Transcranial magnetic stimulation of the brain: What is stimulated? - A consensus and critical position paper.

Transcranial (electro)magnetic stimulation (TMS) is currently the method of choice to non-invasively induce neural activity in the human brain. A single transcranial stimulus induces a time-varying electric field in the brain that may evoke action potentials in cortical neurons. The spatial relationship between the locally induced electric field and the stimulated neurons determines axonal depolarization. The induced electric field is influenced by the conductive properties of the tissue compartments and is strongest in the superficial parts of the targeted cortical gyri and underlying white matter. TMS likely targets axons of both excitatory and inhibitory neurons. The propensity of individual axons to fire an action potential in response to TMS depends on their geometry, myelination and spatial relation to the imposed electric field and the physiological state of the neuron. The latter is determined by its transsynaptic dendritic and somatic inputs, intrinsic membrane potential and firing rate. Modeling work suggests that the primary target of TMS is axonal terminals in the crown top and lip regions of cortical gyri. The induced electric field may additionally excite bends of myelinated axons in the juxtacortical white matter below the gyral crown. Neuronal excitation spreads ortho- and antidromically along the stimulated axons and causes secondary excitation of connected neuronal populations within local intracortical microcircuits in the target area. Axonal and transsynaptic spread of excitation also occurs along cortico-cortical and cortico-subcortical connections, impacting on neuronal activity in the targeted network. Both local and remote neural excitation depend critically on the functional state of the stimulated target area and network. TMS also causes substantial direct co-stimulation of the peripheral nervous system. Peripheral co-excitation propagates centrally in auditory and somatosensory networks, but also produces brain responses in other networks subserving multisensory integration, orienting or arousal. The complexity of the response to TMS warrants cautious interpretation of its physiological and behavioural consequences, and a deeper understanding of the mechanistic underpinnings of TMS will be critical for advancing it as a scientific and therapeutic tool.

Effects of iTBS-rTMS on the Behavioral Phenotype of a Rat Model of Maternal Immune Activation.

Repetitive transcranial magnetic stimulation (rTMS) is considered a promising therapeutic tool for treating neuropsychiatric diseases. Previously, we found intermittent theta-burst stimulation (iTBS) rTMS to be most effective in modulating cortical excitation-inhibition balance in rats, accompanied by improved cortical sensory processing and sensory learning performance. Using an animal schizophrenia model based on maternal immune activation (MIA) we tested if iTBS applied to either adult or juvenile rats can affect the behavioral phenotype in a therapeutic or preventive manner, respectively. In a sham-controlled fashion, iTBS effects in MIA rats were compared with rats receiving vehicle NaCl injection instead of the synthetic viral strand. Prior to iTBS, adult MIA rats showed deficits in sensory gating, as tested with prepulse inhibition (PPI) of the acoustic startle reflex, and deficits in novel object recognition (NOR). No differences between MIA and control rats were evident with regard to signs of anxiety, anhedonia and depression but MIA rats were somewhat superior to controls during the training phase of Morris Water Maze (MWM) test. MIA but not control rats significantly improved in PPI following iTBS at adulthood but without significant differences between verum and sham application. If applied during adolescence, verum but not sham-iTBS improved NOR at adulthood but no difference in PPI was evident in rats treated either with sham or verum-iTBS. MIA and control rat responses to sham-iTBS applied at adulthood differed remarkably, indicating a different physiological reaction to the experimental experiences. Although verum-iTBS was not superior to sham-iTBS, MIA rats seemed to benefit from the treatment procedure in general, since differences-in relation to control rats declined or disappeared. Even if classical placebo effects can be excluded, motor or cognitive challenges or the entire handling procedure during the experiments appear to alleviate the behavioral impairments of MIA rats.

Effects of repetitive transcranial magnetic and deep brain stimulation on long-range synchrony of oscillatory activity in a rat model of developmental schizophrenia.

