ArticlePDF Available

Abstract and Figures

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.
This content is subject to copyright. Terms and conditions apply.
1
Vol.:(0123456789)
Scientic Reports | (2022) 12:20571 | https://doi.org/10.1038/s41598-022-24934-8
www.nature.com/scientificreports
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 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
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
Content courtesy of Springer Nature, terms of use apply. Rights reserved
2
Vol:.(1234567890)
Scientic Reports | (2022) 12:20571 | https://doi.org/10.1038/s41598-022-24934-8
www.nature.com/scientificreports/
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.
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
Content courtesy of Springer Nature, terms of use apply. Rights reserved
3
Vol.:(0123456789)
Scientic Reports | (2022) 12:20571 | https://doi.org/10.1038/s41598-022-24934-8
www.nature.com/scientificreports/
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).
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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
4
Vol:.(1234567890)
Scientic Reports | (2022) 12:20571 | https://doi.org/10.1038/s41598-022-24934-8
www.nature.com/scientificreports/
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
Content courtesy of Springer Nature, terms of use apply. Rights reserved
5
Vol.:(0123456789)
Scientic Reports | (2022) 12:20571 | https://doi.org/10.1038/s41598-022-24934-8
www.nature.com/scientificreports/
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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
6
Vol:.(1234567890)
Scientic Reports | (2022) 12:20571 | https://doi.org/10.1038/s41598-022-24934-8
www.nature.com/scientificreports/
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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
7
Vol.:(0123456789)
Scientic Reports | (2022) 12:20571 | https://doi.org/10.1038/s41598-022-24934-8
www.nature.com/scientificreports/
(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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
8
Vol:.(1234567890)
Scientic Reports | (2022) 12:20571 | https://doi.org/10.1038/s41598-022-24934-8
www.nature.com/scientificreports/
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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
9
Vol.:(0123456789)
Scientic Reports | (2022) 12:20571 | https://doi.org/10.1038/s41598-022-24934-8
www.nature.com/scientificreports/
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
References
1. Seewoo, B. J., Feindel, K. W., Etherington, S. J. & Rodger, J. Frequency-specic eects of low-intensity rTMS can persist for up to
2 weeks post-stimulation: A longitudinal rs-fMRI/MRS study in rats. Brain Stimul. 12, 1526–1536. https:// doi. org/ 10. 1016/j. brs.
2019. 06. 028 (2019).
2. Seewoo, B. J., Feindel, K. W., Etherington, S. J. & Rodger, J. Resting-state fMRI study of brain activation using low-intensity repeti-
tive transcranial magnetic stimulation in rats. Sci. Rep. https:// doi. org/ 10. 1038/ s41598- 018- 24951-6 (2018).
3. Tik, M. et al. Towards understanding rTMS mechanism of action: Stimulation of the DLPFC causes network-specic increase in
functional connectivity. Neuroimage 162, 289–296. https:// doi. org/ 10. 1016/j. neuro image. 2017. 09. 022 (2017).
4. Wang, J. X. et al. Targeted enhancement of cortical-hippocampal brain networks and associative memory. Science (80‑) 345,
1054–1057. https:// doi. org/ 10. 1126/ scien ce. 12529 00 (2014).
5. Abellaneda-Pérez, K. et al. BDNF Val66Met gene polymorphism modulates brain activity following rTMS-induced memory
impairment. Sci. Rep. 12, 1–10. https:// doi. org/ 10. 1038/ s41598- 021- 04175-x (2022).
6. Cheeran, B. et al. A common polymorphism in the brain-derived neurotrophic factor gene (BDNF) modulates human cortical
plasticity and the response to rTMS. J. Physiol. 586, 5717–5725. https:// doi. org/ 10. 1113/ jphys iol. 2008. 159905 (2008).
7. Schintu, S. et al. Callosal anisotropy predicts attentional network changes aer parietal inhibitory stimulation. Neuroimage 226,
117559. https:// doi. org/ 10. 1016/j. neuro image. 2020. 117559 (2021).
8. Mariner, J., Loetscher, T. & Hordacre, B. Parietal cortex connectivity as a marker of shi in spatial attention following continuous
theta burst stimulation. Front. Hum. Neurosci. 15, 1–11. https:// doi. org/ 10. 3389/ fnhum. 2021. 718662 (2021).
9. Fox, M. D. et al. Resting-state networks link invasive and noninvasive brain stimulation across diverse psychiatric and neurological
diseases. Proc. Natl. Acad. Sci. U. S. A. 111, E4367–E4375. https:// doi. org/ 10. 1073/ pnas. 14050 03111 (2014).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
10
Vol:.(1234567890)
Scientic Reports | (2022) 12:20571 | https://doi.org/10.1038/s41598-022-24934-8
www.nature.com/scientificreports/
10. Fox, M. D., Buckner, R. L., White, M. P., Greicius, M. D. & Pascual-Leone, A. Ecacy of transcranial magnetic stimulation targets
for depression is related to intrinsic functional connectivity with the subgenual cingulate. Biol. Psychiatry 72, 595–603. https://
doi. org/ 10. 1016/j. biops ych. 2012. 04. 028 (2012).
