Continuous TBS to the M1 increases GABA concentration when compared with control stimulation, without any significant effect on Glx levels. GABAergic activity therefore may be a mechanism by which long-lasting aftereffects of TBS on corticospinal excitability are generated. This is consistent with preclinical data suggesting the importance of the GABAergic mechanisms in LTD-like phenomena within the neocortex in freely moving animals (Hess et al. 1996
; Komaki et al. 2007
; Trepel and Racine 2000
). More indirectly, it may be related to the decreases in local sensorimotor cortex GABA concentrations during successful motor learning in humans (Floyer-Lea et al. 2006
GABA is produced in neurons by decarboxylation of glutamate by the enzyme glutamic acid decarboxylase (GAD65
). The GAD65
isoform is associated with synaptic vesicles and is likely to be involved in synthesizing GABA for neurotransmission (Martin and Rimvall 1993
; Martin et al. 1991a
). In contrast, the GAD67
isoform is distributed more widely in the cytoplasm and is thought to be important in synthesizing GABA for cytosolic use. GAD is the rate-limiting step in production of GABA. In vitro, neuronal activity is associated with increases in the active isoform of GAD65
(de Graaf et al. 2003
). In neural stem cell–derived cultures, depolarization of the neurons also leads to an increase in the active isoform of GAD65
(Gakhar-Koppole et al. 2008
). Induction of synthesis thus likely enables enhanced GABAergic activity in vivo.
It is important to note that MRS is sensitive only to changes in the overall concentration of a neurotransmitter and cannot inform our understanding of changes within the receptors at the synapses. Specifically, the lack of a change in this measure of Glx does not contradict previous pharmacological studies that show abolition of the effects of cTBS after N
-aspartate (NMDA)-receptor antagonism (Huang et al. 2007
), but instead strongly suggests that these changes are due to changes in the NMDA receptors themselves.
The aftereffects of cTBS on motor cortex are commonly assessed from their effects on the amplitude of electromyographic responses (MEPs) evoked in a small intrinsic hand muscle by a standard single TMS pulse (Huang et al. 2005
). At intensities commonly used for TBS, the effect of TMS is cortical and thus results from stimulation of either excitatory or inhibitory synaptic inputs to layer V pyramidal neurons (Di Lazzaro et al. 1998
). A reduced MEP after cTBS therefore indicates either a reduction in the net efficiency of excitatory stimulation by the TMS pulse or an increase in intracortical inhibition.
Our results here—suggesting increased GABAergic inhibition contributes to the aftereffects of TBS—were unexpected. Previous work has shown that the effects of cTBS are abolished by administration of memantine at concentrations sufficient to antagonize NMDA function in the human brain (Huang et al. 2007
; Teo et al. 2007). Thus it has usually been assumed that the aftereffects are due to an action on glutamatergic transmission, which leads to a reduction in cortical excitability via an LTD-like action on the excitatory synapses activated by the TMS test pulses.
Integration of the neuropharmacological results with the MRS data suggests a new hypothesis regarding cTBS action. cTBS is applied at a low intensity (80% AMT), which is below the threshold for activating the excitatory inputs to pyramidal neurons (Ziemann et al. 1996
). The intensity is the same as that used in a common double-pulse paradigm to assess short-interval intracortical inhibition (SICI) in the motor cortex (Kujirai et al. 1993). To elicit SICI, two TMS pulses are applied through the same coil with the first pulse being subthreshold intensity (usually 80% AMT) and the second pulse large enough on its own to elicit an MEP. If the interval between the pulses is between 1 and 5 ms then the MEP is suppressed. Pharmacological studies suggest the effect is due to activation of the GABAA
-ergic neurons by the first pulse (Di Lazzaro et al. 2000
; Muller-Dahlhaus et al. 2008
; Werhahn et al. 1999
). Whether the GABAA
neurons are activated directly by the TMS pulse or indirectly via an excitatory synaptic input is unclear; there is some evidence that it may be the latter (Bestmann et al. 2003).
Whatever the precise mechanism, these data imply that cTBS (600 stimuli) at 80% AMT activates a population of cortical GABAA-ergic interneurons. The increase in GABAergic activity is then sustained by induction of GAD65 and subsequent increased GABA concentration within the cytoplasm of the GABAA-ergic interneuron.