Aberrant neuronal network activity likely resulting from disturbed interactions of excitatory and inhibitory systems may be a major cause of cognitive deficits in neuropsychiatric diseases, like within the spectrum of schizophrenic phenotypes. In particular, the synchrony and pattern of oscillatory brain activity appears to be disturbed within limbic networks, e.g. between prefrontal cortex and hippocampus. In a rat model of maternal immune activation (MIA), we compared the acute effects of deep brain stimulation within either medial prefrontal cortex or ventral hippocampus with the effects of repetitive transcranial magnetic stimulation (rTMS), using the intermittent theta-burst protocol (iTBS), on oscillatory activity within limbic structures. Simultaneous local field potential recordings were made from medial prefrontal cortex, ventral hippocampus, nucleus accumbens and rostral part of ventral tegmental area before and after deep brain stimulation in anaesthetized rats previously (~3 h) treated with sham or verum rTMS. We found a waxing and waning pattern of theta and gamma activity in all structures which was less synchronous in particular between medial prefrontal cortex and ventral hippocampus in MIA offspring. Deep brain stimulation in medial prefrontal cortex and pre-treatment with iTBS-rTMS partly improved this pattern. Gamma-theta cross-frequency coupling was stronger in MIA offspring and could partly be reduced by deep brain stimulation in medial prefrontal cortex. We can confirm aberrant limbic network activity in a rat MIA model, and at least acute normalizing effects of the neuromodulatory methods. It has to be proven whether these procedures can have chronic effects suitable for therapeutic purposes.

Repetitive transcranial magnetic stimulation reverses reduced excitability of rat visual cortex induced by dark rearing during early critical period.

Early critical period of visual cortex is characterized by enhanced activity-driven neuronal plasticity establishing the specificity of neuronal connections required for optimal processing of sensory signals. Deprivation from visual input by dark rearing (DR) during this period leads to a lasting impairment of visual performance. Previously, we demonstrated that repetitive transcranial magnetic stimulation (rTMS) applied with intermittent theta-burst (iTBS) pattern during the critical period improved the visual performance of the DR rats. In this study, we describe that the excitability of the binocular part of the visual cortex (V1b), as measured in acute brain slices by input-output ratios of field excitatory synaptic potentials (fEPSPs), is lowered in DR rats compared to normal controls. Verum rTMS applied with the iTBS pattern during DR reversed this DR effect, while no rTMS effect was evident in the non-DR (nDR) rats. In addition, verum rTMS reduced the number of neurons expressing the 67 kD isoform of glutamic acid decarboxylase (GAD67), the calcium-binding protein calbindin (CB) and the zinc-finger transcription factor zif268/EGR1, as determined via immunohistochemistry, only in DR rats but not in nDR rats. Moreover, rTMS reduced the number of neurons expressing the calcium-binding protein parvalbumin (PV) only in nDR rats which showed more PV+ neurons compared to DR rats. This study confirms that iTBS-rTMS may be able to prevent or reverse the effects of DR on visual cortex physiology, likely through a modulation of the activity of inhibitory interneurons.

AMPA Induces NO-Dependent cGMP Signals in Hippocampal and Cortical Neurons via L-Type Voltage-Gated Calcium Channels.

The nitric oxide (NO)/cGMP signaling cascade has an established role in synaptic plasticity. However, with conventional methods, the underlying cGMP signals were barely detectable. Here, we set out to confirm the well-known NMDA-induced cGMP increases, to test the impact of AMPA on those signals, and to identify the relevant phosphodiesterases (PDEs) using a more sensitive fluorescence resonance energy transfer (FRET)-based method. Therefore, a "knock-in" mouse was generated that expresses a FRET-based cGMP indicator (cGi-500) allowing detection of cGMP concentrations between 100 nM and 3 µM. Measurements were performed in cultured hippocampal and cortical neurons as well as acute hippocampal slices. In hippocampal and cortical neurons, NMDA elicited cGMP signals half as high as the ones elicited by exogenous NO. Interestingly, AMPA increased cGMP independently of NMDA receptors and dependent on NO synthase (NOS) activation. NMDA- and AMPA-induced cGMP signals were not additive indicating that both pathways converge on the level of NOS. Accordingly, the same PDEs, PDE1 and PDE2, were responsible for degradation of NMDA- as well as AMPA-induced cGMP signals. Mechanistically, AMPAR induced calcium influx through L-type voltage-gated calcium channels leading to NOS and finally NO-sensitive guanylyl cyclase activation. Our results demonstrate that in addition to NMDA also AMPA triggers endogenous NO formation and hence cGMP production.

Prevention of schizophrenia deficits via non-invasive adolescent frontal cortex stimulation in rats.

Schizophrenia is a severe neurodevelopmental psychiatric affliction manifested behaviorally at late adolescence/early adulthood. Current treatments comprise antipsychotics which act solely symptomatic, are limited in their effectiveness and often associated with side-effects. We here report that application of non-invasive transcranial direct current stimulation (tDCS) during adolescence, prior to schizophrenia-relevant behavioral manifestation, prevents the development of positive symptoms and related neurobiological alterations in the maternal immune stimulation (MIS) model of schizophrenia.

