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.

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.

Theta-burst transcranial magnetic stimulation alters cortical inhibition.

Human cortical excitability can be modified by repetitive transcranial magnetic stimulation (rTMS), but the cellular mechanisms are largely unknown. Here, we show that the pattern of delivery of theta-burst stimulation (TBS) (continuous versus intermittent) differently modifies electric activity and protein expression in the rat neocortex. Intermittent TBS (iTBS), but not continuous TBS (cTBS), enhanced spontaneous neuronal firing and EEG gamma band power. Sensory evoked cortical inhibition increased only after iTBS, although both TBS protocols increased the first sensory response arising from the resting cortical state. Changes in the cortical expression of the calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CB) indicate that changes in spontaneous and evoked cortical activity following rTMS are in part related to altered activity of inhibitory systems. By reducing PV expression in the fast-spiking interneurons, iTBS primarily affected the inhibitory control of pyramidal cell output activity, while cTBS, by reducing CB expression, more likely affected the dendritic integration of synaptic inputs controlled by other classes of inhibitory interneurons. Calretinin, the third major calcium-binding protein expressed by another class of interneurons was not affected at all. We conclude that different patterns of TBS modulate the activity of inhibitory cell classes differently, probably depending on the synaptic connectivity and the preferred discharge pattern of these inhibitory neurons.

Continuous and intermittent transcranial magnetic theta burst stimulation modify tactile learning performance and cortical protein expression in the rat differently.

Repetitive transcranial magnetic stimulation (rTMS) can modulate cortical excitability in a stimulus-frequency-dependent manner. Two kinds of theta burst stimulation (TBS) [intermittent TBS (iTBS) and continuous TBS (cTBS)] modulate human cortical excitability differently, with iTBS increasing it and cTBS decreasing it. In rats, we recently showed that this is accompanied by changes in the cortical expression of proteins related to the activity of inhibitory neurons. Expression levels of the calcium-binding protein parvalbumin (PV) and of the 67-kDa isoform of glutamic acid decarboxylase (GAD67) were strongly reduced following iTBS, but not cTBS, whereas both increased expression of the 65-kDa isoform of glutamic acid decarboxylase. In the present study, to investigate possible functional consequences, we applied iTBS and cTBS to rats learning a tactile discrimination task. Conscious rats received either verum or sham rTMS prior to the task. Finally, to investigate how rTMS and learning effects interact, protein expression was determined for cortical areas directly involved in the task and for those either not, or indirectly, involved. We found that iTBS, but not cTBS, improved learning and strongly reduced cortical PV and GAD67 expression. However, the combination of learning and iTBS prevented this effect in those cortical areas involved in the task, but not in unrelated areas. We conclude that the improved learning found following iTBS is a result of the interaction of two effects, possibly in a homeostatic manner: a general weakening of inhibition mediated by the fast-spiking interneurons, and re-established activity in those neurons specifically involved in the learning task, leading to enhanced contrast between learning-induced and background activity.

θ burst and conventional low-frequency rTMS differentially affect GABAergic neurotransmission in the rat cortex.

Modified cortical excitability following repetitive transcranial magnetic stimulation (rTMS) may be related to short- or long-term synaptic plasticity of neuronal excitation but could also affect cortical inhibition. Therefore, in the rat we tested how three different rTMS protocols, intermittent and continuous theta-burst (iTBS, cTBS), and low-frequency 1 Hz stimulation, change the expression of GAD65, GAD67 and GAT-1 which are expressed in cortical inhibitory interneurons in an activity-dependent manner. Acutely (2 h), all protocols reduced the expression of GAD67 in frontal, motor, somatosensory and visual cortex but increased that of GAD65 and GAT-1 to different degree, with iTBS having the strongest acute effect. The initial decrease in GAD67 reversed after 1 day, leading to a strong increase in GAD67 expression for up to 7 days primarily in the frontal cortex in case of iTBS, cTBS and in all studied areas following 1 Hz rTMS. While also GAD65 and GAT-1 expression reversed after 1 day in case of iTBS and cTBS, 1 Hz rTMS induced a steady increase in GAD65 and GAT-1 expression during the 7 days investigated. Our data demonstrate that rTMS affects the expression of activity-dependent proteins of the cortical inhibitory interneurons. Besides common effects of low- (1 Hz) and high-frequency (TBS) stimulation on protein expression, differences in quantity and time course of changes point to differences in the contribution of possible neuronal subsystems. Further studies are needed to distinguish cell-type specific effects.