos+ parvalbumin positive (PV+) neurons, which are
usually GABA-ergic interneurons and play an important role in coordinating and modulating neuronal circuit
activity21,22, particularly in response to rTMS15,23–26. We then conducted a network analysis, correlating c-fos
expression between brain regions to explore how functional connectivity changes during rTMS, and whether
PV+ neurons may contribute to these changes27–30. Additionally, although therapeutic rTMS eects are oen
thought to be cumulative, there is still limited understanding of the dierent outcomes on brain activity of single
and multiple sessions of rTMS31,32. erefore, we included animals that received either acute (single session) or
chronic (14 daily sessions) of rTMS to visualise on a cellular level whether acute and chronic stimulation activate
dierent brain regions and circuits.
Results
Brain wide c‑Fos density changes. LI-rTMS modulated c-Fos expression in various regions throughout
the brain. Of the 73 regions included in analysis, 53 had a signicant omnibus model eect. Several regions
showed a signicant eect of time, indicating dierences between the chronic and acute group, regardless of
stimulation. c-Fos density for regions showing signicant changes are reported in Figs.1 and 2, organised by
hierarchical brain region (see Supplementary File S1 for list of regions and abbreviations). A summary of signi-
cant eects and interaction are reported in Supplementary File S2. Percentage changes in c-Fos density between
sham and LI-rTMS groups for all regions are reported in Supplementary Fig.S1.
Stimulation-induced changes in c-Fos density were present throughout both cortical and subcortical
regions—49 regions showed an eect related to stimulation, and several had signicant stimulation*time inter-
actions. Follow up simple main eect analysis indicated that the main eect of stimulation could be interpreted
for 14 regions, but the majority of stimulation-induced changes were due to altered activity following acute, but
not chronic stimulation. Only 3 regions (ECT, CEA, VMH) showed signicantly dierent c-Fos density following
chronic but not acute stimulation. e direction of c-Fos density changes was varied across brain regions (24
regions show reduced c-Fos density; 24 regions show increased density). e direction of change in c-Fos expres-
sion for broader hierarchical groupings, based on signicant changes in individual regions, is reported in Table1.
e largest dierence in c-Fos expression was upregulation occurring during acute stimulation. Areas with
the largest mean dierence in c-Fos density with acute stimulation were prevalent in the cortex, as well as stri-
atal regions. Downregulation was particularly prevalent in several thalamic regions during acute stimulation.
In relation to the position of the coil, supercial regions positioned below the greatest induced e-eld17 showed
signicant increases in c-Fos with acute LI-rTMS (Fig.3). Videos showing the 3D model of signicantly regulated
regions can be found in supplementary materials (Movie S1–S2), and the 3D objects from the videos, created
using the Scalable Brain Atlas33,34 are available in our the GitHub repository. Chronic stimulation induced sig-
nicant density changes in fewer regions than acute stimulation, with mostly upregulation of c-Fos expression.
e greatest dierence in c-Fos activity was the upregulation of VMH, CEA, and VISC.
Functional connectivity network. In altering brain-wide c-Fos expression, LI-rTMS also manipulated
brain-wide functional network topology (Fig.4a, b). ese changes had minor eects on the density of sta-
tistically signicant positively correlated activity in the network, with stimulation decreasing the density of
such connections in acute conditions by a factor of 0.71 (0.17696to 0.12560) and increasing this density in the
chronic condition by a factor of 1.14 (0.105400to0.120200) (Fig.4c). However, stimulation increased the den-
sity of statistically signicant anti-correlations by factors of 4.38 in the acute condition (0.000457 to0.00200) and
22.88 in the chronic condition (0.00153to 0.003500; Fig.4d).
Together, these results suggest that LI-rTMS shis brain-wide functional coactivation, coinciding with not
statistically signicant correlations becoming increasingly anti-correlated. ese changes in correlation coef-
cient magnitude were most apparent across neuroanatomical regions within broader hierarchical groupings of
the striatum, pallidum, and the amygdala.