Repetitive transcranial magnetic stimulation recovers cortical map plasticity induced by sensory deprivation due to deafferentiation.

KEY POINTS: Partial sensory deprivation (deafferentation) by removing whiskers from the rat snout resulted in a reduced responsiveness of related cortical representations. Repetitive transcranial magnetic stimulation (three blocks of intermittent theta-burst) applied for 5 days in combination with sensory exploration restored the normal responsiveness level of the deafferented barrel cortex. However, intracortical inhibition (lateral and recurrent) appeared to be reduced after repetitive transcranial magnetic stimulation, probably as the cause of improved responsiveness. Repetitive transcranial magnetic stimulation also reduced the asymmetry of the lateral spread of sensory activity. ABSTRACT: Repetitive transcranial magnetic stimulation (rTMS) modulates human cortical excitability. It has the potential to support recovery to normal cortical function when the excitation-inhibition balance is altered (e.g. after a stroke or loss of sensory input). We tested cortical map plasticity on the basis of sensory responses (local field potentials, LFPs) and expression of neuronal activity marker proteins within the barrel cortex of rats receiving either active or sham rTMS after selective unilateral deafferentation by whiskers plucking. Rats received daily rTMS [intermittent theta-burst (iTBS), active or sham] for 5 days before exploring an enriched environment. Our previous studies indicated a disinhibitory effect of iTBS on cortical activity. Therefore, we also expected disinhibitory effects if deafferentation causes depression of sensory responses. Deafferentation resulted in an acute general reduction of sensory responsiveness and enhanced expression of inhibitory activity markers (GAD67, parvalbumin) in the deafferented hemisphere. Active but not sham-iTBS-rTMS normalized these measures. The stronger caudal-to-frontal horizontal spread of activity across barrels was reduced after deafferentation but not restored after active iTBS, despite generally increased responses. Fitting the LFP data with a computational model of different strengths and types of excitatory and inhibitory connections further revealed an iTBS-induced reduction of lateral and recurrent inhibition as the most probable scenario. Whether the disinhibitory effect of iTBS for the restoration of normal cortical function in the acute phase of depression after deafferentiation is also beneficial in humans remains to be demonstrated. As recently discussed, disinhibition appears to be required to open a window for neuronal plasticity.

Neuropeptide Y as a possible homeostatic element for changes in cortical excitability induced by repetitive transcranial magnetic stimulation.

BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) is able to modify cortical excitability. Rat rTMS studies revealed a modulation of inhibitory systems, in particular that of the parvalbumin-expressing (PV+) interneurons, when using intermittent theta-burst stimulation (iTBS). OBJECTIVE: The potential disinhibitory action of iTBS raises the questions of how neocortical circuits stabilize excitatory-inhibitory balance within a physiological range. Neuropeptide Y (NPY) appears to be one candidate. METHODS: Analysis of cortical expression of PV, NPY and vesicular glutamate transporter type 1 (vGluT1) by immunohistochemical means at the level of cell counts, mean neuropil expression and single cell pre-/postsynaptic expression, with and without intraventricular NPY-injection. RESULTS: Our results show that iTBS not only reduced the number of neurons with high-PV expression in a dose-dependent fashion, but also increased the cortical expression of NPY, discussed to reduce glutamatergic transmission, and this was further associated with a reduced vGluT1 expression, an indicator of glutamateric presynaptic activity. Interneurons showing a low-PV expression exhibit less presynaptic vGluT1 expression compared to those with a high-PV expression. Intraventricular application of NPY prior to iTBS prevented the iTBS-induced reduction in the number of high-PV neurons, the reduction in tissue vGluT1 level and that presynaptic to high-PV cells. CONCLUSIONS: We conclude that NPY, possibly via a global but also slow homeostatic control of glutamatergic transmission, modulates the strength and direction of the iTBS effects, likely preventing pathological imbalance of excitatory and inhibitory cortical activity but still allowing enough disinhibition beneficial for plastic changes as during learning.

Real-World Data Collection Regarding Titration Algorithms for Insulin Glargine in Patients With Type 2 Diabetes Mellitus.