11. Cash, R. F. H. et al. Personalized connectivity-guided DLPFC-TMS for depression: Advancing computational feasibility, precision
and reproducibility. Hum. Brain Mapp. 42, 4155–4172. https:// doi. org/ 10. 1002/ hbm. 25330 (2021).
12. Drew, P. J. Vascular and neural basis of the BOLD signal. Curr. Opin. Neurobiol. 58, 61–69. https:// doi. org/ 10. 1016/j. conb. 2019.
06. 004 (2019).
13. Aydin-Abidin, S., Trippe, J., Funke, K., Eysel, U. T. & Benali, A. High- and low-frequency repetitive transcranial magnetic stimula-
tion dierentially activates c-Fos and zif268 protein expression in the rat brain. Exp. Brain Res. 188, 249–261. https:// doi. org/ 10.
1007/ s00221- 008- 1356-2 (2008).
14. Labedi, A., Benali, A., Mix, A., Neubacher, U. & Funke, K. Modulation of inhibitory activity markers by intermittent theta-burst
stimulation in rat cortex is NMDA-receptor dependent. Brain Stimul. 7, 394–400. https:// doi. org/ 10. 1016/j . brs. 2014. 02. 010 (2014).
15. Mix, A., Hoppenrath, K. & Funke, K. Reduction in cortical parvalbumin expression due to intermittent theta-burst stimulation
correlates with maturation of the perineuronal nets in young rats. Dev. Neurobiol. 75, 1–11. https:// doi. org/ 10. 1002/ dneu. 22205
(2015).
16. Rodger, J. & Sherrard, R. M. Optimising repetitive transcranial magnetic stimulation for neural circuit repair following traumatic
brain injury. Neural Regen. Res. 10, 357 (2015).
17. Madore, M. R. et al. Moving back in the brain to drive the eld forward: Targeting neurostimulation to dierent brain regions in
animal models of depression and neurodegeneration. J. Neurosci. Methods 360, 109261. https:// doi. org/ 10. 1016/j. jneum eth. 2021.
109261 (2021).
18. Moretti, J. & Rodger, J. A little goes a long way: Neurobiological eects of low intensity rTMS and implications for mechanisms of
rTMS. Curr. Res. Neurobiol. 3, 100033. https:// doi. org/ 10. 1016/j. crneur. 2022. 100033 (2022).
19. Gersner, R., Kravetz, E., Feil, J., Pell, G. & Zangen, A. Long-term eects of repetitive transcranial magnetic stimulation on markers
for neuroplasticity: Dierential outcomes in anesthetized and awake animals. J. Neurosci. 31, 7521–7526. https:// doi. o rg/ 10. 1523/
JNEUR OSCI. 6751- 10. 2011 (2011).
20. Sykes, M. et al. Dierences in motor evoked potentials induced in rats by transcranial magnetic stimulation under two separate
anesthetics: Implications for plasticity studies. Front. Neural Circuits 10, 1–11. https:// doi. org/ 10. 3389/ fncir. 2016. 00080 (2016).
21. Agetsuma, M., Hamm, J. P., Tao, K., Fujisawa, S. & Yuste, R. Parvalbumin-positive interneurons regulate neuronal ensembles in
visual cortex. Cereb. Cortex 28, 1831–1845. https:// doi. org/ 10. 1093/ cercor/ bhx169 (2018).
22. Caillard, O. et al. Role of the calcium-binding protein parvalbumin in short-term synaptic plasticity. Proc. Natl. Acad. Sci. U. S. A.
97, 13372–13377. https:// doi. org/ 10. 1073/ pnas. 23036 2997 (2000).
23. Hoppenrath, K. & Funke, K. Time-course of changes in neuronal activity markers following iTBS-TMS of the rat neocortex.
Neurosci. Lett. 536, 19–23. https:// doi. org/ 10. 1016/j. neulet. 2013. 01. 003 (2013).
24. Volz, L. J., Benali, A., Mix, A., Neubacher, U. & Funke, K. Dose-dependence of changes in cortical protein expression induced with
repeated transcranial magnetic theta-burst stimulation in the rat. Brain Stimul. 6, 598–606. https:// doi. org/ 10. 1016/j. brs. 2013. 01.
008 (2013).
25. Mulders, W. H. A. M. et al. Low-intensity repetitive transcranial magnetic stimulation over prefrontal cortex in an animal model
alters activity in the auditory thalamus but does not aect behavioural measures of tinnitus. Exp. Brain Res. 237, 883–896. https://
doi. org/ 10. 1007/ s00221- 018- 05468-w (2019).
26. Makowiecki, K., Garrett, A., Harvey, A. R. & Rodger, J. Low-intensity repetitive transcranial magnetic stimulation requires concur-
rent visual system activity to modulate visual evoked potentials in adult mice. Sci. Rep. 8, 1–13. https:// doi. org/ 10. 1038/ s41598-
018- 23979-y (2018).
