Transcranial theta-burst stimulation alters GLT-1 and vGluT1 expression in rat cerebellar cortex
Introduction
Repetitive transcranial magnetic stimulation (rTMS) represents a non-invasive brain stimulation technique developed for safe and painless application in humans, mimicking the invasive electrical stimulation techniques of the brain in animal models. As well as in basic research before, so the researchers in this field have experimented with various forms of repeated brain stimulation with variable magnetic field (Hoogendam et al., 2010). Accordingly, in humans the most extensive studied are the effects of certain rTMS protocols on indicators motor cortex excitability parameters, where the most robust effects demonstrated by the protocol of theta-burst stimulation (TBS) (Huang et al., 2005). Across all variants of the rTMS protocols, including the TBS, the modulation is possible in two directions, an increase or decrease of cortical excitability. The continuous form of theta-burst stimulation (cTBS), may induce a suppression of excitatory synaptic transmission while the intermittent form of theta-burst stimulation (iTBS) may potentiate it (Funke and Benali, 2011, Huang et al., 2005). Results presented in many studies have shown that rTMS induces its effects by mechanisms initially described as similar, but recently pointed out as different from classical long-term potentiation (LTP) and long-term depression (LTD) (Aydin-Abidin et al., 2008, Funke and Benali, 2011, Hayashi et al., 2004, Ogiue-Ikeda et al., 2003).
TMS has been utilized for therapy of various neurological and psychiatric disorders (Caramia et al., 2004, George et al., 2010, Khedr et al., 2010, Kleinjung et al., 2005, Lipton et al., 2010, Rabey et al., 2013, Torres et al., 2015, Vucic et al., 2013, Zanette et al., 2008) but also in healthy subjects for the improvement of cognitive and motor functions (Ridding and Ziemann, 2010).
Animal studies have shown that rTMS can induce changes in neurotransmitter release, expression of proteins engaged in the activity of excitatory and inhibitory systems, signaling pathways and in gene transcription (Aydin-Abidin et al., 2008, Yue et al., 2009, Zhang et al., 2007). Changes were identified in cortical regions and also in regions distant from the stimulation site (George et al., 1996). Precise cellular and molecular mechanisms underlying these properties still remain to be explained.
Synaptic functions of glutamate are regulated on several levels from the transport into synaptic vesicles, release into the cleft to the reuptake by glial cells and neurons. Vesicular glutamate transporters (vGluT1-3) accomplishes the presynaptic transport of glutamate from cytoplasm into the synaptic vesicles (Linguz-Lecznar and Skagiel-Kramska, 2007, Zink et al., 2010)while its uptake from synapses is enabled by excitatory amino acid transporters (EAATs). Over 90% of total glutamate uptake is under EAAT2 (GLT-1 in rodents) activity which is predominantly expressed in astrocytes (Fontana, 2015, Jensen et al., 2015, Zhou and Danbolt, 2013).
Glutamate transport system also supplies glutamate for glutathione (GSH) synthesis. This molecule protects cells through a chelation mechanism which promotes the removal of reactive species and maintenance of the redox state of other thiols (Dringen et al., 1999, Meister, 1991). Various enzymes enable GSH to express its functions including glutathione reductase (GR), which catalyzes regeneration of oxidized form of GSH to reduced form and glucose-6-phosphate-dehydrogenase (G6PD) which replenishes NADPH (Aoyama and Nakaki, 2013, Jovanovic et al., 2014). All of the transporter subtypes possess cysteine residues with thiol groups (SH), important for defense against reactive species. These functional groups regulate glutamate transport via the S-glutathionylation process (Munir et al., 2000). Cerebral pool of this antioxidant is mainly localized in astroglia (Dringen et al., 1999).
Efficiency and morphology of synapses exposed to TMS are strongly connected with astrocytic functions, and these data provide a strong case for the involvement of astrocytes in mediating effect of TMS on synaptic structure and efficacy (Croarkin et al., 2016). Due to the extensive function of glial fibrillary acidic protein (GFAP) in vesicle trafficking and autophagy, synaptic plasticity, glutamate transport, glutamine synthesis and many others, it has been widely used for assessing the safety of various treatments (Middeldorp and Hol, 2011, Vedam-Mai et al., 2012, Verkhratsky and Parpura, 2010, Zamanian et al., 2012).
