3T MR Spectroscopy Reveals an Imbalance between Excitatory and Inhibitory Neurotransmitters in Amyotrophic Lateral Sclerosis

Abstract

Objective

To determine whether there are reductions in γ-aminobutyric acid (GABA) and elevations of glutamate + glutamine (Glx) levels in different brain regions of patients with amyotrophic lateral sclerosis (ALS) using proton magnetic resonance spectroscopy (¹H-MRS).

Design

3T short echo time and GABA-edited ¹H-MRS centered on the left motor cortex and left subcortical white matter. Short echo time ¹H-MRS was also performed centered on the pons. Data were analyzed using logistic regression, t-tests, and Pearson correlations. Post hoc analyses were performed to investigate differences between riluzole-naïve and riluzole-treated ALS patients.

Participants

Twenty-nine ALS patients and thirty age- and gender-matched healthy controls (HCs).

Results

ALS patients had significantly lower levels of GABA in the motor cortex compared to HCs (P<.01). ALS patients also had significantly lower levels of N-acetylaspartate in the motor cortex (P<.01), subcortical white matter (P<.05), and pons (P<.01) and higher levels of myo-inositol in the motor cortex (P<.001) and subcortical white matter (P<.01) compared to HCs. Compared to riluzole-treated ALS patients, riluzole-naïve ALS patients had higher levels of Glx in the motor cortex (P<.05) and pons (P<.01), higher levels of creatine in the motor cortex (P<.001) and subcortical white matter (P=.05), and higher levels of N-acetylaspartate in the motor cortex (P<.01).

Conclusion

There are reduced levels of GABA in the motor cortex of ALS patients. There are elevations of Glx in riluzole-naïve ALS patients compared to ALS riluzole-treated patients. These results point to an imbalance between excitatory and inhibitory neurotransmission, contributing to the pathogenesis of ALS.

Over a hundred years have passed since Jean-Martin Charcot first described amyotrophic lateral sclerosis (ALS). However, the underlying pathophysiology is not well understood, diagnoses are often delayed, and effective treatments are still needed. ALS is a progressive degenerative motor neuron disease involving the motor cortex, corticospinal tract, brain stem, and spinal anterior horn neurons.¹ The disease is uniformly fatal, although the clinical presentation and course are quite heterogeneous, with median survival times between two and four years.² Patients present most commonly with combined upper motor neuron (UMN) and lower motor neuron (LMN) features, although earlier in the course of their disease only UMN or LMN signs may be present. Evaluation of LMN pathology in ALS is commonly supplemented by electromyography, whereas UMN pathology is solely assessed on clinical grounds, hindering diagnosis.³ Riluzole, the only FDA-approved medication for ALS, has limited efficacy, extending life expectancy by only 3 to 6 months on average.^{4, 5} Riluzole is postulated to modulate excitatory neurotransmission, although the exact in vivo pharmacologic actions are not well understood.⁶

To further the understanding of the disease process and perhaps to diagnose the disease at an earlier stage, advanced magnetic resonance imaging techniques, including magnetic resonance spectroscopy (MRS), have been applied to study UMN changes in ALS. Conventional in vivo MRS at 3T can quantify various brain metabolites including N-acetylaspartate (NAA), choline (Cho), creatine (Cr), myo-inositol (mI) and glutamate + glutamine (Glx). γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter, is difficult to quantify using conventional MRS, but can be measured using spectral-editing techniques.^{7, 8} Given our limited prior knowledge of in vivo GABA changes in ALS, direct interrogation of GABA may lead to new understanding of this complex disease and provide opportunities for the development of new disease-modifying treatments.

The most common finding reported in MRS studies of ALS is reduced NAA in the motor cortex, which is generally interpreted as neuronal loss.⁹ Although riluzole is thought to modulate excitatory neurotransmission, only two published MRS studies have explored the effect of riluzole on brain metabolites, and measured only Cho, Cr, and NAA.^{10, 11} We recently published the first MRS study reporting a decrease in GABA levels in the motor cortex in a small cohort of ALS patients (n = 10), suggesting reductions in inhibitory neurotransmission.¹² The primary purpose of the present study was to determine whether there are reductions in inhibitory neurotransmission, as measured by GABA, and increases in excitatory neurotransmission, as measured by Glx, in ALS patients compared to healthy controls (HCs). GABA and Glx levels were measured in the left motor cortex and left subcortical white matter, as well as Glx levels in the pons. A secondary aim was to determine differences between brain metabolite profiles of riluzole-treated vs. riluzole treatment-naïve ALS patients.