27. Wheeler, A. L. et al. Identication of a functional connectome for long-term fear memory in mice. PLoS Comput. Biol. https:// doi.
org/ 10. 1371/ journ al. pcbi. 10028 53 (2013).
28. Vetere, G. et al. Chemogenetic interrogation of a brain-wide fear memory network in mice. Neuron 94, 363-374.e4. https:// doi.
org/ 10. 1016/j. neuron. 2017. 03. 037 (2017).
29. Scott, G. A. et al. Disrupted neurogenesis in germ-free mice: Eects of age and sex. Front. Cell Dev. Biol. 8, 1–11. https:// doi. org/
10. 3389/ fcell. 2020. 00407 (2020).
30. Hodges, T. E., Lee, G. Y., Noh, S. H. & Galea, L. A. M. Sex and age dierences in cognitive bias and neural activation in response
to cognitive bias testing. Neurobiol. Stress 18, 100458. https:// doi. org/ 10. 1016/j. ynstr. 2022. 100458 (2022).
31. Valero-Cabré, A., Pascual-Leone, A. & Rushmore, R. J. Cumulative sessions of repetitive transcranial magnetic stimulation (rTMS)
build up facilitation to subsequent TMS-mediated behavioural disruptions. Eur. J. Neurosci. 27, 765–774. h ttps:// doi. org/ 10. 1111/j.
1460- 9568. 2008. 06045.x (2008).
32. Bäumer, T. et al. Repeated premotor rTMS leads to cumulative plastic changes of motor cortex excitability in humans. Neuroimage
20, 550–560. https:// doi. org/ 10. 1016/ S1053- 8119(03) 00310-0 (2003).
33. Lein, E. S. et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176. https:// doi. org/ 10. 1038/
natur e05453 (2007).
34. Bakker, R., Tiesinga, P. & Kötter, R. e scalable brain atlas: Instant web-based access to public brain atlases and related content.
Neuroinformatics 13, 353–366. https:// doi. org/ 10. 1007/ s12021- 014- 9258-x (2015).
35. Chen, C. C., Lu, J., Yang, R., Ding, J. B. & Zuo, Y. Selective activation of parvalbumin interneurons prevents stress-induced synapse
loss and perceptual defects. Mol. Psychiatry 23, 1614–1625. https:// doi. org/ 10. 1038/ mp. 2017. 159 (2018).
36. Boyer, M. et al. Invivo low-intensity magnetic pulses durably alter neocortical neuron excitability and spontaneous activity. J.
Physiol. 600, 4019–4037. https:// doi. org/ 10. 1113/ JP283 244 (2022).
37. Tang, A. D. et al. Low-intensity repetitive magnetic stimulation lowers action potential threshold and increases spike ring in layer
5 pyramidal neurons invitro. Neuroscience 335, 64–71. https:// doi. org/ 10. 1016/j. neuro scien ce. 2016. 08. 030 (2016).
38. Lonergan, M. E., Gaord, G. M., Jarome, T. J. & Helmstetter, F. J. Time-dependent expression of arc and Zif268 aer acquisition
of fear conditioning. Neural Plast. 2010, 8–11. https:// doi. org/ 10. 1155/ 2010/ 139891 (2010).
39. Fujiki, M., Yee, K. M. & Steward, O. Non-invasive high frequency repetitive transcranial magnetic stimulation (hfrTMS) robustly
activates molecular pathways implicated in neuronal growth and synaptic plasticity in select populations of neurons. Front. Neu
rosci. 14, 1–17. https:// doi. org/ 10. 3389/ fnins. 2020. 00558 (2020).
40. Bestmann, S., Baudewig, J., Siebner, H. R., Rothwell, J. C. & Frahm, J. Subthreshold high-frequency TMS of human primary motor
cortex modulates interconnected frontal motor areas as detected by interleaved fMRI-TMS. Neuroimage 20, 1685–1696. https://
doi. org/ 10. 1016/j. neuro image. 2003. 07. 028 (2003).
41. Bestmann, S., Baudewig, J., Siebner, H. R., Rothwell, J. C. & Frahm, J. Functional MRI of the immediate impact of transcranial
magnetic stimulation on cortical and subcortical motor circuits. Eur. J. Neurosci. 19, 1950–1962. https:// doi. org/ 10. 1111/j. 1460-
9568. 2004. 03277.x (2004).
42. Kim, Y. et al. Mapping social behavior-induced brain activation at cellular resolution in the mouse. Cell R ep. 10, 292–305. https://
doi. org/ 10. 1016/j. celrep. 2014. 12. 014 (2015).
43. Charles James, J. & Funke, K. Repetitive transcranial magnetic stimulation reverses reduced excitability of rat visual cortex induced
by dark rearing during early critical period. Dev. Neurobiol. 80, 399–410. https:// doi. org/ 10. 1002/ dneu. 22785 (2020).