The primary objective of this study was to collect data regarding the effectiveness of different dose titration algorithms (TAs) for optimization or initiation of basal insulin supported oral therapy (BOT) in patients with type 2 diabetes. A total of 50 patients were enrolled in this trial (17 women, 33 men, age 63 ± 8 years, HbA1c 7.9 ± 0.8%). The investigator decided on an individual basis to apply any of 4 standard TAs: standard (S: fasting glucose target 90-130 mg/dL, n = 39), standard-fast titration (S-FT: 90-130 mg/dL, larger dose increments at FBG < 180 mg/dl, n = 1), less tight (LT: 110-150 mg/dL, n = 5), and tight (T: 70-100 mg/dL, n = 5). During the next 30 days daily contacts were used to adapt the insulin dose. The majority of all patients (70%) achieved a stable insulin glargine dose within 5 ± 6 days after initiation of the dose titration. HbA1c improved from 7.9 ± 0.8% to 7.5 ± 0.7% (P < .001). In total, 1300 dose decisions were made (1192 according to the TA and 108 by the physicians independently from the TA in 29 patients [58% of study population]). Reasons for TA-overruling dosing decisions were hypoglycemic events (14 mild/4 moderate) in 9 patients. In the majority of these cases (89.8%), the physician recommended continuation of the previous dose or a higher dose. The majority of FBG values were within the respective target range after 4 weeks. In conclusion, the insulin glargine TAs delivered safe dose recommendations with a low risk of hypoglycemia, which successfully led to a stable dose in the vast majority of patients.

Intermittent Theta-Burst Transcranial Magnetic Stimulation Alters Electrical Properties of Fast-Spiking Neocortical Interneurons in an Age-Dependent Fashion.

Modulation of human cortical excitability by repetitive transcranial magnetic stimulation (rTMS) appears to be in part related to changed activity of inhibitory systems. Our own studies showed that intermittent theta-burst stimulation (iTBS) applied via rTMS to rat cortex primarily affects the parvalbumin-expressing (PV) fast-spiking interneurons (FSIs), evident via a strongly reduced PV expression. We further found the iTBS effect on PV to be age-dependent since no reduction in PV could be induced before the perineuronal nets (PNNs) of FSIs start to grow around postnatal day (PD) 30. To elucidate possible iTBS-induced changes in the electrical properties of FSIs and cortical network activity during cortical critical period, we performed ex vivo-in vitro whole-cell patch clamp recordings from pre-labeled FSIs in the current study. FSIs of verum iTBS-treated rats displayed a higher excitability than sham-treated controls at PD29-38, evident as higher rates of induced action potential firing at low current injections (100-200 pA) and a more depolarized resting membrane potential. This effect was absent in younger (PD26-28) and older animals (PD40-62). Slices of verum iTBS-treated rats further showed higher rates of spontaneous excitatory postsynaptic currents (sEPSCs). Based on these and previous findings we conclude that FSIs are particularly sensitive to TBS during early cortical development, when FSIs show an activity-driven step of maturation which is paralleled by intense growth of the PNNs and subsequent closure of the cortical critical period. Although to be proven further, rTMS may be a possible early intervention to compensate for hypo-activity related mal-development of cortical neuronal circuits.

Repetitive magnetic stimulation induces plasticity of inhibitory synapses.

Repetitive transcranial magnetic stimulation (rTMS) is used as a therapeutic tool in neurology and psychiatry. While repetitive magnetic stimulation (rMS) has been shown to induce plasticity of excitatory synapses, it is unclear whether rMS can also modify structural and functional properties of inhibitory inputs. Here we employed 10-Hz rMS of entorhinohippocampal slice cultures to study plasticity of inhibitory neurotransmission on CA1 pyramidal neurons. Our experiments reveal a rMS-induced reduction in GABAergic synaptic strength (2-4 h after stimulation), which is Ca(2+)-dependent and accompanied by the remodelling of postsynaptic gephyrin scaffolds. Furthermore, we present evidence that 10-Hz rMS predominantly acts on dendritic, but not somatic inhibition. Consistent with this finding, a reduction in clustered gephyrin is detected in CA1 stratum radiatum of rTMS-treated anaesthetized mice. These results disclose that rTMS induces coordinated Ca(2+)-dependent structural and functional changes of specific inhibitory postsynapses on principal neurons.

Effects of chronic iTBS-rTMS and enriched environment on visual cortex early critical period and visual pattern discrimination in dark-reared rats.