Methods

Twenty-nine ALS patients were recruited from our institution’s ALS clinic. Thirty age- and gender-matched HCs were also recruited. All ALS participants met the El Escorial Criteria for probable (n = 15), probable laboratory-supported (n = 10), or definite (n = 4) ALS.¹³ ALS and HC participants were right-handed. Subjects were excluded if they had a history of central nervous system infection, head injury, or cerebrovascular disease; active substance abuse; or contraindication for MRI. Our institutional review board approved all study protocols, and informed consent was obtained from all of the participants. UMN scores were graded by combining the Ashworth Spasticity Scale, presence of pathological reflexes, and the Pseudobulbar Affect Scoring (scale range 0–33, with a higher score reflecting higher disease burden). The GABA data from the prior published study from 10 ALS subjects and 9 HCs are included in the present larger study. Characteristics of the participants are presented in Table 1.

Table 1.

Participant Characteristics

	Healthy Controls	ALS Patients
Numbers	30	29
Age, yr, mean ± SD (range)	59.3 ± 9.9 (29–79)	59.5 ± 10.2 (32–78)
Male:female	20:10	17:12
Onset	NA	7 bulbar, 22 limb
Disease Duration, mo, mean ± SD (range)	NA	28.6 ± 14.5 (4–64)
UMN Score, mean ± SD (range)	NA	15.6 ± 7.0 (1–27)
ALSFRS-R Score, mean ± SD (range)	NA	34.1 ± 8.2 (18–47)

Abbreviations: ALS = amyotrophic lateral sclerosis, ALSFRS-R = revised Amyotrophic Lateral Sclerosis Functional Rating Scale, UMN = upper motor neuron.

Magnetic resonance spectroscopy

Nineteen subjects (10 ALS, 9 HCs) were imaged on a Philips Achieva 3T system (Best, Netherlands) using an 8-channel receive head coil. Forty subjects (19 ALS, 21 HCs) were imaged on a Philips Ingenia 3T system using a 15-channel receive head coil (Best, Netherlands). T₁-weighted 3D-MPRAGE images were used to specify the placement of 3.0 cm × 3.0 cm × 2.0 cm voxels in the left motor cortex (MC) and the left subcortical white matter (SCWM) located caudally to the motor cortex for single-voxel point resolved spectroscopy (PRESS) and Mescher-Garwood point resolved spectroscopy (MEGA-PRESS) data acquisitions.⁷ A 1.4 cm × 1.4 cm × 1.8 cm voxel was placed centrally in the pons for PRESS data acquisition (Figure 1).

Figure 1.

Voxel placement and MEGA-PRESS spectrum. T1-weighted images showing single voxel placement centered on pons in the sagittal (A) and axial projections (B); the left motor cortex in the sagittal (C) and axial projections (D) and on the left subcortical white matter located caudal to the motor cortex in the sagittal (E) and axial projections (F). Representative magnetic resonance spectroscopy spectrum from the pons using conventional PRESS technique (G). Representative magnetic resonance spectroscopy spectrum from the left motor cortex using MEGA-PRESS editing technique (H). Combined measure of glutamine and glutamate (Glx) is resolved at 3.8 ppm, γ-aminobutyric acid (GABA) at 3.0 ppm with an inverted N-acetylaspartate (NAA) peak at 2.0 ppm.

Conventional PRESS acquisition

PRESS spectra (TR/TE=2000/35 ms) were acquired using ‘VAPOR’ water suppression: 32 averages were performed for the MC and the SCWM voxel and 96 averages were performed for the pons voxel. Conventional PRESS data were analyzed using the LCModel (version 6.1-4A; S. Provencher, PhD, Oakville, Ontario, Canada).¹⁴ Metabolite concentrations from LCModel were only used for statistical analysis if the Cramér-Rao lower bounds were less than 20% for the MC and the SCWM voxels and less than 25% for the pons voxel. Cerebral spinal fluid correction was performed for each voxel based on the MPRAGE images using the program SPM5 (Wellcome Trust Centre for Neuroimaging, London, England).