Content courtesy of Springer Nature, terms of use apply. Rights reserved
11
Vol.:(0123456789)
Scientic Reports | (2022) 12:20571 | https://doi.org/10.1038/s41598-022-24934-8
www.nature.com/scientificreports/
44. Kimbrough, A. et al. Characterization of the brain functional architecture of psychostimulant withdrawal using single-cell whole-
brain imaging. Eneuro https:// doi. org/ 10. 1523/ ENEURO. 0208- 19. 2021 (2021).
45. Chen, G., Chen, G., Xie, C. & Li, S.-J. Negative functional connectivity and its dependence on the shortest path length of positive
network in the resting-state human brain. Brain Connect. 1, 195–206. https:// doi. org/ 10. 1089/ brain. 2011. 0025 (2011).
46. Murphy, K., Birn, R. M., Handwerker, D. A., Jones, T. B. & Bandettini, P. A. e impact of global signal regression on resting state
correlations: Are anti-correlated networks introduced?. Neuroimage 44, 893–905. https:// doi. org/ 10. 1016/j. neuro image. 2008. 09.
036 (2009).
47. Benali, A. et al. eta-burst transcranial magnetic stimulation alters cortical inhibition. J. Neurosci. 31, 1193–1203. https:// doi.
org/ 10. 1523/ JNEUR OSCI. 1379- 10. 2011 (2011).
48. Makinson, C. D. et al. Regulation of thalamic and cortical network synchrony by Scn8a. Neuron 93, 1165-1179.e6. https:// doi. org/
10. 1016/j. neuron. 2017. 01. 031 (2017).
49. Jang, H. J. et al. Distinct roles of parvalbumin and somatostatin interneurons in gating the synchronization of spike times in the
neocortex. Sci. Adv. https:// doi. org/ 10. 1126/ sciadv. aay53 33 (2020).
50. Sohal, V. S., Zhang, F., Yizhar, O. & Deisseroth, K. Parvalbumin neurons and gamma rhythms enhance cortical circuit performance.
Nature 459, 698–702. https:// doi. org/ 10. 1038/ natur e07991 (2009).
51. Bartova, L. et al. Reduced default mode network suppression during a working memory task in remitted major depression. J.
Psychiatr. Res. 64, 9–18. https:// doi. org/ 10. 1016/j. jpsyc hires. 2015. 02. 025 (2015).
52. Taylor, J. E. et al. Depressive symptoms reduce when dorsolateral prefrontal cortex-precuneus connectivity normalizes aer
functional connectivity neurofeedback. Sci. Rep. 12, 2581. https:// doi. org/ 10. 1038/ s41598- 022- 05860-1 (2022).
53. Baggio, H.-C. et al. Cognitive impairment and resting-state network connectivity in Parkinsons disease. Hum. Brain Mapp. 36,
199–212. https:// doi. org/ 10. 1002/ hbm. 22622 (2015).
54. Baldassarre, A., Ramsey, L. E., Siegel, J. S., Shulman, G. L. & Corbetta, M. Brain connectivity and neurological disorders aer
stroke. Curr. Opin. Neurol. 29, 706–713. https:// doi. org/ 10. 1097/ WCO. 00000 00000 000396 (2016).
55. Manning, J. et al. Altered resting-state functional connectivity of the frontal-striatal reward system in social anxiety disorder. PLoS
ONE 10, e0125286. https:// doi. org/ 10. 1371/ journ al. pone. 01252 86 (2015).
56. Hale, M. W. et al. Multiple anxiogenic drugs recruit a parvalbumin-containing subpopulation of GABAergic interneurons in the
basolateral amygdala. Prog. Neuro‑Psychopharmacol. Biol. Psychiatry 34, 1285–1293. https:// doi. org/ 10. 1016/j. pnpbp. 2010. 07. 012
(2010).
57. Xie, Y., Chen, S., Wu, Y. & Murphy, T. H. Prolonged decits in parvalbumin neuron stimulation-evoked network activity despite
recovery of dendritic structure and excitability in the somatosensory cortex following global ischemia in mice. J. Neurosci. 34,
14890–14900. https:// doi. org/ 10. 1523/ JNEUR OSCI. 1775- 14. 2014 (2014).
58. Sauer, J.-F., Strüber, M. & Bartos, M. Impaired fast-spiking interneuron function in a genetic mouse model of depression. Elife
https:// doi. org/ 10. 7554/ eLife. 04979 (2015).
59. Li, L.-B. et al. e theta-related ring activity of parvalbumin-positive neurons in the medial septum-diagonal band of broca
complex and their response to 5-HT 1A Receptor stimulation in a rat model of Parkinson’s disease. Hippocampus 24, 326–340.
https:// doi. org/ 10. 1002/ hipo. 22226 (2014).
60. Kim, M. S. et al. Ecacy of cumulative high-frequency rTMS on freezing of gait in Parkinsons disease. Restor. Neurol. Neurosci.
33, 521–530. https:// doi. org/ 10. 3233/ RNN- 140489 (2015).
61. De Raedt, R., Vanderhasselt, M.-A. & Baeken, C. Neurostimulation as an intervention for treatment resistant depression: From
research on mechanisms towards targeted neurocognitive strategies. Clin. Psychol. Rev. 41, 61–69. https:// doi. org/ 10. 1016/j. cpr.