Brain‑wide parvalbumin and c‑Fos co‑expression. It has previously been demonstrated that LI-rTMS
may inuence parvalbumin interneurons underneath the coil e-eld, as LI-rTMS can induce increases in corti-
cal parvalbumin expression25,26. e altered brain-wide c-Fos expression patterns and network topology suggest
that the eects of LI-rTMS extend beyond the site of the stimulation. To determine whether LI-rTMS aects
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activity of parvalbumin interneurons and whether changes also extended beyond the stimulation target, the
brain-wide co-expression of parvalbumin and c-Fos was assessed. C-Fos is an excellent marker of activity in PV+
neurons, with > 95% of these cells expressing c-Fos following direct chemogenetic activation35. Representative
images of parvalbumin and c-Fos co-expression are shown in Fig.5d, e. e 2-way ANOVA for acute stimula-
tion showed no signicant eects or interaction. However, for chronic stimulation, there was a main eect of
stimulation (F (1, 56) = 4.146, p = 0.0465), but no region eect or interaction (Fig.5a). Animals that received
chronic stimulation showed signicantly reduced % c-Fos+/PV+ cells. (Fig.5b). e eect of chronic stimulation,
as reported by Hedge’s G, was most prevalent in the amygdala followed by the striatum (Fig.5c).
Discussion
Excitatory 10Hz LI-rTMS caused widespread regulation of neuronal activity during stimulation. Both upregula-
tion and downregulation of c-Fos expression occurred throughout the brain. e most prominent changes were
during acute stimulation, particularly with upregulation of neuronal activity however, there were more limited
activity changes with chronic stimulation. Changes to neuronal activity were present both underneath and away
from the coil, suggesting direct and indirect induction of activity. LI-rTMS was also able to modulate functional
connectivity on a brain-wide scale. LI-rTMS increased the extent to which regional c-Fos expression density
was anti-correlated, with the most prominent changes occurring in the striatum, pallidum, and amygdala. is
increase in anti-correlated activity was increasingly prominent with chronic stimulation. Potentially underlying
the dierence between acute and chronic stimulation eects, the activity of parvalbumin-positive interneurons
across the brain decreased signicantly with chronic LI-rTMS. ese changes were most prominent in the stria-
tum and amygdala, further corroborating the hypothesis that LI-rTMS manipulates parvalbumin interneuron
activity to drive changes in functional connectivity. Overall, we show that LI-rTMS over the visual cortex appears
to induce signicant and widespread changes to the neuronal activity and functional connectivity of the brain,
particularly in subcortical areas outside of the induced LI-rTMS e-eld.
In the acute stimulation group, cortical regions directly beneath the coil showed prominent increases in
c-Fos density compared to sham, suggesting that 10Hz LI-rTMS excites neurons within the induced e-eld. Our
interpretation is supported by previous electrophysiological experiments showing that 10Hz LI-rTMS triggers
action potentials and increases neuronal ring in the barrel cortex (S1) during stimulation36. However, in the
period immediately aer stimulation, there is electrophysiological evidence for both increases and decreases in
neuronal activity: Boyer etal.36 found a reduced neuronal ring rate 10–20min post-stimulation in the barrel
Figure1. C-Fos cell density in mice in the acute stimulation group for regions which had a signicant eect
related to stimulation across all groups. Violin graphs represent c-Fos cell density (cells/mm2) for mice that
received acute active or sham stimulation organised by hierarchical brain regions. Red lines indicate the median
value. Shaded boxes to the le of the region name indicate whether there was a signicant stimulation eect for
the region.
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Figure2. C-Fos cell density in mice in the chronic stimulation group for regions which had a signicant eect
related to stimulation across all groups. Violin graphs represent c-Fos cell density (cells/mm2) for mice that
received acute active or sham stimulation organised by hierarchical brain regions. Red lines indicate the median
value. Shaded boxes to the le of the region name indicate whether there was a signicant stimulation eect for
the region.
