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Published in final edited form as: J Neurosci Methods. 2020 Apr 5;339:108709. doi: 10.1016/j.jneumeth.2020.108709

Focal transcranial magnetic stimulation in awake rats: enhanced glucose uptake in deep cortical layers

Samantha Cermak 1, Qinglei Meng 1, Kevin Peng 1, Simone Baldwin 1, Carlos A Mejías-Aponte 2, Yihong Yang 1, Hanbing Lu 1
PMCID: PMC8917821  NIHMSID: NIHMS1585704  PMID: 32259609

Abstract

Background:

Transcranial magnetic stimulation (TMS) is an emerging neuromodulation tool. However, preclinical models of TMS are limited.

Objective:

To develop a method for performing TMS in awake rats and to characterize neuronal response to TMS by mapping glucose uptake following TMS administration.

Methods:

A headpost was implanted into rat skull serving as a refence to guide TMS target. Motor threshold measurement was used as the metric to assess the consistency in TMS delivery across animals and across sessions. Using a fluorescent glucose analogue (2-NBDG) as a marker of neuronal activity, we mapped glucose uptake in response to TMS of the rat motor cortex.

Results:

The average motor threshold (n=41) was 34.6±6.3% of maximum stimulator output (MSO). The variability of motor threshold across animals was similar to what has been reported in human studies. Furthermore, there was no significant difference in motor threshold measured across 3 separate days. Enhancement in fluorescent signals were TMS dose (power)-dependent, which centered around the motor cortex, covering an area medial-laterally 2 mm, rostral-caudally 4 mm at 55% MSO, and 3 mm at 35% MSO. The count of total cells with significant fluorescent signal was: 107±23 (55% MSO), 73±11 (35% MSO) and 42±11 (sham, 5% MSO).

Conclusions:

Our method allows for consistent motor threshold assessment for longitudinal studies. Notably, cells with fluorescent signal enhancement were consistently aggregated in deep cortical layers, with minimal enhancement in superficial layers

Comparisons with Existing Method(s):

To our knowledge, this is the first study of focal TMS in awake rodents.

Keywords: Glucose, 2-NBDG, focal TMS, fluorescence, 2-DG

INTRODUCTION

Transcranial magnetic stimulation (TMS) can modulate brain activity and has shown promise in therapeutic treatment of neurological and psychiatric disorders(Chen et al., 1997; Fitzgerald et al., 2006; Levitt et al., 2019; Ridding and Ziemann, 2010; Siebner et al., 2009; Sommer et al., 2018; Wassermann and Zimmermann, 2012; Ziemann et al., 2008). However, the way in which TMS alters brain activity and how this relates to therapeutic outcomes are largely unknown(Hamada et al., 2013; Lisanby, 2017). Animal models permit invasive manipulations, and could provide value in investigating the mechanism of TMS at the molecular, cellular and neuronal circuit levels (Banerjee et al., 2017; Gersner et al., 2011; Mueller et al., 2014; Murphy et al., 2016; Vahabzadeh-Hagh et al., 2012; Zhang et al., 2007). Unfortunately, most previous rodent studies have employed a large TMS coil, and lacked face-validity when translating the results from rodents to humans, due largely to the coil-to-brain size ratio(Cohen et al., 1990). Specifically, human TMS can induce single thumb twitch when directed toward the motor cortex; the same TMS coil likely stimulates the entire rat brain. There have been efforts to develop rodent-specific TMS coils (Boonzaier et al., 2019; Parthoens et al., 2016; Tang et al., 2016). Rotenberg et al., (Rotenberg et al., 2010) achieved unilateral limb twitch using a 2.5 cm figure-of-eight coil. Our lab recently developed a coil capable of suprathreshold focal stimulation, eliciting single limb twitch during contralateral motor cortex stimulation of anesthetized mice(Meng et al., 2018).

TMS induces electrical current in the targeted brain region, causing complex excitation and inhibition in the stimulus loci and in the interconnected brain networks(Drysdale et al., 2017; Luber et al., 2017; Paus, 1999). Anesthetic agents, depending on their molecular targets, differentially affect the balance of the excitatory and inhibitory processes in the neuronal system, and likely confound the outcomes of TMS treatment. Indeed, Gersner et al. (Gersner et al., 2011) compared neuronal plasticity in awake and isoflurane anesthetized rats following daily sessions of high frequency repetitive TMS for 10 days, and found that brain-derived neurotrophic factor increased in awake rats but decreased in anesthetized rats. The sharp contrast in brain response to TMS between these conditions underscores the importance of brain state during TMS. Thus, for animal results to be translational and ultimately improve clinical outcomes, it would be ideal to perform TMS studies in an awake state, avoiding confounds from anesthesia.

