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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.
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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
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 structures14; these signicant changes to network connectivity may underpin the therapeu-
tic eects of rTMS511. 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
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:;
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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 rTMS1315. 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 rTMS15,2326. 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 changes2730. 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.
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 Hedges G, was most prevalent in the amygdala followed by the striatum (Fig.5c).
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 Hedges 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 ignored4446. 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
scale4850. 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, Parkinsons disease53, stroke54, and anxiety55. ese same
conditions have also been demonstrated to have altered parvalbumin interneuron activity5659. Many of the
symptoms of these conditions have also been shown to improve with rTMS treatment6063. 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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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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Scientic Reports | (2022) 12:20571 |
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 networks2730. 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 Pearsons 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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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,
Competing interests
e authors declare no competing interests.
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Scientic Reports | (2022) 12:20571 |
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... Different stimulation patterns affect distinct interneuron populations appropriate to their observed effects on cortical excitability (e.g., MEP). According to the pattern applied, rTMS modulates the inhibitory interneuron expression of the immediate early genes c-fos and zif268 [46,61,62], GABA-synthesising enzymes GAD65 and GAD67 [46,60,63], and calcium-binding proteins [59,63,64]. Stimulation patterns that increase cortical excitability, such as 10 Hz and iTBS stimulation, not only induce Ca 2+ -dependent signalling [56,58,59,65,66] but also depress inhibitory circuits by reducing parvalbumin (PV) expression in fast-spiking interneurons (FSIs) [62,[67][68][69] and destabilising GABA receptors to reduce GABAergic synaptic strength [66]. ...
... According to the pattern applied, rTMS modulates the inhibitory interneuron expression of the immediate early genes c-fos and zif268 [46,61,62], GABA-synthesising enzymes GAD65 and GAD67 [46,60,63], and calcium-binding proteins [59,63,64]. Stimulation patterns that increase cortical excitability, such as 10 Hz and iTBS stimulation, not only induce Ca 2+ -dependent signalling [56,58,59,65,66] but also depress inhibitory circuits by reducing parvalbumin (PV) expression in fast-spiking interneurons (FSIs) [62,[67][68][69] and destabilising GABA receptors to reduce GABAergic synaptic strength [66]. In contrast, cTBS and 1 Hz ("inhibitory" protocols) predominantly alter calbindin (CB) expression [67,68]. ...
... In vivo, Zif268 expression was increased in almost all cortical areas after iTBS but only in the primary motor and sensory cortices after 10 Hz rTMS [61]. A recent study also showed that c-fos increases can be found in connected cortical regions [62]. ...
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Neurological and psychiatric diseases generally have no cure, so innovative non-pharmacological treatments, including non-invasive brain stimulation, are interesting therapeutic tools as they aim to trigger intrinsic neural repair mechanisms. A common brain stimulation technique involves the application of pulsed magnetic fields to affected brain regions. However, investigations of magnetic brain stimulation are complicated by the use of many different stimulation parameters. Magnetic brain stimulation is usually divided into two poorly connected approaches: (1) clinically used high-intensity stimulation (0.5–2 Tesla, T) and (2) experimental or epidemiologically studied low-intensity stimulation (μT–mT). Human tests of both approaches are reported to have beneficial outcomes, but the underlying biology is unclear, and thus optimal stimulation parameters remain ill defined. Here, we aim to bring together what is known about the biology of magnetic brain stimulation from human, animal, and in vitro studies. We identify the common effects of different stimulation protocols; show how different types of pulsed magnetic fields interact with nervous tissue; and describe cellular mechanisms underlying their effects—from intracellular signalling cascades, through synaptic plasticity and the modulation of network activity, to long-term structural changes in neural circuits. Recent advances in magneto-biology show clear mechanisms that may explain low-intensity stimulation effects in the brain. With its large breadth of stimulation parameters, not available to high-intensity stimulation, low-intensity focal magnetic stimulation becomes a potentially powerful treatment tool for human application.