Table 1. General direction of change in c-Fos expression following Acute or Chronic LI-rTMS, organised
across higher order brain classications. % Δ in c-Fos density compared to group sham per region, averaged
across hig her order brain classication: − = − 200–0%; + = 0–200%; + + = 201–500%; + + + > 500%. # signicant
regions = # signicantly modulated regions changing in the labelled direction/total # of signicantly modulated
regions within the higher order classication; * = Higher order classications consisting of predominantly
signicantly down-regulated regions, with a minority of highly upregulated regions. Overall, the degree of
upregulation was greater than downregulation, but the regional downregulation was more numerous is several
higher-order brain classications. n.s = no signicant regions.
Higher-order brain classications
Acute Chronic
Δ c-Fos+ density # Signicant regions Δ c-Fos+ density # Signicant regions
Isocortex + + + 11/15 + 3/5
Hippocamp al Formation + + 2/4 + 1/1
Amygdala + + + 1/3* + + 2/2
Striatum + + 3/3 n.s
Pallidum − 1/1 n.s
Cerebellum + + + 1/1 + 1/1
alamus − 6/6 + + 1/1
Hypothalamus + + 1/6* + + 5/6
Midbrain + 2/6* + 2/2
Hindbrain + + + 1/1 n.s
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Figure3. Spatial representation supercial brain regions with signicantly modulated c-Fos density following
acute (A) or chronic (B) LI-rTMS compared to the LI-rTMS-induced e-eld. Le hemisphere: Simulated
e-eld in mV/mm induced by the LI-rTMS coil placed above lambda with a current of 1.83mA/μs17. Right
hemisphere: Top-down view of brain regions with signicantly upregulated (yellow) or downregulated (pink)
overall c-Fos density following acute (A) or chronic (B) LI-rTMS compared to sham controls. Videos showing
the 3D model of signicantly regulated regions can be found in supplementary materials (Movie S1–S2), and
for exploration in a 3D space, 3D objects for import into the Scalable Brain Atlas Composer34 are included on
our GitHub Repository. Brain regions and outlines use data obtained from the Scalable Brain Atlas Composer34
which uses the Allen Brain Atlas template33.
Figure4. Brain-wide functional network topology and correlation density analyses. Functional network
correlation matrices for sham (bottom le corner) and active LI-rTMS (top right corner) for acute (A) and
chronic (B) stimulation groups. Matrices depict the coactivation of 115 neuroanatomical regions, with each row
and column represent a single region and the intersection of rows and columns depicting the magnitude of the
correlation between pairs of regions. Regions are also more broadly organised as isocortex (ISO), hippocampal
formation (HPF), amygdala, (AMYG), striatum (STR), pallidum (PAL), cerebellum (CB), thalamus (TH),
hypothalamus (HY), midbrain (MB), and hindbrain (HB). e prevalence of anticorrelations (depicted in
blue) in the amygdala, pallidum, and striatum is increased considerably with LI-rTMS stimulation. Network
density values, dened as the proportion of actual functional connections relative to the potential number of
connections in a fully saturated network, show little change in (C) statistically signicant positively correlated
activity. (D) However, there was an increase in the density of anti-correlated activity in the network. See S1 File
for list of regions in the order than they are presented in the correlation matrices.
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cortex, decreasing neuronal excitability, while Tang etal.37 found that LI-rTMS lowered action potential thresh-
olds invitro in motor cortical neurons, increasing neuronal excitability. e dierent experimental preparations
Figure5. Percentage c-Fos/parvalbumin (PV) co-expression with acute or chronic LI-rTMS organised by brain
region. (A, B) Percentage of parvalbumin cells co-expressing c-Fos in each region with acute (A) or chronic (B)
LI-rTMS or sham controls. Error bars represent ± SEM (C) e absolute value (ABV) of Hedge’s G in both the
acute and chronic groups. (D, E) Representational images of parvalbumin (red), c-Fos (greyscale) and DAPI
(blue) from the acute (D) and chronic (E) groups. Scale bars represent (i) 1000µm or (ii) 25µm.