Considering animal studies showing the existence of LTP and LTD mechanisms in the cerebellum, aim of the study is to examine the influence of different TBS protocols on GLT-1 and vGluT1 expression, as well as on different enzymes and molecules included in antioxidative protection in rats cerebellar tissue and plasma.
Section snippets
Animals
The experimental animals were treated according to the Guidelines for Animal Study, No. 323-07-7363/2014-05/1 (Ministry of Agriculture and Environmental Protection – Veterinary Directorate). Male young Wistar rats, 4 weeks old, with body mass 200 ± 50 g, were used for experiments. The rats were housed in cages under standardized housing conditions (ambient temperature of 23 ± 2 °C, relative humidity of 55 ± 3% and a light/dark cycle of 13/11 h). They had free access to standard laboratory
Immunofluorescence assessment
Photomicrographs of fluorescent immunoreactivity of vGluT1 and GFAP in the cerebellar tissue are shown in Fig. 2. Immunoreactivity of GLT-1 and GFAP positive tissue sections is represented in Fig. 3.
Compared to Sham RS group (55.82 ± 7.98 average grey value) in cTBS RS (44.93 ± 4.94 average grey value; p < 0.05) vGluT1 immunoreactivity decreased. Comparing SS (67.26 ± 8.78 average grey value) vs. RS (44.93 ± 4.94 average grey value) protocols of cTBS, there was significant difference of vGluT1
Discussion
Although GLT-1 seems to play a minor role in the clearing of synaptically released glutamate due to its low density in cerebellum (Takatsuru et al., 2007), our results show that single iTBS induces rise of its expression (Fig. 4B) suggesting increased need for glutamate removal from the synaptic cleft. Unchanged vGluT1 (Fig. 4A) expression after single or repeated iTBS indicates no differences of glutamate turnover in neurons. Given the necessity for rapid removal of synaptically released
Conclusions
Current study provides insight into subcellular mechanisms by which rTMS structured as iTBS and cTBS exert its effects on the cerebellum. Intensity of GLT-1 and vGluT1 expression vary depending on stimulus pattern and duration of stimulation which has been confirmed by greater intensity of changes in cTBS groups. Increased expression of GLT-1 due to single iTBS with unchanged expression due to repeated iTBS indicates astrocytic preconditioning achieved by electrical stimulation.
Decreased
Acknowledgment
Authors would like to acknowledge support from Ivana Bjelobaba and Irena Lavrnja from Department of Neurobiology Institute for Biological Research „Sinisa Stankovic”. This study was supported by Ministry of Defense of the Republic of Serbia (Projects No.: MFVMA/6/15-17, MFVMA/4/16-18).
References (62)
- et al.
Brain excitability changes in the relapsing and remitting phases of multiple sclerosis: a study with transcranial magnetic stimulation
Clin. Neurophysiol.
(2004) - et al.
Transcranial magnetic stimulation potentiates glutamatergic neurotransmission in depressed adolescents
Psychiatry Res.
(2016) - et al.
Chronic psychosocial stress and concomitant repetitive transcranial magnetic stimulation: effects on stress hormone levels and adult hippocampal neurogenesis
Biol. Psychiatry
(2002) - et al.
Ischemic tolerance and endogenous neuroprotection
Trends Neurosci.
(2003) - et al.
Physiology of repetitive transcranial magnetic stimulation of the human brain
Brain Stimul.
(2010) - et al.
Theta burst stimulation of the human motor cortex
Neuron
(2005) - et al.
Excitatory amino acid transporters: recent insights into molecular mechanisms, novel modes of modulation and new therapeutic possibilities
Curr. Opin. Pharmacol.
(2015) - et al.
Long-term effects of repetitive transcranial magnetic stimulation (rTMS) in patients with chronic tinnitus
Otolaryngol. Head. Neck Surg.
(2005) - et al.
Safety aspects of chronic low-frequency transcranial magnetic stimulation based on localized proton magnetic resonance spectroscopy and histology of the rat brain
J. Psychiatr. Res.
(2003) - et al.
Single-pulse transcranial magnetic stimulation for acute treatment of migraine with aura: a randomised, double-blind, parallel-group, sham-controlled trial
Lancet Neurol.
(2010)
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
Glutathione deficiency produced by inhibition of its synthesis, and its reversal; applications in research and therapy
Pharmacol. Ther.
GFAP in health and disease
Prog. Neurobiol.
Substrate-induced up-regulation of Na(+)-dependent glutamate transport activity
Neurochem. Int.