... Functional connectivity within the networks was determined by calculating a correlation matrix of all ROI pairs (Perry et al., 2016;Junod et al., 2019). While functional connectivity using fMRI or electrophysiological data involves within animal correlation or coherence of time series activity across regions (Shibasaki, 2008;Kelly et al., 2012;Xu et al., 2022), functional connectivity assessment based on correlations of static interregional metabolic or immediate early gene activity across animals, as used here, has been shown to be a reliable assay across multiple paradigms (Teles et al., 2015;Zuloaga et al., 2015;Perry et al., 2016;Tanimizu et al., 2017;Junod et al., 2019;Moretti et al., 2022;Santos et al., 2023;Stefaniuk et al., 2023). ...
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Developmental exposure to ethanol is a leading cause of cognitive, emotional and behavioral problems, with fetal alcohol spectrum disorder (FASD) affecting more than 1:100 children. Recently, comorbid sleep deficits have been highlighted in these disorders, with sleep repair a potential therapeutic target. Animal models of FASD have shown non-REM (NREM) sleep fragmentation and slow-wave oscillation impairments that predict cognitive performance. Here we use a mouse model of perinatal ethanol exposure to explore whether reduced sleep pressure may contribute to impaired NREM sleep, and compare the function of a brain network reported to be impacted by insomnia–the Salience network–in developmental ethanol-exposed mice with sleep-deprived, saline controls. Mice were exposed to ethanol or saline on postnatal day 7 (P7) and allowed to mature to adulthood for testing. At P90, telemetered cortical recordings were made for assessment of NREM sleep in home cage before and after 4 h of sleep deprivation to assess basal NREM sleep and homeostatic NREM sleep response. To assess Salience network functional connectivity, mice were exposed to the 4 h sleep deprivation period or left alone, then immediately sacrificed for immunohistochemical analysis of c-Fos expression. The results show that developmental ethanol severely impairs both normal rebound NREM sleep and sleep deprivation induced increases in slow-wave activity, consistent with reduced sleep pressure. Furthermore, the Salience network connectome in rested, ethanol-exposed mice was most similar to that of sleep-deprived, saline control mice, suggesting a sleep deprivation-like state of Salience network function after developmental ethanol even without sleep deprivation.
... In fact, intact functional connectivity of the RSC has been suggested to be a key indicator of healthy cognitive aging 5 . While anticorrelated activity is often overlooked in functional connectivity studies, it has been speculated that it may relate to activity driven by inhibitory interneurons 24,25 . If so, changes in anti-correlated activity would be predicted to occur based on the loss and disruption of PV-IN activity observed here. ...
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Alzheimer's Disease is a common and debilitating neurodegenerative disorder with no cure and few treatment options. Impaired metabolism in the retrosplenial cortex during prodromal stages of the disease has been proposed as a strong predictor of future cognitive impairments. The retrosplenial cortex is also one of the earlier brain regions to exhibit functional impairments in Alzheimer's Disease. Therefore, understanding Alzheimer's related deficits in the retrosplenial cortex may be critical in understanding the origins of cognitive impairment and could provide early treatment targets. Here, we investigated alterations in retrosplenial cortex structure and function in a mouse model of Alzheimer's Disease. We identified a novel sex dependent early impairment in parvalbumin-interneuron activity which, is sufficient to induce cognitive impairments and, dysregulate functional connectivity of the retrosplenial cortex. Reversal of cognitive deficits by stimulation of parvalbumin interneurons in retrosplenial cortex suggests that this may serve as a promising novel therapeutic strategy.
... did not observe an anticorrelated amygdala cluster, suggesting that the extended amygdala may undergo continued remodeling over the course of abstinence. Comparison of modules and hubs identified in the 2 studies is difficult due to methodological differences; while the prior study employed arbitrary correlation thresholds, we used a data-driven approach and incorporated both positive and negative correlations because the latter are important for network responsiveness (28). Modular organization, where neural elements form strong within-module connections and weak intermodule connections, is a feature of complex networks that increases efficiency and supports specialized processing (29). ...