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(in vivo vs. invitro) make the results hard to compare, and in our study we saw increased c-Fos density in both
motor and somatosensory cortices with acute LI-rTMS. However, because of the timing of euthanasia (90min
aer the start of stimulation) and the poor time resolution of c-Fos, it is likely that our results primarily reect
the increase in activity during stimulation. In addition, the c-Fos mRNA and protein produced in response to
increased neuronal activity are stable and are not removed by a subsequent decrease in activity. erefore our
results cannot be used to assess any increases or decreases in activity that may occur between the end of stimula-
tion and euthanasia. Inclusion of a second immediate early gene (IEG) marker (e.g. Arc, zif268) could provide
additional resolution as dierent IEGs’ peak expression time varies38. e pattern of rTMS-induced IEG activity
also diers across region and cortical layer (e.g. c-Fos vs Arc39, c-Fos vs. zif26813) and may oer further insight
into changes in neuronal excitability that occur at dierent times during and post stimulation. It will also be
important in the future to follow up our results with electrophysiology to obtain a spatially and temporally precise
measure of neuronal function following LI-rTMS.
Interestingly, despite the low-intensity stimulation, areas outside of the induced e-eld also show signicant
regulation of neuronal activity, such as upregulation of c-Fos expression in striatal regions, and downregula-
tion in several thalamic regions. ese results support fMRI experiments in rats demonstrating that the acute
eects of LI-rTMS on neuronal activity extend beyond the site of stimulation1,2, and are consistent with clinical
neuroimaging studies of conventional rTMS in humans40,41. LI-rTMS may thus acutely modulate interconnected
regions via activation of downstream pathways.
Perhaps surprisingly, chronic LI-rTMS induced fewer changes to neuronal activity compared to acute LI-
rTMS. However, chronic stimulation did result in more signicant changes to brain-wide functional connectivity
network topology, and these changes were most prevalent beyond the site of stimulation. e dierent outcomes
of acute and chronic stimulation suggest that LI-rTMS eects are cumulative and may involve homeostatic
mechanisms that prevent over-activation of neurons, as well as plasticity mechanisms that alter functional con-
nectivity across brain regions. ese processes are likely to underpin the long-term benecial outcomes of
therapeutic rTMS in depression and OCD and highlight the potential to optimise TMS treatment targets and
protocols for specic dysfunctional networks.
Although our experimental design controlled for the procedural eects of rTMS by delivering sham stimula-
tion, handling per se has been shown to have an eect on a range of brain and behavioural markers42, raising the
possibility that our results reect an interaction of LI-rTMS with the animals’ response to handling. For example,
our main interpretation of the changes observed in the chronic animals is that LI-rTMS reduced parvalbumin
activity, particularly in the striatum and amygdala. An alternative interpretation could be that c-Fos/parvalbumin
co-expression is signicantly increased by handling alone, and LI-rTMS accelerated a return to baseline (Naïve)
levels, potentially through increased plasticity. A previous study in the visual cortex has shown parvalbumin
expression, compared to naïve controls, increases following a sham rTMS group, but not active excitatory rTMS
which may support this alternative interpretation43. Future studies should include a naïve baseline control to rule
out or conrm such alternate interpretations and more precisely assess the eect of electromagnetic stimulation.
e signicance and origins of anti-correlations in c-Fos-based functional connectivity networks have been
largely ignored44–46. Excitingly, the present study provides the rst evidence for a link between parvalbumin
interneurons and network anticorrelations. While previous studies have established that rTMS and LI-rTMS alter
expression of parvalbumin15,43,47 our study extends this work by showing that LI-rTMS signicantly decreased the
activity of parvalbumin interneurons. ese largely GABAergic cell populations exert control over the activity
of many more-abundant glutamatergic neuronal populations. erefore, by altering the expression and activity
of parvalbumin interneurons, LI-rTMS has the potential to modulate network synchronicity on a brain-wide
scale48–50. is is in line with what we observed: regions in which parvalbumin interneuron activity was most
prevalently modulated with chronic LI-rTMS (striatum and amygdala) coincide with the neuroanatomical
regions in which neuronal c-Fos expression density became most prevalently anti-correlated. ese results
suggest that decreased parvalbumin interneuron activity promotes anti-correlated activity and is a potential
mechanism through which LI-rTMS is able to modulate brain-wide functional connectivity.