The effect of repetitive transcranial magnetic stimulation on long-term potentiation in rat hippocampus depends on stimulus intensity
Brain Res.
Astroglial glutamate transporters coordinate excitatory signaling and brain energetics
Neurochem. Int.
Contribution of glutamate transporter GLT-1 to removal of synaptically released glutamate at climbing fiber-Purkinje cell synapses
Neurosci. Lett.
Extremely low-frequency electromagnetic fields activate the antioxidant pathway Nrf2 in a Huntington's disease-like rat model
Brain Stimul.
Effect of transcranial magnetic stimulation on oxidative stress induced by 3-nitropropionic acid in cortical synaptosomes
Neurosci. Res.
Dose-dependence of changes in cortical protein expression induced with repeated transcranial magnetic theta-burst stimulation in the rat
Brain Stimul.
Long-term potentiation protects rat hippocampal slices from the effects of acute hypoxia
Brain Res.
The effects of chronic repetitive transcranial magnetic stimulation on glutamate and gamma-aminobutyric acid in rat brain
Brain Res.
The effect of repetitive transcranial magnetic stimulation on motor performance, fatigue and quality of life in amyotrophic lateral sclerosis
J. Neurol. Sci.
Reduced expression of glutamate transporters vGluT1, EAAT2 and EAAT4 in learned helpless rats, an animal model of depression
Neuropharmacology
Tissue Glutathione: Handbook of Methods for Oxygen Radical Research
Impaired glutathione synthesis in neurodegeneration
Int. J. Mol. Sci.
High- and low-frequency repetitive transcranial magnetic stimulation differentially activates c-Fos and zif268 protein expression in the rat brain
Exp. Brain Res.
Methods of Enzymatic Analysis
NMDA preconditioning protects against seizures and hippocampal neurotoxicity induced by quinolinic acid in mice
Epilepsia
N-methyl-D-aspartate preconditioning improves short-term motor deficits outcome after mild traumatic brain injury in mice
J. Neurosci. Res.
Theta-burst repetitive transcranial magnetic stimulation suppresses specific excitatory circuits in the human motor cortex
J. Physiol.
Cited by (13)
Intermittent theta burst stimulation ameliorates cognitive impairment and hippocampal gliosis in the Streptozotocin-induced model of Alzheimer's disease
2022, Behavioural Brain ResearchCitation Excerpt :At the end of each trial, the maze was cleaned with 70% ethanol to eliminate olfactory cues. Theta burst stimulation (TBS) was applied in the form of iTBS by a MagStim Rapid2 device and a 25-mm figure-of-eight coil (The MagStim Company, Whitland, Dyfed, UK) as previously described [36–38]. Briefly, the protocol was implemented according to Huang [39], and the iTBS block was consisting of 20 trains of 10 bursts (3 pulses at a frequency of 50 Hz), repeated at 5 Hz (lasting 192 s with 10 s intervals between trains), so each block contained a total of 600 pulses.
Theta burst stimulation ameliorates symptoms of experimental autoimmune encephalomyelitis and attenuates reactive gliosis
2020, Brain Research BulletinCitation Excerpt :The studies combining rTMS with functional imaging and neurophysiological techniques were able to show that rTMS modifies cerebral blood flow, glucose metabolism, and neuronal excitability in both stimulated area and interconnected brain regions (Conca et al., 2002). Animal studies have described more closely that rTMS affect release of neurotransmitters, expression of SNARE proteins in excitatory and inhibitory synapses, intracellular signaling pathways and gene expression (Mancic et al., 2016). With regard to MS, several pilot studies with MS patients have demonstrated the efficacy of electromagnetic stimulation for the improvement of spasticity, hand dexterity, fatigue lower urinary tract dysfunction and gait (Centonze et al., 2007; Mori et al., 2011; Nasios et al., 2018).
Neuropeptide Y as a possible homeostatic element for changes in cortical excitability induced by repetitive transcranial magnetic stimulation
2018, Brain StimulationCitation Excerpt :The lower number of pulses applied may have been not sufficient to significantly raise the NPY expression. On the other hand, a recent study by Mancic et al. [36] described a moderate reduction in cortical vGluT1 expression after repeated iTBS but no effect after a single iTBS block. Interestingly, iTBS reduced the cortical vGluT1 expression primarily within the supragranular cortical layers (layers 2/3), those layers exhibiting the highest density of NPY+ interneurons (including PV+ FSI, see Ref. [37]).