Background: High-level alcohol consumption causes neuroplastic changes in the brain that promote pathological drinking behavior. Some of these changes have been characterized in defined brain circuits and cell types, but unbiased approaches are needed to explore broader patterns of adaptations. Methods: We employed whole-brain c-fos mapping and network analysis to assess patterns of neuronal activity during alcohol withdrawal and following reaccess in a well-characterized model of alcohol dependence. Mice underwent four cycles of chronic intermittent ethanol (CIE) to increase voluntary alcohol consumption, and a subset underwent forced swim stress (FSS) to further escalate consumption. Brains were collected either 24 hours (withdrawal) or immediately following a one-hour period of alcohol reaccess. C-fos counts were obtained for 110 brain regions using iDISCO and ClearMap. We then classified mice as high or low drinkers (HD or LD) and used graph theory to identify changes in network properties associated with high-drinking behavior. Results: During withdrawal, CIE mice displayed widespread increased c-fos expression relative to AIR mice, independent of FSS. Reaccess drinking reversed this increase. Network modularity, a measure of segregation into communities, was increased in HD mice after alcohol reaccess relative to withdrawal. The cortical amygdala (COA) showed increased cross-community coactivation during withdrawal in HD mice, and COA silencing in CIE mice reduced voluntary drinking. Conclusions: Alcohol withdrawal in dependent mice causes changes in brain network organization that are attenuated by reaccess drinking. Olfactory brain regions, including COA, drive some of these changes and may play an important but underappreciated role in alcohol dependence.
... This temporal delay coincides with a considerably wide tagging window, with detectable protein expression as early as 30 minutes after induction and remains elevated above baseline until 2 h after neuronal activity [50]. A wide tagging window can be beneficial in that the activity from an entire testing session can be captured, a feature which helped c-Fos become the most studied IEG for IEG-based functional connectivity analyses [51][52][53][54][55]. However, because of this wide tagging window, care must be taken to ensure that experimenters limit the extent to which they cause erroneous neuronal activity. ...
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Studying how spatially discrete neuroanatomical regions across the brain interact is critical to advancing our understanding of the brain. Traditional neuroimaging techniques have led to many important discoveries about the nature of these interactions, termed functional connectivity. However, in animal models these traditional neuroimaging techniques have generally been limited to anesthetized or head-fixed setups or examination of small subsets of neuroanatomical regions. Using the brain-wide expression density of immediate early genes (IEG), we can assess brain-wide functional connectivity underlying a wide variety of behavioural tasks in freely behaving animal models. Here, we provide an overview of the necessary steps required to perform IEG-based analyses of functional connectivity. We also outline important considerations when designing such experiments and demonstrate the implications of these considerations using an IEG-based network dataset generated for the purpose of this review.
Repetitive transcranial magnetic stimulation (rTMS) is a widely used therapeutic tool in neurology and psychiatry, but its cellular and molecular mechanisms are not fully understood. Standardizing stimulus parameters, specifically electric field strength and direction, is crucial in experimental and clinical settings. It enables meaningful comparisons across studies and facilitating the translation of findings into clinical practice. However, the impact of biophysical properties inherent to the stimulated neurons and networks on the outcome of rTMS protocols remains not well understood. Consequently, achieving standardization of biological effects across different brain regions and subjects poses a significant challenge. This study compared the effects of 10 Hz repetitive magnetic stimulation (rMS) in entorhino-hippocampal tissue cultures from mice and rats, providing insights into the impact of the same stimulation protocol on similar neuronal networks under standardized conditions. We observed the previously described plastic changes in excitatory and inhibitory synaptic strength of CA1 pyramidal neurons in both mouse and rat tissue cultures, but a higher stimulation intensity was required for the induction of rMS-induced synaptic plasticity in rat tissue cultures. Through systematic comparison of neuronal structural and functional properties and computational modeling, we found that morphological parameters of CA1 pyramidal neurons alone are insufficient to explain the observed differences between the groups. However, axon morphologies of individual cells played a significant role in determining activation thresholds. Notably, differences in intrinsic cellular properties were sufficient to account for the 10 % higher intensity required for the induction of synaptic plasticity in the rat tissue cultures. These findings demonstrate the critical importance of axon morphology and intrinsic cellular properties in predicting the plasticity effects of rTMS, carrying valuable implications for the development of computer models aimed at predicting and standardizing the biological effects of rTMS.
Alzheimer's disease (AD) is the most common form of dementia, and both the incidence of this disease and its associated cognitive decline disproportionally effect women. While the etiology of AD is unknown, recent work has demonstrated that the balance of excitatory and inhibitory activity across the brain may serve as a strong predictor of cognitive impairments in AD. Across the cortex, the most prominent source of inhibitory signalling is from a class of parvalbumin-expressing interneurons (PV+). In this mini-review, the impacts of sex- and age-related factors on the function of PV+ neurons are examined within the context of vulnerability to AD pathology. These primary factors of influence include changes in brain metabolism, circulating sex hormone levels, and inflammatory response. In addition to positing the increased vulnerability of PV+ neurons to dysfunction in AD, this mini-review highlights the critical importance of presenting sex stratified data in the study of AD.