e ability to modulate anticorrelated activity has numerous clinical implications, particularly through the
lens of parvalbumin interneuron modulation. e magnitude of anticorrelated functional connectivity is damp-
ened in several conditions, including depression51,52, Parkinson’s disease53, stroke54, and anxiety55. ese same
conditions have also been demonstrated to have altered parvalbumin interneuron activity56–59. Many of the
symptoms of these conditions have also been shown to improve with rTMS treatment60–63. Our results suggest
that a possible mechanism through which LI-rTMS is able to ameliorate these symptoms is through its brain-
wide modulation of parvalbumin interneuron activity and anticorrelated functional connectivity. Our research
provides novel insight into how LI-rTMS changes functional connectivity at the cellular level, and forms part
of a growing translational pipeline of preclinical and clinical neuromodulation studies that continue to inform
human treatments. For example, our nding of changes in functional connectivity and parvalbumin interneuron
activity in the amygdala and striatum provides new evidence that rTMS may be eective for treating disorders
associated with aberrant activity in these regions. In addition, c-Fos density changes with acute LI-rTMS dem-
onstrate that even short-term exposure to low-levels of electromagnetic elds can induce changes to neuronal
activity throughout the brain, including in subcortical regions. While subthreshold rTMS eects remain poorly
characterised in humans, low intensity magnetic elds are delivered as part of conventional, high-intensity rTMS,
because magnetic eld intensity decays with distance from the coil18. erefore, our research showing changes
with low-intensity elds is directly relevant to improving rTMS clinical outcomes in disorders characterised by
dysregulation of subcortical circuitry.
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Methods
Animals. All experiments were approved and performed in accordance with e University of Western Aus-
tralia Animal Ethics Committee (AEC 100/1639) and complied with ARRIVE guidelines. Twenty-two wildtype
C57Bl6/J (Jackson) (8wks) mice supplied by Animal Resources Centre were used and housed in 12-h day/night
cycle (7am–7pm). Mice were organised into acute or chronic stimulation groups, and either sham or LI-rTMS
conditions (Acute Sham: n = 6, 3 males; Acute LI-rTMS: n = 6, 3 males; Chronic Sham: n = 5, 3 males; Chronic
LI-rTMS: n = 5, 2 males). e two chronic groups originally numbered 6 animals but two brains were damaged
during freezing for cryosectioning, so had be removed from the study. Mice were allocated to each group ran-
domly, aer balancing for sex. Mice were acclimatised to the animal holding facility for at least a week before the
beginning of experimental procedures.
Procedure. Coil support attachment surgery. To allow stimulation to be delivered accurately on freely mov-
ing animals, surgery to attach a coil support to the skull was performed, as described previously64. e coil sup-
port consisted of a plastic pipette tip attached to the exposed skull over the stimulation target area with dental
cement and trimmed to < 10mm in length. e skin was then sutured over the cement base of the coil support.
Post-operatively, mice were housed individually with the cage hopper removed to prevent damage to the sup-
port, with Hydrogel (HydroGel, ClearH2O) and food provided adlibitum.
Stimulation. e coil support allows a custom LI-rTMS coil to be xed in place during stimulation by attaching
it to the support with an alligator clip. From the h day following surgery, mice were habituated to the coil by
attaching a dummy coil to the support for 5–10min each day for 3days prior to beginning stimulation. Stimula-
tion was delivered with a custom animal LI-rTMS coil (300 copper windings, external diameter, 8mm; internal
diameter 5mm; see Supplementary Fig.S2) delivering approximately 21 mT at the base of the coil. e coil was
powered by an electromagnetic pulse generator (e-cell™) programmed to deliver 10Hz stimulation for 10min
(6000 pulses). Stimulation was applied to freely moving animals in their home cage either once (acute group),
or daily for 14days (chronic). Stimulation times were between 13:00–15:00 conducted in a randomised order
each day. For sham stimulation, the coil was attached to the support, but with the pulse generator switched o.