A major technical challenge in employing awake rats for TMS is that rodents do not readily comply with motion restrictions. This is particularly important when the stimulus target is focal. In the present study, we report methods that permit consistent TMS in awake rats. Furthermore, 2 NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose), a fluorescent glucose analogue(Yoshioka et al., 1996), has been used to assess glucose metabolic activity in-vitro(O'Neil et al., 2005; Rouach et al., 2008) and in-vivo at a cellular resolution(Ito et al., 2006; Itoh et al., 2004; Millon et al., 2011; Tsytsarev et al., 2012; Tsytsarev et al., 2013; Yao et al., 2013). Compared with classical methods, such as fluorodeoxyglucose (FDG) or 2-Deoxy-d-glucose (2-DG), 2-NBDG is non-radioactive; enhancement in fluorescent signal reflects glucose uptake in individual cells(Itoh et al., 2004). Using fluorescent glucose uptake as a marker of brain activity, we mapped brain regions activated by TMS of the rat motor cortex. Enhancement in cellular glucose uptake in the targeted and surrounding regions reflects the focality of the stimulation. Results reveal that TMS at 10 Hz enhanced glucose uptake in deep cortical layers. The methods and findings reported in this article provide insight towards the use of rodent models to investigate the neurobiological effects of TMS.

MATERIALS AND METHODS

We developed a strategy to align the coil to the rat head and achieve consistent TMS in a targeted brain area in awake rats. Specific procedures are detailed below.

Headpost implantation

The headpost and coil guide were made with 3D printing technology (Printer model: Pro2 Plus 3D printer; filament type: premium polylactic acid. Both from Raised3D Technologies, California, USA) (see Fig. 1). Based on the pre-determined focal spot of the coil (the location with the highest electric field strength), we identified the coordinates for headpost implantation (−8.5 mm from bregma, mediolateral 0.0 mm). The headpost served as a fixed reference point. The coil guide had a “U” shape opening to fit the TMS coil, and was detachable from the headpost. The headpost and coil guide direct the focal spot of the coil to the stimulation target, the hindlimb motor cortex.

Figure 1.

Figure 1.

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Illustration of the experimental procedures for consistent TMS targeting across sessions and across animals. A-C: A headmount post is surgically implanted onto rat skull. A detachable coil guide is placed over the post. The coil guide fits the TMS coil so that the coil can be consistently positioned to the same region across sessions. D: The location of the headmount is calculated such that the focal spot of the TMS coil is above the target region (e.g. motor cortex). E shows a rat with headmount post implantation. F: A rat receiving TMS. The TMS coil is precisely positioned relative to the rat head with the aid of the coil guide.

A total of 41 male Sprague-Dawley rats (Charles River Laboratories), weighing 300-400 g, underwent standard stereotaxic procedures for surgical implantation of the headpost to the skull. A 4mm midline incision was made from the posterior eye edge toward the interaural line. The skull was cleaned thoroughly using 3% hydrogen peroxide and saline. The headpost was positioned onto the designated coordinates, and two plastic screws were inserted into the rat skull lateral to the headpost to secure the headpost in place. The skull was also scored lightly with a drill. The headpost and the plastic screws were secured onto the skull using a thin layer of dental cement. Animals recovered from surgery for at least one week before TMS administration. All procedures were approved by NIDA Animal Care and Use Committee.

TMS Habituation

The rodent-specific TMS coil reported previously (Meng et al., 2018) was interfaced to a commercial MagStim Rapid2 stimulator (The Magstim Co. Ltd). The coil was cooled by dry ice, with the capability of administering repetitive TMS (rTMS) at 10 Hz (higher frequencies were not tested because the Magstim system produced errors with 2400 pulses, likely due to excessive heating in the driver system). A micromanipulator was customized to hold the coil in place. The micromanipulator permitted accurate adjustment of the coil along X, Y and Z directions at 10 micrometer resolution, plus an angle adjustment with respect to rat skull surface.