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There are no FDA-approved treatments for the chronic sequelae of concussion. Repetitive magnetic transcranial stimulation (rTMS) has been explored as a therapy but outcomes have been inconsistent. To address this we developed a personalized rTMS (PrTMS) protocol involving continual rTMS stimulus frequency adjustment and progressive activation of multiple cortical sites, guided by spectral electroencephalogram (EEG)-based analyses and psychological questionnaires. We acquired pilot clinical data for 185 symptomatic brain concussion patients who underwent the PrTMS protocol over an approximate 6 week period. The PrTMS protocol used a proprietary EEG spectral frequency algorithm to define an initial stimulation frequency based on an anteriorly graded projection of the measured occipital alpha center peak, which was then used to interpolate and adjust regional stimulation frequency according to weekly EEG spectral acquisitions. PrTMS improved concussion indices and normalized the cortical alpha band center frequency and peak EEG amplitude. This potentially reflected changed neurotransmitter, cognitive, and perceptual status. PrTMS may be a promising treatment choice for patients with persistent concussion symptoms. This clinical observational study was limited in that there was no control group and a number of variables were not recorded, such as time since injury and levels of depression. While the present observations are indeed preliminary and cursory, they may suggest further prospective research on PrTMS in concussion, and exploration of the spectral EEG as a concussion biomarker, with the ultimate goals of confirmation and determining optimal PrTMS treatment parameters.
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Introduction Overreliance on habit is linked with disorders, such as drug addiction and obsessive-compulsive disorder, and there is increasing interest in the use of repetitive transcranial magnetic stimulation (rTMS) to alter neuronal activity in the relevant pathways and for therapeutic outcomes. In this study, we researched the brains of ephrin-A2A5−/− mice, which previously showed perseverative behavior in progressive-ratio tasks, associated with low cellular activity in the nucleus accumbens. We investigated whether rTMS treatment had altered the activity of the dorsal striatum in a way that suggested altered hierarchical recruitment of brain regions from the ventral striatum to the dorsal striatum, which is linked to abnormal habit formation. Methods Brain sections from a limited number of mice that underwent training and performance on a progressive ratio task with and without low-intensity rTMS (LI-rTMS) were taken from a previous study. We took advantage of the previous characterization of perseverative behavior to investigate the contribution of different neuronal subtypes and striatal regions within this limited sample. Striatal regions were stained for c-Fos as a correlate of neuronal activation for DARPP32 to identify medium spiny neurons (MSNs) and for GAD67 to identify GABA-ergic interneurons. Results and discussion Contrary to our hypothesis, we found that neuronal activity in ephrin-A2A5−/− mice still reflected the typical organization of goal-directed behavior. There was a significant difference in the proportion of neuronal activity across the striatum between experimental groups and control but no significant effects identifying a specific regional change. However, there was a significant group by treatment interaction which suggests that MSN activity is altered in the dorsomedial striatum and a trend suggesting that rTMS increases ephrin-A2A5−/− MSN activity in the DMS. Although preliminary and inconclusive, the analysis of this archival data suggests that investigating circuit-based changes in striatal regions may provide insight into chronic rTMS mechanisms that could be relevant to treating disorders associated with perseverative behavior.
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Memory storage and retrieval are shaped by past experiences. Prior learning and memory episodes have numerous impacts on brain structure from micro to macroscale. Previous experience with specific forms of learning increases the efficiency of future learning. It is less clear whether such practice effects on one type of memory might also have transferable effects to other forms of memory. Different forms of learning and memory rely on different brain-wide networks but there are many points of overlap in these networks. Enhanced structural or functional connectivity caused by one type of learning may be transferable to another type of learning due to overlap in underlying memory networks. Here, we investigated the impact of prior chronic spatial training on the task-specific functional connectivity related to subsequent contextual fear memory recall in mice. Our results show that mice exposed to prior spatial training exhibited decreased brain-wide activation compared to control mice during the retrieval of a context fear memory. With respect to functional connectivity, we observed changes in several network measures, notably an increase in global efficiency. Interestingly, we also observed an increase in network resilience based on simulated targeted node deletion. Overall, this study suggests that chronic learning has transferable effects on the functional connectivity networks of other types of learning and memory. The generalized enhancements in network efficiency and resilience suggest that learning itself may protect brain networks against deterioration.