Tissue processing. Tissue collection. Animals were euthanised with sodium pentobarbitone (0.1ml i.p.,
Lethabarb, Virbac, Australia) on the nal day of stimulation, 90min aer the beginning of stimulation to cap-
ture the peak c-Fos expression during stimulation. Animals were then transcardially perfused with saline (0.9%
NaCl, w/v) and paraformaldehyde (4% in 0.1M phosphate buer, w/v), the brains were dissected out and post-
xed in paraformaldehyde for 24h and transferred to 30% sucrose in phosphate buer solution (PBS) (w/v)
for cryoprotection. Coronal sections (30μm) were cryosected into 5 series. One of the resulting series was
divided in half, wherein sections were alternatingly sorted for either brain-wide c-Fos labelling or an analysis of
parvalbumin and c-Fos co-expression. is division resulted in a spacing of 300μm between sections in each
immunohistochemistry procedure.
Immunohistochemistry. Brain-wide c-Fos expression. Tissue sections were stained with c-Fos (Rabbit poly-
clonal c-Fos antibody, 1:5000, Abcam, ab190289) and NeuN (mouse monoclonal anti-NeuN, 1:2000, Millipore,
MAB377). Free-oating sections were washed (30min per wash) with PBS and permeabilised with two washes
of 0.1% Triton-X in PBS (PBS-T). Sections were incubated for 2h in blocking buer of 3% bovine serum albu-
min (BSA, Sigma) and 2% donkey serum (Sigma) diluted in PBS-T. Primary antibodies were incubated in fresh
blocking buer at 4°C for 18h, washed with PBS-T and then incubated with secondary antibodies for 2h (don-
key anti-rabbit lgG Alexa Fluor 488, Invitrogen, ermo Fisher, A21206; donkey anti-Mouse lgG Alexa Fluor
555, Invitrogen, ermo Fisher, A21202, 1:600 in blocking buer). Sections were washed twice with PBS before
being mounted onto gelatin subbed slides, coverslipped with mounting medium (Dako, Glostrup, Denmark)
and sealed with nail polish.Slides were stored at 4°C in a light-controlled environment until imaging.
Parvalbumin and c-Fos co-expression. Tissue sections were washed three times (10min per wash) in 0.1M
PBS before being incubated in a primary antibody solution of mouse anti-PV (1:2000, EnCor Biotechnology
Inc., MCA-3C9), rabbit anti-c-Fos (1:2000, EnCor Biotechnology Inc., RPCA-c-Fos), 3% normal goat serum,
and 0.03% Triton-X100 for 48h. Tissue sections were washed three more times in 0.1M PBS before secondary
antibody incubation. e secondary antibody solution was composed of 1:500 goat anti-mouse Alexa Fluor 594
(Invitrogen, ermo Fisher, A11005) and 1:500 goat anti-rabbit Alexa Fluor 647 (Jackson ImmunoResearch,
111-605-003) in PBS for 24h. Sections were then transferred to 1:1000 DAPI solution for 20min before three
nal PBS washes. Labelled sections were mounted to plain glass slides and coverslipped with PVA-DABCO
mounting medium.
Imaging. For the analysis of brain-wide c-Fos expression density, tissue sections were imaged using a Nikon
C2 Confocal microscope (Nikon, Tokyo, Japan). e entire section was imaged via multiple images taken at
10× magnication and z-stacks separated by 5μm. Images were automatically stitched together with a 10% over-
lap using NIS Elements soware (Nikon, Tokyo, Japan).
Images of c-Fos and parvalbumin co-expression were collected using an OLYMPUS VS120-L100-W
slide-scanning microscope (Richmond Hill, ON, Canada). Images of a single z-plane were collected using a
10 × objective.
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Image processing. Quantication of c-Fos labelling was segmented and registered using a semi-automated pipe-
line described in Terstege etal.65. Briey, c-Fos labelled cells were segmented using Ilastik, a machine-learning
based pixel classication program66. Ilastik output images were then registered to the Allen Mouse Brain Atlas
using Whole Brain, an R based soware67 and used in combination with custom ImageJ soware designed to cal-
culate region volumes and output accurate c-Fos density counts per region. To minimise bias, Ilastik was trained
on a range of images from dierent animals and groups and experimenters were blinded to experimental group
when registering images.