2014. 10. 006 (2015).
62. Watanabe, K. et al. Comparative study of ipsilesional and contralesional repetitive transcranial magnetic stimulations for acute
infarction. J. Neurol. Sci. 384, 10–14. https:// doi. org/ 10. 1016/j. jns. 2017. 11. 001 (2018).
63. Diefenbach, G. J., Assaf, M., Goethe, J. W., Gueorguieva, R. & Tolin, D. F. Improvements in emotion regulation following repetitive
transcranial magnetic stimulation for generalized anxiety disorder. J. Anxiety Disord. 43, 1–7. https:// doi. org/ 10. 1016/j. janxd is.
2016. 07. 002 (2016).
64. Poh, E. Z., Harvey, A. R., Makowiecki, K. & Rodger, J. Online LI-rTMS during a Visual Learning Task: dierential Impacts on
Visual Circuit and Behavioural Plasticity in Adult Ephrin-A2A5-/- Mice. ENeuro https:// doi. org/ 10. 1523/ ENEURO. 0163- 17. 2018
(2018).
65. Terstege, D. J., Durante, I. M. & Epp, J. R. Brain-wide neuronal activation and functional connectivity are modulated by prior
exposure to repetitive learning episodes. Front. Behav. Neurosci. 16, 1–28. https:// doi. org/ 10. 3389/ fnbeh. 2022. 907707 (2022).
66. Berg, S. et al. Ilastik: Interactive machine learning for (bio)image analysis. Nat. Methods 16, 1226–1232. https:// doi. org/ 10. 1038/
s41592- 019- 0582-9 (2019).
67. Fürth, D. et al. An interactive framework for whole-brain maps at cellular resolution. Nat. Neurosci. 21, 139–149. https:// doi. org/
10. 1038/ s41593- 017- 0027-7 (2018).
68. Terstege, D. J., Oboh, D. O. & Epp, J. R. FASTMAP: Open-source exible atlas segmentation tool for multi-area processing of
biological images. ENeuro 9, 1–12. https:// doi. org/ 10. 1523/ ENEURO. 0325- 21. 2022 (2022).
69. Rubinov, M. & Sporns, O. Complex network measures of brain connectivity: Uses and interpretations. Neuroimage 52, 1059–1069.
https:// doi. org/ 10. 1016/j. neuro image. 2009. 10. 003 (2010).
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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
12
Vol:.(1234567890)
Scientic Reports | (2022) 12:20571 | https://doi.org/10.1038/s41598-022-24934-8
www.nature.com/scientificreports/
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 022- 24934-8.
Correspondence and requests for materials should be addressed to J.M.orJ.R.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
Open Access is article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. e images or other third party material in this
article are included in the articles Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
© e Author(s) 2022
Content courtesy of Springer Nature, terms of use apply. Rights reserved
1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com
... Following demyelination, when the density of cFos + neun + cells was significantly elevated in M1 and V2, it again appeared unchanged by a single session of iTBS ( Fig. 5f-j). As rTMS, delivered in an iTBS pattern, is most effective at modulating the activity of a subset of cortical neurons, particularly the activity of interneurons [55][56][57][58], we also examined the effect of CPZ demyelination and iTBS on the density of cFos + PV + interneurons in M1 and V2 (Fig. 5k-r). CPZ-demyelination significantly increased the density of cFos + PV + interneurons in both regions (Fig. 5s) but this effect was further exacerbated by iTBS (Fig. 5s). ...
... Our results match those of others showing that PV + interneurons are strongly modulated by rTMS [55,58,[76][77][78] although the direction of modulation is highly dependent on TMS intensity, frequency and number of sessions, as well as brain state -for example the effect can be different in awake versus anaesthetised rodents [53]. As PV + interneurons also regulate brain wide oscillations [65,[79][80][81], they likely mediate the effects of LI-rTMS on brain network synchronisation in rodents [57] and theta oscillations in humans [82,83]. Therefore, we propose that iTBS alters the activity of PV + interneurons and promotes remyelination, however, it is unclear whether the restoration of normal network activity supports remyelination or visa versa. ...
... rTMS can modulate neuronal activity in the brain [52,57], however, the outcome of rTMS protocols is heavily influenced by the baseline level of neuronal activity within the targeted region [93]. Structural differences between M1 and V2 cortical circuitry [94] may influence the neuronal response to demyelination. ...