Early cortical critical period resembles a state of enhanced neuronal plasticity enabling the establishment of specific neuronal connections during first sensory experience. Visual performance with regard to pattern discrimination is impaired if the cortex is deprived from visual input during the critical period. We wondered how unspecific activation of the visual cortex before closure of the critical period using repetitive transcranial magnetic stimulation (rTMS) could affect the critical period and the visual performance of the experimental animals. Would it cause premature closure of the plastic state and thus worsen experience-dependent visual performance, or would it be able to preserve plasticity? Effects of intermittent theta-burst stimulation (iTBS) were compared with those of an enriched environment (EE) during dark-rearing (DR) from birth. Rats dark-reared in a standard cage showed poor improvement in a visual pattern discrimination task, while rats housed in EE or treated with iTBS showed a performance indistinguishable from rats reared in normal light/dark cycle. The behavioral effects were accompanied by correlated changes in the expression of brain-derived neurotrophic factor (BDNF) and atypical PKC (PKCζ/PKMζ), two factors controlling stabilization of synaptic potentiation. It appears that not only nonvisual sensory activity and exercise but also cortical activation induced by rTMS has the potential to alleviate the effects of DR on cortical development, most likely due to stimulation of BDNF synthesis and release. As we showed previously, iTBS reduced the expression of parvalbumin in inhibitory cortical interneurons, indicating that modulation of the activity of fast-spiking interneurons contributes to the observed effects of iTBS.

Multiple blocks of intermittent and continuous theta-burst stimulation applied via transcranial magnetic stimulation differently affect sensory responses in rat barrel cortex.

KEY POINTS: Theta-burst stimulation (TBS) applied via transcranial magnetic stimulation is able to modulate human cortical excitability. Here we investigated in a rat model how two different forms of TBS, intermittent (iTBS) and continuous (cTBS), affect sensory responses in rat barrel cortex. We found that iTBS but less cTBS promoted late (>18 ms) sensory response components while not affecting the earliest response (8-18 ms). The effect increased with each of the five iTBS blocks applied. cTBS somewhat reduced the early response component after the first block but had a similar effect as iTBS after four to five blocks. We conclude that iTBS primarly modulates the activity of (inhibitory) cortical interneurons while cTBS may first reduce general neuronal excitability with a single block but reverse to iTBS-like effects with application of several blocks. ABSTRACT: Cortical sensory processing varies with cortical state and the balance of inhibition to excitation. Repetitive transcranial magnetic stimulation (rTMS) has been shown to modulate human cortical excitability. In a rat model, we recently showed that intermittent theta-burst stimulation (iTBS) applied to the corpus callosum, to activate primarily supragranular cortical pyramidal cells but fewer subcortical neurons, strongly reduced the cortical expression of parvalbumin (PV), indicating reduced activity of fast-spiking interneurons. Here, we used the well-studied rodent barrel cortex system to test how iTBS and continuous TBS (cTBS) modulate sensory responses evoked by either single or double stimuli applied to the principal (PW) and/or adjacent whisker (AW) in urethane-anaesthetized rats. Compared to sham stimulation, iTBS but not cTBS particularly enhanced late (>18 ms) response components of multi-unit spiking and local field potential responses in layer 4 but not the very early response (<18 ms). Similarly, only iTBS diminished the suppression of the second response evoked by paired PW or AW-PW stimulation at 20 ms intervals. The effects increased with each of the five iTBS blocks applied. With cTBS a mild effect similar to that of iTBS was first evident after 4-5 stimulation blocks. Enhanced cortical c-Fos and zif268 expression but reduced PV and GAD67 expression was found only after iTBS, indicating increased cortical activity due to lowered inhibition. We conclude that iTBS but less cTBS may primarily weaken a late recurrent-type cortical inhibition mediated via a subset of PV+ interneurons, enabling stronger late response components believed to contribute to the perception of sensory events.

Reduction in cortical parvalbumin expression due to intermittent theta-burst stimulation correlates with maturation of the perineuronal nets in young rats.