For analyses of c-Fos and parvalbumin co-expression, cells expressing c-Fos and parvalbumin labels were
segmented independently using Ilastik. e Ilastik binary object prediction images were further processed using
a custom ImageJ plug-in to identify instances of co-expression and output a binary image containing only these
c-Fos and parvalbumin co-expressing cells. Finally, both these co-expression images and the Ilastik object predic-
tion images of parvalbumin labelling were mapped to a custom neuroanatomical atlas based on a higher-order
region organization of the Allen Mouse Brain Atlas using FASTMAP68. is approach facilitated the accurate
assessment of the percentage of parvalbumin interneurons which were expressing c-Fos across several higher-
order brain regions.
Data analysis. Brain‑wide c‑Fos density. To assess general activation of regions across the brain, c-Fos+
cells were quantied in 115 neuroanatomical regions. is regional organization encompassed the entire mouse
brain and was selected based on experimenter ability to delineate these neuroanatomical regions of interest in
NeuN-stained tissue (see Supplementary File S1 for list of regions and abbreviations).
We compared c-Fos expression density (c-Fos+ cells/mm2) in a negative binomial generalised linear model
with a log link for each region of interest. Fixed factors were Stimulation and Time. Data from all animals were
included however, values with a Cook’s Distance > 0.5 were excluded and regions with less than three values in
any group were excluded from analysis, resulting in 73 regions analysed for density (listed in Supplementary
File S2). To account for multiple comparisons across regions we used a false discovery rate approach (Q = 0.01)
for the omnibus eects. For regions that had signicant omnibus eects, we followed up with analysis of the
main eects and interaction. If there was a signicant interaction eect, we ran simple main eect analyses in
order to interpret the changes.
Functional connectivity networks. e impact of regional changes in c-Fos expression density on brain dynam-
ics was examined through the scope of functional connectivity networks27–30. Networks were constructed by
cross-correlating regional c-Fos expression density within each group to generate pairwise correlation matrices.
Correlations were ltered by statistical signicance (α < 0.005) and a false discovery rate of 95%. e number
of pairwise correlations exhibiting anticorrelated activity and the mean Pearson’s correlation coecient were
assessed for each network. Network density, dened as the proportion of actual functional connections relative
to the potential number of connections in a fully saturated network was also assessed69.
Brain‑wide parvalbumin and c‑Fos co‑expression. Regional co-expression of c-Fos and parvalbumin was
expressed as a percentage of the total number of parvalbumin interneurons present in each region. ese data
were compared separately for acute and chronic groups using Two-Factor ANOVA, with factors of Stimulation
and Region.
Data availability
All datasets generated for this study and the scripts developed for its analysis can be found at the following
GitHub repository [https:// github. com/ dters tege/ Publi catio nRepo/ tree/ main/ Moret ti2022].
Received: 1 September 2022; Accepted: 22 November 2022
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Acknowledgements
Funding for this study was provided in part by an NSERC Discovery Grant (RGPIN-2018-05135) to JRE. JM
was supported by an Australian Government Research Training Program (RTP) scholarship, and Byron Kakulas
Prestige scholarship. DJT received fellowships from NSERC and the Canadian Open Neuroscience Platform. JR
was supported by a fellowship from Multiple Sclerosis Western Australia (MSWA). We acknowledge the Hotch-
kiss Brain Institute Advanced Microscopy Platform and the Cumming School of Medicine for support and use
of the Olympus VS120-L100-W slide scanning microscope.
Author contributions
JM: Conceptualization, Methodology, Formal Analysis, Investigation, Visualisation, Writing—original dra,
Writing—review and editing. DJT: Methodology, Soware, Formal Analysis, Investigation, Visualisation, Writ-
ing—original dra, Writing—review and editing. EZP: Methodology, Investigation, Writing—review and edit-
ing. JRE: Writing—review and editing, Supervision, Resources. JR: Writing—review and editing, Supervision,
Resources.
Competing interests
e authors declare no competing interests.
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