Article
Full-text available
In people with multiple sclerosis (MS), newborn and surviving oligodendrocytes (OLs) can contribute to remyelination, however, current therapies are unable to enhance or sustain endogenous repair. Low intensity repetitive transcranial magnetic stimulation (LI-rTMS), delivered as an intermittent theta burst stimulation (iTBS), increases the survival and maturation of newborn OLs in the healthy adult mouse cortex, but it is unclear whether LI-rTMS can promote remyelination. To examine this possibility, we fluorescently labelled oligodendrocyte progenitor cells (OPCs; Pdgfrα-CreER transgenic mice) or mature OLs (Plp-CreER transgenic mice) in the adult mouse brain and traced the fate of each cell population over time. Daily sessions of iTBS (600 pulses; 120 mT), delivered during cuprizone (CPZ) feeding, did not alter new or pre-existing OL survival but increased the number of myelin internodes elaborated by new OLs in the primary motor cortex (M1). This resulted in each new M1 OL producing ~ 471 µm more myelin. When LI-rTMS was delivered after CPZ withdrawal (during remyelination), it significantly increased the length of the internodes elaborated by new M1 and callosal OLs, increased the number of surviving OLs that supported internodes in the corpus callosum (CC), and increased the proportion of axons that were myelinated. The ability of LI-rTMS to modify cortical neuronal activity and the behaviour of new and surviving OLs, suggests that it may be a suitable adjunct intervention to enhance remyelination in people with MS.
... According to the pattern applied, rTMS modulates the inhibitory expression of the immediate early genes c-fos and zif268, enzymes GAD65 and GAD67 involved in gammaamino-butyric acid (GABA) synthesis, and calcium-binding proteins [62]. Stimulation patterns that increase cortical excitability, such as 10 Hz and iTBS stimulation, induce Ca 2+ -dependent signaling, depress inhibitory circuits by reducing parvalbumin expression in fast-spiking interneurons and destabilizing GABA receptors to reduce GABAergic synaptic strength. ...
... Stimulation patterns that increase cortical excitability, such as 10 Hz and iTBS stimulation, induce Ca 2+ -dependent signaling, depress inhibitory circuits by reducing parvalbumin expression in fast-spiking interneurons and destabilizing GABA receptors to reduce GABAergic synaptic strength. In contrast, inhibitory protocols like 1 Hz and cTBS alter calbindin expression [62]. Ca 2+ activates calmodulin dependent kinase II and calcineurin, that regulate the initiation and maintenance of LTP and LTD, respectively. ...
Article
Full-text available
Traumatic brain injury (TBI) is one of the growing public health problems and a leading cause of disabilities and mortality worldwide. After the mechanical impact to the head, patients of all ages suffer from cognitive and neurological deficits, as well as psychological disorders to different extents. In the last years, the use of electrical impulses and magnetic currents to achieve therapeutic effects have shown promising results and became potential treatments for TBI. Potential mechanisms of action described so far include long term potentiation and depression of neuronal synapses, stimulation of neurotransmitters and growth factors release, and reduction of neuroinflammation, apoptosis and excitotoxicity, among others. Although promising results have been obtained in pre-clinical experiments and limited clinical studies, the high rate of variability in technical parameters and the limited number of patients enrolled have made difficult to clearly define the optimal conditions to obtain reliable therapeutic effects with these stimulation techniques in TBI patients. The present review aims to describe the molecular processes taking place in the brain after the injury, as well as to describe some of the neurostimulation treatments currently under development for TBI management.
... Notwithstanding, a widely held model describing ASC's pathophysiology involves the brain's excitatory-inhibitory disbalance (E/I disbalance) [6,14,18] driven by impairments Our team expanded the conventional technique to what we term personalized rTMS (PrTMS), which is based on the stimulation of a relatively larger portion of the brain cortical mantle, a general concept that has been suggested by numerous rTMS studies and position papers [31][32][33]. Contrary to standard rTMS protocols, PrTMS is not confined to one or two cortical sites but rather stimulates the DLPFC, orbitofrontal cortex, medial posterior cortex, and the central motor strip, thereby covering substantially larger areas and different functional territories of the cortex, against the backdrop of reduced TMS amplitude (power) [34]. ...
... This report describes the results obtained with PrTMS treatment of ASC patients in our clinic. The superiority and safety of the lower-power TMS approach exposing more of the cortical mantle to the stimulating magnetic field are suggested by a growing body of evidence [31][32][33][34]. None of the subjects developed seizures or reported any other side effects, and PrTMS induced the most rapid and marked responses in the oldest age group, viz., ASQ responders (median age = 19), with 55% of subject scores showing a reduction of 15% or more, and 44%, called 'responders', exhibiting a reduction to or below the ASC diagnostic threshold. ...
Article
Full-text available
Autism spectrum condition (ASC) is a neurodevelopmental condition that is only partly responsive to prevailing interventions. ASC manifests core challenges in social skills, communication, and sensory function and by repetitive stereotyped behaviors, along with imbalances in the brain’s excitatory (E) and inhibitory (I) signaling. Repetitive transcranial magnetic stimulation (rTMS) has shown promise in ASC and may be a useful addition to applied behavioral analysis (ABA), a gold-standard psychotherapeutic intervention. We report an open-label clinical pilot (initial) study in which ABA-treated ASC persons (n = 123) received our personalized rTMS protocol (PrTMS). PrTMS uses low TMS pulse intensities and continuously updates multiple cortical stimulation locales and stimulation frequencies based on the spectral EEG and psychometrics. No adverse effects developed, and 44% of subjects had ASC scale scores reduced to below diagnostic cutoffs. Importantly, in PrTMS responders, the spectral EEG regression flattened, implying a more balanced E/I ratio. Moreover, with older participants, alpha peak frequency increased, a positive correlate of non-verbal cognition. PrTMS may be an effective ASC intervention, offering improved cognitive function and overall symptomatology. This warrants further research into PrTMS mechanisms and specific types of subjects who may benefit, along with validation of the present results and exploration of broader clinical applicability.