However, this simple formulation does not account for the decrease in SICI (a TMS measure of relative GABAergic transmission) found with cTBS (Huang et al. 2005
). To account for this, we propose an extension of this simple formulation into a mixed model in which effects are mediated by changes in both glutamatergic and GABAergic signaling within local excitation–inhibition networks (Logothetis 2008). The excitability of the cortical output neurons (as reflected by the MEP) is controlled by associative mechanisms of LTD, whereby both NMDA-receptor–dependent mechanisms and GABA input control excitability (Tsumoto 1992
There is one more important aspect that may help to explain our present findings. LTD of GABAA
synapses is predominantly presynaptic, being mediated through endocannabinoids (Tsumoto 1992
). On the other hand, LTD at glutamatergic synapses is more likely to involve postsynaptic changes (Hess and Donoghue 1996
). Previous work shows that cTBS reduces the effectiveness of excitatory inputs to MEP generators in motor cortex, as well as the effectiveness of the circuits mediating short-intracortical inhibition (SICI) (Huang et al. 2008
). In both cases, cTBS is likely to provoke activity in GABAergic and glutamatergic circuits that may be followed by an increased synthesis of both transmitters. However, on termination of cTBS, presynaptic LTD at GABAergic neurons prevents further GABA release. This leads to increased presynaptic GABA levels that in turn increase the MRS signal. On the other hand, presynaptic release of glutamate is unchanged and no accumulation of glutamate should occur. Consequently, no changes in Glx should occur.
This explanation would make the previous pharmacological studies—suggesting that the aftereffects of cTBS are NMDA-receptor dependent (Huang et al. 2007
)—consistent with the results from this study showing an increase in GABA activity. The reduction in SICI can be accounted for because this phasic test of relative GABAA
activity in the paired-pulse paradigm would be less effective in the context of a baseline of increased, ongoing inhibition. Although this cannot be proved from these data, this model provides a framework for further testing in future experiments.
There are limitations to our experiment. The measure of total GABA within the voxel gives no information concerning subcellular localization and thus our interpretation can only be speculative. Because sensitivity did not allow a full relaxation time characterization, it remains possible that the increases in apparent concentration arise simply from redistribution of GABA from pools in which it is relatively immobilized, such as by association with MMs, and therefore MR “invisible” (Matthews et al. 1986). However, such large relaxation time changes with short-term changes in functional state would be unprecedented as far as we are aware. It is also possible that the result sreflect a change in the volume of the cells within the voxel. However, we have controlled for this by considering the GABA:NAA ratio rather than using GABA absolute quantification. In addition, there is no significant change in the creatine concentration after stimulation.
There are some questions raised by the current study that should be addressed in future investigations. First, due to the constraints of the technique we have acquired data only from the brain tissue in the stimulated sensorimotor cortex and therefore a future study is needed to clearly distinguish the local and more general effects of tDCS on neurotransmitter levels.
In addition, we are unable to determine the direct relationship between neurotransmitter changes as assessed by MRS and neurophysiological changes as assessed by TMS. However, at this point the experiments required to investigate this relationship remain technically challenging. Subjects were moved out of the scanner for stimulation and there was a delay of about 20 min between the end of stimulation and the start of MRS measurements, which then demanded a further 20 min. Shorter-lived neurochemical changes would not be able to be defined. However, the neurophysiological aftereffects of 600 pulses of cTBS, as applied in the present study, are relatively stable for ≤1 h (Huang et al. 2005
), suggesting that the dynamics of the underlying neurochemical changes also occur over a similar period. In addition it would be of interest to investigate the effects of intermittent TBS in a similar study. There is evidence that iTBS and cTBS affect different intracortical circuits (Di Lazzaro et al. 2005
), so different effects might be expected.
A significant variability in the Glx measure was seen in both stimulation conditions. In our experience this is often seen in MRS studies and may represent an interaction between the resonance and the neighboring water peak. However, although this interaction would be expected to add variance to the signal, as seen here, no trend toward a significant change in Glx measures with cTBS was seen.