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Magnetic brain stimulation is a promising treatment for neurological and psychiatric disorders. However, a better understanding of its effects at the individual neuron level is essential to improve its clinical application. We combined focal low‐intensity repetitive transcranial magnetic stimulation (LI‐rTMS) to the rat somatosensory cortex with intracellular recordings of subjacent pyramidal neurons in vivo . Continuous 10 Hz LI‐rTMS reliably evoked firing at ∼4–5 Hz during the stimulation period and induced durable attenuation of synaptic activity and spontaneous firing in cortical neurons, through membrane hyperpolarization and a reduced intrinsic excitability. However, inducing firing in individual neurons by repeated intracellular current injection did not reproduce the effects of LI‐rTMS on neuronal properties. These data provide a novel understanding of mechanisms underlying magnetic brain stimulation showing that, in addition to inducing biochemical plasticity, even weak magnetic fields can activate neurons and enduringly modulate their excitability. image Key points Repetitive transcranial magnetic stimulation (rTMS) is a promising technique to alleviate neurological and psychiatric disorders caused by alterations in cortical activity. Our knowledge of the cellular mechanisms underlying rTMS‐based therapies remains limited. We combined in vivo focal application of low‐intensity rTMS (LI‐rTMS) to the rat somatosensory cortex with intracellular recordings of subjacent pyramidal neurons to characterize the effects of weak magnetic fields at single cell level. Ten minutes of LI‐rTMS delivered at 10 Hz reliably evoked action potentials in cortical neurons during the stimulation period, and induced durable attenuation of their intrinsic excitability, synaptic activity and spontaneous firing. These results help us better understand the mechanisms of weak magnetic stimulation and should allow optimizing the effectiveness of stimulation protocols for clinical use.
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Cognitive symptoms of depression, including negative cognitive bias, are more severe in women than in men. Current treatments to reduce negative cognitive bias are not effective and sex differences in the neural activity underlying cognitive bias may play a role. Here we examined sex and age differences in cognitive bias and functional connectivity in a novel paradigm. Male and female rats underwent an 18-day cognitive bias procedure, in which they learned to discriminate between two contexts (shock paired context A, no-shock paired context B), during either adolescence (postnatal day (PD 40)), young adulthood (PD 100), or middle-age (PD 210). Cognitive bias was measured as freezing behaviour in response to an ambiguous context (context C), with freezing levels akin to the shock paired context coded as negative bias. All animals learned to discriminate between the two contexts, regardless of sex or age. However, adults (young adults, middle-aged) displayed a greater negative cognitive bias compared to adolescents, and middle-aged males had a greater negative cognitive bias than middle-aged females. Females had greater neural activation of the nucleus accumbens, amygdala, and hippocampal regions to the ambiguous context compared to males, and young rats (adolescent, young adults) had greater neural activation in these regions compared to middle-aged rats. Functional connectivity between regions involved in cognitive bias differed by age and sex, and only adult males had negative correlations between the frontal regions and hippocampal regions. These findings highlight the importance of examining age and sex when investigating the underpinnings of negative cognitive bias and lay the groundwork for determining what age- and sex-specific regions to target in future cognitive bias studies.
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To better understand complex systems, such as the brain, studying the interactions between multiple brain regions is imperative. Such experiments often require delineation of multiple brain regions on microscopic images based on preexisting brain atlases. Experiments examining the relationships of multiple regions across the brain have traditionally relied on manual plotting of regions. This process is very intensive and becomes untenable with a large number of regions of interest (ROIs). To reduce the amount of time required to process multi-region datasets, several tools for atlas registration have been developed; however, these tools are often inflexible to tissue type, only supportive of a limited number of atlases and orientation, require considerable computational expertise, or are only compatible with certain types of microscopy. To address the need for a simple yet extensible atlas registration tool, we have developed FASTMAP, a Flexible Atlas Segmentation Tool for Multi-Area Processing. We demonstrate its ability to register images efficiently and flexibly to custom mouse brain atlas plates, to detect differences in the regional numbers of labels of interest, and to conduct densitometry analyses. This open-source and user-friendly tool will facilitate the atlas registration of diverse tissue types, unconventional atlas organizations, and a variety of tissue preparations.