We first placed the removable coil guide over the headpost, and held the rat underneath the TMS coil. The coil guide aligned the rat head to the TMS coil, and its focal spot targeted the intended brain region (motor cortex). Habituation was performed by applying TMS stimulation at 3% of maximum stimulator output (MSO) at 10 Hz for 5 min. Rats were habituated until they stopped showing signs of acute stress, which included increase urination, defecation(Barone et al., 1990), and struggles to escape. These signs were prominent in the first 3 habituation days. After 7 days of training, most rats did not show signs of stress during the entire TMS training session. Many rats still occasionally struggled, which was handled by repositioning them under the TMS coil. Rats that continued to exhibit signs of stress after the habituation period were excluded from the study.

Motor threshold measurement

On the experimental day, we used the headpost and coil guide to direct the focal spot of the coil to the motor cortex of the left hindlimb. The motor threshold was defined as the stimulator’s output power level at which a hindlimb twitch was elicited 50% of the time. We applied single TMS pulses, starting at the power level of 20%. We carefully observed any paw/limb movement following each pulse. If no motor threshold was elicited, the power level was increased or decreased by 3-5% of MSO at a time. This was done until stimulation was elicited 5-6 time out of a total of ten times, separated by several seconds between each interval. To evaluate the consistency of the motor threshold assessment, we performed the same measurement on a subgroup of rats (N= 7) on 3 consecutive days.

Mapping glucose uptake using fluorescent 2-NBDG

We adapted the protocol by Itoh et al. who first applied fluorescent 2-NBDG to map glucose uptake in rats (Itoh et al., 2004). After 7 days of recovery from headpost surgery and 10 days of habituation to the TMS environment as described above, each rat fasted for 16 hours, followed by tail vein catheterization under 2% isoflurane. Rats received 2-NBDG infusion via the tail vein (MW: 342.3 g/mol, GLPBio, California, USA), dosage: 2 ml, 0.4% 2-NBDG (or 23.3 μmol) in saline. Animals were subsequently released to home cage, and recovered from anesthesia for 20 min. Again, we directed the focal spot of the coil to the left motor cortex of the hindlimb region using the headpost and coil guide. Stimulation parameters were 2 sec ON, 6 sec OFF at 10 Hz, a total of 2400 pulses. Three power levels were applied: active TMS: 55% and 35% MSO; sham control: 5% MSO.

Following TMS administration, rats were deeply anesthetized under isoflurane, and perfused transcardially with 0.1M PBS solution followed by 4% paraformaldehyde. Brains were harvested and sectioned at 40 μm using standard histological procedures. Slices were imaged under an Olympus Macroscope MVX10 using GFP lighting with 25× magnification. Cells were counted using a customized MATLAB script which will be available upon request. The script created a binary mask to distinguish fluorescent cells. Fluorescent cells were converted to hue saturation values, and the image contrast was increased. Then, a binarized image was created of the fluorescent cells, and the cells were extracted by size. These cells were quantified and output the number of fluorescent cells in the image. The binarized and original images were then carefully examined to conform the proper cell identifications.

RESULTS

Motor Threshold between and within rats

The motor threshold measured across 41 rats was 34.6±6.3% of MSO, as illustrated in Fig. 3. We performed repeated measurements on a subgroup of rats on 3 separate days. Results are shown in Fig. 2B. One-way ANOVA analysis revealed no significant difference in motor threshold measurements across these days (F(2, 21)=0.28, p=0.76): 35.6 ± 8.2% of MSO on day1; 34.4 ± 9.8% on day2; 32.5 ± 7.1% on day3. These data indicate that motor threshold readouts are reasonably consistent in awake rats using the procedures we have developed.

Figure 3.

Figure 3.

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2-NBDG uptake after TMS of hindlimb motor cortex in awake rats. (A) TMS: 55% maximum stimulator output (MSO); (B) sham: 5% MSO. (C) and (D) are enlarged from areas indicated by white boxes in A and B, respectively. White arrows indicate cells with high florescence intensity. The 2 red arrows in A indicate artifacts that happened to exist in this section. For visualization purpose, these two artifacts serve as landmarks to identify regions activated by TMS (E, F). Rat brain atlas was overlaid onto this section (E), showing primary motor cortex (M1) and neighboring sensory cortex of the hindlimb (S1HL) were stimulated (pink in F). The activated region in the medial-lateral direction is about 2 mm as indicated by the coordinate axis in F. Note cells with high florescence are in deep cortical layers (presumably layer 5, A).