... (3) Repetitive magnetic transcranial stimulation exerts therapeutic effects by regulating certain gene expression and protein synthesis ( Figure 1). Some studies have shown that rTMS can regulate cortical network activities by modulating gene expression of c-Fos, GABAergic and glutamatergic and inhibiting calcium-binding proteins, CKD-95, etc. [53][54][55][56] These genes and proteins are closely related to recovery after brain injury, can improve the brain regions damaged by PTSD and promote their functional recovery, and can also help us determine the effectiveness of PTSD treatment. ...
Article
Full-text available
Post-traumatic stress disorder (PTSD) is a psychiatric disorder that develops and persists after an individual experiences a major traumatic or life-threatening event. While phar-macological treatment and psychological interventions can alleviate some symptoms, pharmacotherapy is time-consuming with low patient compliance, and psychological interventions are costly. Repetitive Transcranial Magnetic Stimulation (rTMS) is a safe and effective technique for treating PTSD, with advantages such as high compliance, low cost, and simplicity of implementation. It can even simultaneously improve depressive symp-toms in some patients. Current research indicates that high-frequency rTMS shows better therapeutic effects compared to low-frequency rTMS, with no significant difference in the likelihood of adverse reactions between the two. Theta Burst Stimulation (TBS) exhibits similar efficacy to high-frequency rTMS, with shorter duration and significant improve-ment in depressive symptoms. However, it carries a slightly higher risk of adverse reac-tions compared to traditional high-frequency rTMS. Combining rTMS with psychological therapy appears to be more effective in improving PTSD symptoms, with early onset of effects and longer duration, albeit at higher cost and requiring individualized patient con-trol. The most common adverse effect of treatment is headache, which can be improved by stopping treatment or using analgesics. Despite these encouraging data, several aspects remain unknown. Given the highly heterogeneous nature of PTSD, defining unique treat-ment methods for this patient population is quite challenging. There are also considerable differences between trials regarding stimulation parameters, therapeutic effects, and the role of combined psychological therapy, which future research needs to address.
... Prior work found that increased anti-correlations are associated with lower activity of parvalbumin inhibitory interneurons [100,101]. To determine whether sex or fluid influence the proportions of negative and positive correlations in a network, we counted of the number of negative and positive correlations with the signcount function of the wordspace package (version 0.2-8 [102]). ...
Preprint
Full-text available
Graphical Summary Binge-like ethanol drinking engages more regions and NAcc inputs in female relative to male mice. Left: Comparison of regions with both greater c-Fos expression and c-Fos+GFP colocalization in female relative to male ethanol drinking mice. Right: NAcc inputs engaged by binge-like ethanol drinking compared to water drinking mice, sex collapsed. Thalamic (TH) regions include left parasubthalamic nucleus, left anteromedial nucleus of the thalamus, left central medial nucleus of the thalamus, left medial group of the dorsal thalamus, left subparafascicular nucleus, left peripeduncular nucleus, and right paraventricular nucleus of the thalamus. EW, Edinger-Westphal nucleus; GU, gustatory areas. Bottom Middle: NAcc inputs with greater engagement in male than female ethanol drinking mice (left and right main olfactory bulbs (MOB). Bottom Right: NAcc inputs with greater engagement in female than male ethanol drinking mice. Amygdala (AMY) regions include left anterior amygdalar area and left intercalated amygdalar area. Hippocampal (HPF) regions include right dentate gyrus, right Field CA1, right Field CA2, and right Field CA3. Hypothalamic (HY) regions include left and right lateral hypothalamic area, left and right periventricular hypothalamic nucleus, preoptic part, right dorsomedial nucleus of the hypothalamus, right posterior hypothalamic nucleus, and right ventromedial hypothalamic nucleus. Midbrain (MB) regions include left and right midbrain reticular nucleus, retrorubral area, left and right superior colliculus, motor related, left nucleus of the brachium of the inferior colliculus, left nucleus of the posterior commissure, left olivary pretectal nucleus, left posterior pretectal nucleus, left superior colliculus, sensory related, and right substantia nigra, reticular parts. Pontine (P) regions include left superior central nucleus raphe, left supratrigeminal nucleus, right nucleus raphe pontis, right pontine reticular nucleus, and right superior olivary complex. Thalamic (TH) regions include left and right lateral dorsal nucleus of the thalamus, right dorsal part of the lateral geniculate complex, right lateral posterior nucleus of the thalamus, and right parasubthalamic nucleus. CB, Cerebellum; MA, magnocellular nucleus; SSp-m, primary somatosensory cortex, mouth; Created with BioRender.com. See Table S18 for additional information.