We recently showed that intermittent theta-burst stimulation (iTBS) using transcranial magnetic stimulation strongly reduces the number of rat neocortical interneurons expressing glutamic acid decarboxylase 67 kDa (GAD67) and parvalbumin (PV), indicating changed activity of fast-spiking (FS) interneurons. In advance of in vitro studies intended to characterize changes in electrical properties of FS interneurons under these conditions, we tested whether the iTBS effect is age-dependent. Conscious Sprague-Dawley rats aged between 28 and 90 days received three blocks of iTBS at 15 min intervals. We found that iTBS-related reduction in PV+ cells was absent up to an age of 32 days, then gradually increased, and approached a maximum of about 40% reduction at an age of about 40 days. The relative number of cells expressing PV (PV+, 8-9%) did not change with age in sham-controls and also the increase in cortical c-Fos expression induced by iTBS was not principally age-dependent. However, a prominent growth of the perineuronal nets, typically surrounding the PV+ cells, exactly paralleled the increase in the iTBS effect. Based on these findings, we conclude that the functional development of the inhibitory network of PV+ interneurons with regard to intracortical synaptic connectivity is not sufficiently matured in rats younger than 35 d to enable activity-dependent modifications during iTBS. Outgrowth of the perineuronal nets and associated maturation of excitatory cortical inputs, as is characteristic for the critical cortical period, may take place before PV+ interneurons can be sufficiently activated via repetitive transcranial magnetic stimulation, allowing plastic changes of molecular phenotype and likely also synaptic plasticity.

Synergistic effects of noradrenergic modulation with atomoxetine and 10 Hz repetitive transcranial magnetic stimulation on motor learning in healthy humans.

BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) is able to induce changes in neuronal activity that outlast stimulation. The underlying mechanisms are not completely understood. They might be analogous to long-term potentiation or depression, as the duration of the effects seems to implicate changes in synaptic plasticity. Norepinephrine (NE) has been shown to play a crucial role in neuronal plasticity in the healthy and injured human brain. Atomoxetine (ATX) and other NE reuptake inhibitors have been shown to increase excitability in different systems and to influence learning processes. Thus, the combination of two facilitative interventions may lead to further increase in excitability and motor learning. But in some cases homeostatic metaplasticity might protect the brain from harmful hyperexcitability. In this study, the combination of 60 mg ATX and 10 Hz rTMS over the primary motor cortex was used to examine changes in cortical excitability and motor learning and to investigate their influence on synaptic plasticity mechanisms. RESULTS: The results of this double-blind placebo-controlled study showed that ATX facilitated corticospinal and intracortical excitability in motor cortex. 10 Hertz rTMS applied during a motor task was able to further increase intracortical excitability only in combination with ATX. In addition, only the combination of 10 Hz rTMS and ATX was capable of enhancing the total number of correct responses and reaction time significantly, indicating an interaction effect between rTMS and ATX without signs of homeostatic metaplasticity. CONCLUSION: These results suggest that pharmacologically enhanced NE transmission and 10 Hz rTMS exert a synergistic effect on motor cortex excitability and motor learning in healthy humans.

Modulation of inhibitory activity markers by intermittent theta-burst stimulation in rat cortex is NMDA-receptor dependent.

BACKGROUND: Intermittent theta-burst stimulation (iTBS) applied via transcranial magnetic stimulation has been shown to increase cortical excitability in humans. In the rat brain it strongly reduced the number of neurons expressing the 67-kD isoform of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD67) and those expressing the calcium-binding proteins parvalbumin (PV) and calbindin (CB), specific markers of fast-spiking (FS) and non-FS inhibitory interneurons, respectively, an indication of modified cortical inhibition. OBJECTIVE: Since iTBS effects in humans have been shown to be NMDA receptor sensitive, we wondered whether the iTBS-induced changes in the molecular phenotype of interneurons may be also sensitive to glutamatergic synaptic transmission mediated by NMDA receptors. METHODS: In a sham-controlled fashion, five iTBS-blocks of 600 stimuli were applied to rats either lightly anesthetized by only urethane or by an additional low (subnarcotic) or high dose of the NMDA receptor antagonist ketamine before immunohistochemical analysis. RESULTS: iTBS reduced the number of neurons expressing GAD67, PV and CB. Except for CB, a low dose of ketamine partially prevented these effects while a higher dose almost completely abolished the iTBS effects. CONCLUSIONS: Our findings indicate that iTBS modulates the molecular, and likely also the electric, activity of cortical inhibitory interneurons and that the modulation of FS-type but less that of non-FS-type neurons is mediated by NMDA receptors. A combination of iTBS with pharmacological interventions affecting distinct receptor subtypes may thus offer options to enhance its selectivity in modulating the activity of distinct cell types and preventing others from being modulated.

Strain differences in the effect of rTMS on cortical expression of calcium-binding proteins in rats.