Figure 2.

Figure 2.

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The average motor threshold across animals was 34.6±6.3% of maximum stimulator output. No significant difference in motor threshold measured in a subgroup of rats across 3 different days.

Glucose uptake in response to TMS of the motor cortex

Robust fluorescent 2-NBDG signal enhancement was seen in all rats that received TMS. Figure 3 shows fluorescent images from a typical TMS rat and sham control rat. Green dots in A are cells with high fluorescence signal. The two red arrows in A indicate artifacts that happened to occur in this histological slice. For visualization purposes, these two artifacts, however, can serve as anatomical landmarks to identify regions activated by TMS (E, F). The Paxinos and Watson rat brain atlas (Paxinos and Watson, 2007) was overlaid onto this section to identify the activated regions (E): the primary motor cortex (M1) and neighboring sensory cortex of the hindlimb (S1HL) (pink in F). The activated region in the medial-lateral direction is about 2 mm as indicated by the scale line in F. We applied 55% MSO (160% motor threshold) in this experiment to compensate for the relatively low sensitivity of the 2-NBDG method. As shown in F, even at this high TMS intensity, the activated region is only about 2 mm medial-laterally (M1 and S1HL), based on the atlas. The fluorescent signal in B reflects basal glucose uptake during the TMS administration in awake rats, which involved occasional struggle and movement. Notably, of all the rats we imaged, cells with high florescence accumulation consistently aggregated in deep cortical layers (see Fig. 3C).

The above section approximately corresponds to −0.36 mm from bregma based on rat brain atlas(Paxinos and Watson, 2007). We also counted 2-NBDG enhancement in 4 neighboring sections separated by approximately 1 mm apart, at A-P −2.36mm, −1.36mm, +0.64mm, and +1.64mm. Results are shown in Fig. 4. Two-way ANOVA (brain section × TMS power) reveals significant TMS power effects (d.f. = 2, F = 9.04, p<0.0005); cell counts in the high TMS power condition were significantly higher than in the sham condition (p<0.0003); cell counts between the high and medium TMS power conditions were marginally significant (p=0.09). There was significant main effect of brain section (location) (d.f. =4, F=4.26, p<0.005), with the cell counts in S3 significantly higher than in S1 (p<0.002); cell counts in S4 and S5 were marginally higher than in S1 (p=0.07). Furthermore, there was no significant difference in brain section × TMS power interaction (p=0.58), suggesting that the brain areas exhibiting significant glucose uptake were similarly modulated by TMS power.

Figure 4.

Figure 4.

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Significant differences in the number of cells showing fluorescent signal enhancement. Rats were divided into 3 groups, receiving high, medium and low TMS power, respectively (160%, 100% and 10% motor threshold). Cells in 5 neighboring sections (S1, S2, S3, S4, S5), separated by 1 mm apart, were counted. S3 corresponds to −0.36 mm from bregma. **, p<0.0003; *, p<0.002.

DISCUSSION

While rodents are the most widely used animals in basic neuroscience, they do not readily comply with motion restrictions and the TMS environment, posing unique challenges for using rodents as the experimental subjects for investigating the neurobiology of TMS. Many previous studies applied anesthesia to mitigate this problem. In the present study, we introduce a method that permits TMS on awake rats, avoiding confounding factors associated with anesthesia entirely. The use of headpost and coil guide allows for consistent TMS targeting across rats and across sessions, as supported by the motor threshold measurements across 41 rats; the habituation procedures reduce stress associated with TMS administration. Using fluorescent deoxyglucose 2-NBDG, we mapped brain regions with enhanced glucose uptake in response to TMS of the motor cortex. Results demonstrate that our TMS coil activated a region of about 2 mm medial-laterally, and 3-4 mm rostral-caudally, depending on the TMS power, consistent with our previous estimate based on known neuroanatomy(Meng et al., 2018; Tennant et al., 2011).