... Currently, one of the most relevant scientific directions in biology and medicine is the study of modern environmentally friendly and economical technologies using physical factors. A special role in this direction is played by low-intensity electromagnetic radiation (EMR) of the extremely high frequency (EHF) or millimeter range [1][2][3]. Due to its high biological efficacy, the EMR of EHF is used in medical practice to treat a wide range of diseases, including the cardiovascular system (CVS) [4]. In particular, over the past years, extensive experience has been accumulated in the use of millimeter radiation for the treatment of stable and unstable angina pectoris, coronary heart disease, hypertension, and myocardial infarction [5,6]. ...
... Animal models, both in vivo and in vitro, have provided important insights into mechanisms by which rTMS modifies neuronal circuit excitability and plasticity (Vlachos et al., 2012;Tokay et al., 2014;Lenz et al., 2016;Hong et al., 2020;Romero et al., 2022;Eichler et al., 2023). It has been shown for example that rTMS affects the functional and structural properties of excitatory and inhibitory synapses (Tokay et al., 2009;Vlachos et al., 2012;Lenz et al., 2016), and that it facilitates the reorganisation of abnormal cortical circuits (Tang et al., 2021;Moretti et al., 2022). High frequency rTMS enhances plasticity in the primary motor cortex and mitigates cognitive deficits of aged mice (Ma et al., 2019;Cambiaghi et al., 2021). ...
Article
Full-text available
Introduction 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, is crucial in experimental and clinical settings. It enables meaningful comparisons across studies and facilitates 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. Methods 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. Results 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. Although morphologies of mouse and rat CA1 neurons showed no significant differences, simulations confirmed that axon morphologies significantly influence individual cell 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. Conclusion 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.
... One potential advantage of utilizing rotating permanent magnets is the ability to create portable, cost-effective devices compared to conventional TMS [20]. Depending on the magnet strength and rotational frequency, the E-field strengths in the sTMS, TRPMS, and Watterson multipolar systems are comparable to other forms of low field stimulation, including low field magnetic stimulation (LFMS) [31], transcranial current stimulation (tCS) [32,33], and low-intensity repetitive magnetic stimulation (LI-rMS) [34,35]. Low field stimulation has been shown to induce changes at the cellular and molecular levels. ...
Article
Full-text available
Neurostimulation devices that use rotating permanent magnets are being explored for their potential therapeutic benefits in patients with psychiatric and neurological disorders. This study aims to characterize the electric field (E-field) for ten configurations of rotating magnets using finite element analysis and phantom measurements. Various configurations were modeled, including single or multiple magnets, and bipolar or multipolar magnets, rotated at 10, 13.3, and 350 revolutions per second (rps). E-field strengths were also measured using a hollow sphere (r=9.2 cm) filled with a 0.9% sodium chloride solution and with a dipole probe. The E-field spatial distribution is determined by the magnets’ dimensions, number of poles, direction of the magnetization, and axis of rotation, while the E-field strength is determined by the magnets’ rotational frequency and magnetic field strength. The induced E-field strength on the surface of the head ranged between 0.0092 and 0.52 V/m. In the range of rotational frequencies applied, the induced E-field strengths were approximately an order or two of magnitude lower than those delivered by conventional transcranial magnetic stimulation. The impact of rotational frequency on E-field strength represents a confound in clinical trials that seek to tailor rotational frequency to individual neural oscillations. This factor could explain some of the variability observed in clinical trial outcomes.
... One potential advantage of utilizing rotating permanent magnets is the ability to 366 create portable, cost-effective devices compared to conventional TMS [20]. Depending on 367 the magnet strength and rotational frequency, the E-field strengths in the sTMS, TRPMS, 368 and Watterson multipolar systems are comparable to other forms of low field stimulation, 369 including low field magnetic stimulation (LFMS) [27], transcranial current stimulation 370 (tCS) [28,29], and low-intensity repetitive magnetic stimulation (LI-rMS) [30,31]. Low field 371 stimulation has been shown to induce changes at the cellular and molecular levels. ...
Preprint
Neurostimulation devices that use rotating permanent magnets are being explored for their potential therapeutic benefits in patients with psychiatric and neurological disorders. This study aims to characterize the electric field (E-field) for ten configurations of rotating magnets using finite element analysis and phantom measurements. Various configurations were modeled, including single or multiple magnets, bipolar or multipolar magnets, rotated at 10, 13.3, and 400 Hz. E-field strengths were also measured using a hollow sphere filled with a 0.9% sodium chloride solution and with a dipole probe. The E-field spatial distribution is determined by the magnets' dimensions, number of poles, direction of the magnetization, and axis of rotation, while the E-field strength is determined by the magnets' rotational frequency and magnetic field strength. The induced E-field strength on the surface of the head ranged between 0.0092 and 0.59 V/m. At the range of rotational frequencies applied, the induced E-field strengths were approximately an order or two of magnitude lower than those delivered by conventional transcranial magnetic stimulation. The impact of rotational frequency on E-field strength represents a previously unrecognized confound in clinical trials that seek to personalize stimulation frequency to individual neural oscillations and may represent a mechanism to explain some clinical trial results.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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).
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
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.