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Repetitive transcranial magnetic stimulation (rTMS) is a widespread technique in neuroscience and medicine, however its mechanisms are not well known. In this review, we consider intensity as a key therapeutic parameter of rTMS, and review the studies that have examined the biological effects of rTMS using magnetic fields that are orders of magnitude lower that those currently used in the clinic. We discuss how extensive characterisation of “low intensity” rTMS has set the stage for translation of new rTMS parameters from a mechanistic evidence base, with potential for innovative and effective therapeutic applications. Low-intensity rTMS demonstrates neurobiological effects across healthy and disease models, which include depression, injury and regeneration, abnormal circuit organisation, tinnitus etc. Various short and long-term changes to metabolism, neurotransmitter release, functional connectivity, genetic changes, cell survival and behaviour have been investigated and we summarise these key changes and the possible mechanisms behind them. Mechanisms at genetic, molecular, cellular and system levels have been identified with evidence that low-intensity rTMS and potentially rTMS in general acts through several key pathways to induce changes in the brain with modulation of internal calcium signalling identified as a major mechanism. We discuss the role that preclinical models can play to inform current clinical research as well as uncover new pathways for investigation.
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Depressive disorders contribute heavily to global disease burden; This is possibly because patients are often treated homogeneously, despite having heterogeneous symptoms with differing underlying neural mechanisms. A novel treatment that can directly influence the neural circuit relevant to an individual patient’s subset of symptoms might more precisely and thus effectively aid in the alleviation of their specific symptoms. We tested this hypothesis in a proof-of-concept study using fMRI functional connectivity neurofeedback. We targeted connectivity between the left dorsolateral prefrontal cortex/middle frontal gyrus and the left precuneus/posterior cingulate cortex, because this connection has been well-established as relating to a specific subset of depressive symptoms. Specifically, this connectivity has been shown in a data-driven manner to be less anticorrelated in patients with melancholic depression than in healthy controls. Furthermore, a posterior cingulate dominant state—which results in a loss of this anticorrelation—is expected to specifically relate to an increase in rumination symptoms such as brooding. In line with predictions, we found that, with neurofeedback training, the more a participant normalized this connectivity (restored the anticorrelation), the more related (depressive and brooding symptoms), but not unrelated (trait anxiety), symptoms were reduced. Because these results look promising, this paradigm next needs to be examined with a greater sample size and with better controls. Nonetheless, here we provide preliminary evidence for a correlation between the normalization of a neural network and a reduction in related symptoms. Showing their reproducibility, these results were found in two experiments that took place several years apart by different experimenters. Indicative of its potential clinical utility, effects of this treatment remained one-two months later. Clinical trial registration: Both experiments reported here were registered clinical trials (UMIN000015249, jRCTs052180169).
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The BDNF Val66Met gene polymorphism is a relevant factor explaining inter-individual differences to TMS responses in studies of the motor system. However, whether this variant also contributes to TMS-induced memory effects, as well as their underlying brain mechanisms, remains unexplored. In this investigation, we applied rTMS during encoding of a visual memory task either over the left frontal cortex (LFC; experimental condition) or the cranial vertex (control condition). Subsequently, individuals underwent a recognition memory phase during a functional MRI acquisition. We included 43 young volunteers and classified them as 19 Met allele carriers and 24 as Val/Val individuals. The results revealed that rTMS delivered over LFC compared to vertex stimulation resulted in reduced memory performance only amongst Val/Val allele carriers. This genetic group also exhibited greater fMRI brain activity during memory recognition, mainly over frontal regions, which was positively associated with cognitive performance. We concluded that BDNF Val66Met gene polymorphism, known to exert a significant effect on neuroplasticity, modulates the impact of rTMS both at the cognitive as well as at the associated brain networks expression levels. This data provides new insights on the brain mechanisms explaining cognitive inter-individual differences to TMS, and may inform future, more individually-tailored rTMS interventions.