Variability in motor threshold

We observed substantial variability in motor threshold measurements: of the 41 data points shown in Fig. 2, the highest was 45% MSO, the lowest was 20% MSO, with a standard deviation of 6.3% MSO. There was no significant difference in the motor threshold measurement across 3 separate days. This variability is generally similar to what has been reported in humans with a relatively large sample size (N=151) (Wassermann, 2002).

Notably, human studies have observed lower motor thresholds for active muscles than for resting ones(Fitzgerald et al., 2006; Wassermann, 2002). For rats, even after substantial habituation, one cannot be certain that their limb muscles are at a resting state. This may have contributed to the variability in our motor threshold measurements. This problem appears unique to TMS in awake animals. We thus suggest applying average motor threshold to gauge TMS power in future TMS studies involving awake animals --- this might be a reasonable solution to reduce inter-subject variability resulting from motor threshold assessment.

Enhanced glucose uptake in deep cortical layers

We also found that cells with high florescence are consistently aggregated in deep cortical layers (presumably layer 5, see Fig. 2A). TMS activation of layer 5 pyramidal neurons are expected because these cells have long apical dendrites that terminate at layers 2-3 (Douglas and Martin, 2004), and are preferably activated by TMS, as suggested by a recent theoretical simulation(Aberra et al., 2018). This finding is consistent with a recent calcium imaging study (Murphy et al., 2016). Additionally, Murthy et al. also showed that TMS induced transient calcium increases in layers 1-3. They suggested that calcium signals in layers 1-3 were the results of disinhibition of post-synaptic interneurons, resulting from pre-synaptic GABA release due to the activation of inter-areal axons that lie in layers 1-3. The lack of glucose uptake in top cortical layers is somewhat surprising, and may be due to several reasons:

  1. limited sensitivity of the 2-NBDG method. There must be enough 2-NBDG accumulation in a cell before the fluorescent signal is detectable. The signal in small cell bodies of interneurons activated by TMS may not be strong enough to be detectable.

  2. TMS preferentially activated layer 5 pyramidal neurons as recently suggest by Aberra et al. (Aberra et al., 2018) based on simulations using biophysically realistic neuron models. The motor pyramidal neurons have large cell bodies.

Further work is needed to understand the reasons for the lack of increase in glucose uptake in superficial cortical layers. Nevertheless, we detected enhancement in glucose uptake in deep cortical layers.

Technical limitations

In the present study, motor threshold was estimated by observing limb/paw twitches. Estimating motor threshold based on motor evoked potential (MEP) readout has been widely used in human TMS. Pridmore et al. specifically compared motor threshold measurement based on two methods: one involved neurophysiology techniques and the other was visualization of movement. (Pridmore et al., 1998). They found that these two methods had a < 10% difference in terms of total stimulator output. They concluded that determination of motor threshold with a neurophysiological and visualization of movement method produced similar results. MEP is relatively straightforward to obtain in humans. But it is less straightforward to obtain in awake rats or mice due to motion, and necessitates surgical implantation of micro electromyographic (EMG) electrodes to specific muscles (Akay et al., 2006; Fortier et al., 1987; Tysseling et al., 2013). In our pilot studies, we implanted micro EMG electrodes into tibialis anterior and lateral gastrocnemius of both hindlimbs. The rats recovered from surgery for one week. We then measured motor threshold by measuring MEP through these microwires; In the meantime, we also carefully observed limb/paw movement. We found that the motor threshold measured by EMG and by visual observation of limb movement were very similar. Nevertheless, MEP readout is reproducible and quantitative, and should be employed in studies that require careful comparison of motor thresholds.

In summary, we have developed a method that permits repeated TMS in awake rats. We have applied this method to map glucose uptake in response to TMS of the motor cortex, and have found that areas with enhanced glucose uptake were largely restricted in the deep cortical layers. This work provides the basis for the use of rodent models to investigate the neurobiological effects of TMS in the future.

Highlights.

  • A method for performing focal TMS on awake rats

  • Headpost and coil guide permit consistent targeting of the same brain region

  • Brain regions activated by TMS were mapped using fluorescent glucose indicator 2-NBDG

ACKNOWLEDGEMENT

This work was supported by the NIDA Intramural Research Program, NIH.

Footnotes

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Conflict of interest statement

H. Lu is a co-inventor in the provisional US patent (No. 62/791,753).

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