Using a rat model to study the cellular effects of repetitive transcranial magnetic stimulation (rTMS) with regard to changes in cortical excitability, we previously described opposite effects of continuous and intermittent theta-burst stimulation (cTBS, iTBS) on the expression of the calcium-binding proteins (CaBP) parvalbumin (PV), calbindin (CB) and calretinin (CR) in Dark Agouti rats (DA). While iTBS significantly reduced the number of cortical PV+ cells but did not affect the CB+ cells, cTBS resulted in a decrease in CB+ cells with no effects on PV+ cells. We concluded that activity of these classes of cortical interneurons is differently modulated by iTBS and cTBS. When testing two further rat strains, Sprague-Dawley (SD) and Long Evans (LE), we obtained deviating results. In SD, iTBS reduced PV and CB expression, while cTBS only reduced PV expression. In contrast, reanalysed DA showed reduced CB expression after cTBS and reduced PV expression after iTBS, while LE shows an intermediate reaction. CR expression was unaffected in any case. Interestingly, we found significantly different basal expression patterns of the CaBPs for the strains, with DA and LE showing much higher numbers of PV+, CB+ and CR+ cells than SD, and with particularly higher number of CB+ and CR+ cells in DA compared to the other two strains. These findings demonstrate that inhibitory systems may be either differently developed in rats belonging to diverse strains or show different basal levels of activity and CaBP expression and may therefore be differently sensitive to the rTMS protocols.

Dose-dependence of changes in cortical protein expression induced with repeated transcranial magnetic theta-burst stimulation in the rat.

BACKGROUND: Theta Burst stimulation (TBS) applied via transcranial magnetic stimulation (TMS) effectively modulates human neocortical excitability but repeated applications of the same TBS protocol at short intervals may be not simply accumulative. OBJECTIVE: Our aim was to investigate the impact of multiple blocks of either intermittent (iTBS) or continuous TBS (cTBS) on the expression of neuronal activity marker proteins in rat cortex. METHODS: Up to four iTBS- or cTBS-blocks of 600 stimuli were applied to urethane-anesthetized rats followed by immunohistochemical and Western blot analyses. RESULTS: The effects of iTBS and cTBS were similar but slightly differed with regard to the number of stimuli applied. The expression of the 65-kD isoform of glutamic acid decarboxylase (GAD65) increased with each stimulation block, while that of the 67-kD isoform (GAD67), and that of the calcium-binding proteins (CaBP) Parvalbumin (PV) and Calbindin (CB) and that of the immediate early gene c-Fos progressively decreased. Both TBS protocols increased the expression of the vesicular glutamate transporter 1 (VGLUT1) with 1200-1800 stimuli but then decreased them after the 4th block. CONCLUSION: Our findings indicate that repeated TBS elicits no simple accumulative dose-dependent effect for all activity-markers but distinct profiles with threshold characteristics and a waxing-and-waning effect especially for the markers of inhibitory activity CB and GAD67. Interestingly, somatic activity markers, such as c-Fos for mainly excitatory and GAD67, CB and PV for inhibitory neurons, decreased with repeated stimulation while synaptic activity markers mainly increased which could be a result of the artificial stimulation of axons.

Time-course of changes in neuronal activity markers following iTBS-TMS of the rat neocortex.

In a rat model of transcranial magnetic stimulation we could recently show that intermittent theta-burst stimulation (iTBS) affects the neocortical expression of the immediate early gene products c-Fos and zif268 as well as that of the two glutamic acid decarboxylase isoforms GAD65 and GAD67 and that of the calcium-binding proteins calbindin (CB) and parvalbumin (PV), known as markers of excitatory and inhibitory activity. We now analyzed in more detail the time course of changes in the expression of these proteins at 10, 20, 40, 80 and 160min following a single block of iTBS consisting of 600 stimuli. Initial increase in c-Fos, zif268 and GAD65 (20min) signals transient activation of excitatory and inhibitory neurons, thereafter first followed by a decrease in markers of activity of inhibitory neurons (GAD67, PV, CB: 20-80min) and then by a late decrease in c-Fos and GAD65 expression (160min). The results demonstrate that one iTBS block may have an after-effect of at least two different phases, an early phase with increased neuronal activity (c-Fos, zif268) but also the likelihood of increased GABA-release (GAD65), followed by a late phase (>40min) of reduced neuronal activity in excitatory and inhibitory systems which may indicate a state of reduced excitability.