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Non-invasive brain stimulation is a useful tool to probe brain function and provide therapeutic treatments in disease. When applied to the right posterior parietal cortex (PPC) of healthy participants, it is possible to temporarily shift spatial attention and mimic symptoms of spatial neglect. However, the field of brain stimulation is plagued by issues of high response variability. The aim of this study was to investigate baseline functional connectivity as a predictor of response to an inhibitory brain stimulation paradigm applied to the right PPC. In fourteen healthy adults (9 female, aged 24.8 ± 4.0 years) we applied continuous theta burst stimulation (cTBS) to suppress activity in the right PPC. Resting state functional connectivity was quantified by recording electroencephalography and assessing phase consistency. Spatial attention was assessed before and after cTBS with the Landmark Task. Finally, known determinants of response to brain stimulation were controlled for to enable robust investigation of the influence of resting state connectivity on cTBS response. We observed significant inter-individual variability in the behavioral response to cTBS with 53.8% of participants demonstrating the expected rightward shift in spatial attention. Baseline high beta connectivity between the right PPC, dorsomedial pre-motor region and left temporal-parietal region was strongly associated with cTBS response (R2 = 0.51). Regression analysis combining known cTBS determinants (age, sex, motor threshold, physical activity, stress) found connectivity between the right PPC and left temporal-parietal region was the only significant variable (p = 0.011). These results suggest baseline resting state functional connectivity is a strong predictor of a shift in spatial attention following cTBS. Findings from this study help further understand the mechanism by which cTBS modifies cortical function and could be used to improve the reliability of brain stimulation protocols.
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Repetitive transcranial magnetic stimulation (rTMS) of the dorsolateral prefrontal cortex (DLPFC) is an established treatment for refractory depression, however, therapeutic outcomes vary. Mounting evidence suggests that clinical response relates to functional connectivity with the subgenual cingulate cortex (SGC) at the precise DLPFC stimulation site. Critically, SGC-related network architecture shows considerable interindividual variation across the spatial extent of the DLPFC, indicating that connectivity-based target personalization could potentially be necessary to improve treatment outcomes. However, to date accurate personalization has not appeared feasible, with recent work indicating that the intraindividual reproducibility of optimal targets is limited to 3.5 cm. Here we developed reliable and accurate methodologies to compute individualized connectivity-guided stimulation targets. In resting-state functional MRI scans acquired across 1,000 healthy adults, we demonstrate that, using this approach, personalized targets can be reliably and robustly pinpointed, with a median accuracy of ~2 mm between scans repeated across separate days. These targets remained highly stable, even after 1 year, with a median intraindividual distance between coordinates of only 2.7 mm. Interindividual spatial variation in personalized targets exceeded intraindividual variation by a factor of up to 6.85, suggesting that personalized targets did not trivially converge to a group-average site. Moreover, personalized targets were heritable, suggesting that connectivity-guided rTMS personalization is stable over time and under genetic control. This computational framework provides capacity for personalized connectivity-guided TMS targets to be robustly computed with high precision and has the flexibly to advance research in other basic research and clinical applications.
Background Repetitive transcranial magnetic stimulation is a promising noninvasive therapeutic tool for a variety of brain-related disorders. However, most therapeutic protocols target the anterior regions, leaving many other areas unexplored. There is a substantial therapeutic potential for stimulating various brain regions, which can be optimized in animal models. New Method We illustrate a method that can be utilized reliably to stimulate the anterior or posterior brain in freelymoving rodents. A coil support device is surgically attached onto the skull, which is used for consistent coil placement over the course of up to several weeks of stimulation sessions. Results Our methods provide reliable stimulation in animals without the need for restraint or sedation. We see little aversive effects of support placement and stimulation. Computational models provide evidence that moving the coil support location can be utilized to target major stimulation sites in humans and mice. Summaryof Findings with This Method Animal models are key to optimizing brain stimulation parameters, but research relies on restraint or sedation for consistency in coil placement. The method described here provides a unique means for reliable targeted stimulation in freely moving animals. Research utilizing this method has uncovered changes in biochemical and animal behavioral measurements as a function of brain stimulation. Conclusions The majority of research on magnetic stimulation focuses on anterior regions. Given the substantial network connectivity throughout the brain, it is critical to develop a reliable method for stimulating different regions. The method described here can be utilized to better inform clinical trials about optimal treatment localization, stimulation intensity and number of treatment sessions, and provides a motivation for exploring posterior brain regions for both mice and humans.