MEGA-PRESS acquisition

A MEGA-PRESS experiment for editing GABA was performed with the following parameters: TE=68ms (TE₁=15ms, TE₂=53ms); TR=1.8s; 256 averages; frequency selective editing pulses (14ms) applied at 1.9 ppm (ON) and 7.46 ppm (OFF). Slice-selective refocusing was performed using amplitude-modulated pulse ‘GTST1203’ (length=7ms, bandwidth=1.2 kHz). MEGA-PRESS spectroscopy was analyzed using in-house post-processing software in Matlab 2012a (Mathworks, Natick, Massachusetts) with Gaussian curve fitting to the GABA and inverted N-acetylaspartate (NAA) peaks. GABA levels were expressed relative to the NAA signal in the edited spectra.¹⁵ The GABA/NAA ratio was then multiplied by the NAA concentration determined from LCModel analysis of a short-TE PRESS spectrum of the same voxel to provide an estimate of GABA concentration. Note that metabolite concentration is in ‘institutional units’ since it does not correct for various factors, including editing efficiency and relaxation times.

Statistical analyses

Logistic regression analyses was performed between disease status and individual metabolites using scanner type (Achieva or Ingenia) as a covariate, and found no significant effects of scanner type (z > 0.05 for all metabolites). Two-tailed independent sample t-tests were performed to determine differences in brain metabolites between ALS patients and HCs. Pearson correlations were performed for associations between brain metabolites and clinical status (UMN scores, disease duration, ALSFRS-R). A subset analysis was also performed to compare riluzole-treated ALS patients versus riluzole-naïve ALS patients. Stata v.11 (StataCorp, College Station, TX) was used for the statistical analysis. The significance threshold was set a priori at a P-value of 0.05.

Results

For the conventional PRESS spectra, two ALS patients had inadequate signal-to-noise ratio from the pons voxel and were excluded, and the pons Glx value from one ALS patients was excluded due to a high Cramér-Rao bounds value. For the MEGA-PRESS spectra, GABA spectra in the motor cortex of two ALS patients and one HC and the subcortical white matter of one ALS patient had inadequate signal-to-noise ratio. Four of the ALS patients were unable to complete the entire imaging protocol and did not have PRESS or MEGA-PRESS MRS of the SCWM performed.

MEGA-PRESS results

As shown in Figure 2, ALS patients demonstrated significantly lower levels of GABA in the left MC compared to HCs (P = .002). There were no significant GABA group-level differences in the left SCWM between the ALS patients and the HCs (P = .30). There was a significant correlation between the MC GABA and disease duration (r = 0.39, P = .05).

Figure 2.

Decreased γ-aminobutyric acid (GABA) levels in the motor cortex (MC) of amyotrophic lateral sclerosis (ALS) patients. Circles represent GABA levels in the left motor cortex (A) and subcortical white matter (SWCM) located caudal to the motor cortex (B) for individual healthy controls (HC) and ALS patients. Horizontal bars indicate the mean. ALS patients have reduced levels of GABA in the left motor cortex compared to healthy controls. There is no difference between ALS patient and healthy control GABA levels in the left subcortical white matter. IU = institutional units.

Conventional PRESS results

Results are summarized in Table 2. The levels of NAA were significantly lower in the MC (P = .008), SCWM (P = .02) and pons (P = .003) in ALS patients compared to HCs. The levels of mI were significantly higher in the MC (P = .0003) and SCWM (P = .002) in ALS patients compared to healthy controls. There were significant correlations between MC NAA and ALSFRS-R score (r = 0.39, P < .05), SCWM mI and disease duration (r = 0.43, P < .05), and pons Glx and UMN score (r = −0.63, P < .001). There were no significant correlations between GABA and Glx levels within the same voxel location or between voxel locations.

Table 2.

Conventional PRESS Results

	Motor Cortex		Subcortical White Matter		Pons
	HC	ALS	HC	ALS	HC	ALS
Cho	1.15 ± 0.18	1.23 ± 0.14	1.34 ± 0.21	1.41 ± 0.17	2.45 ± 0.25	2.46 ± 0.34
Cr	4.93 ± 0.33	5.09 ± 0.59	4.21 ± 0.35	4.36 ± 0.32	4.39 ± 0.57	4.18 ± 1.00
Glx	5.62 ± 1.00	5.88 ± 1.37	4.34 ± 1.13	4.05 ± 0.89	6.83 ± 1.77	7.11 ± 2.38
mI	3.18 ± 0.46	3.79 ± 0.74***	3.10 ± 0.40	3.53 ± 0.57**	4.64 ± 0.76	4.18 ± 1.00
NAA	8.20 ± 0.48	7.72 ± 0.81**	7.73 ± 0.57	7.30 ± 0.69*	8.50 ± 0.84	7.76 ± 0.95**

Values are mean ± SD of respective metabolites expressed in institutional units.

Abbreviations: ALS = amyotrophic lateral sclerosis, Cho = Choline, Cr = Creatine, Glx = Glutamine + Glutamate, HC = healthy control, mI = myo-Inositol, NAA = N-acetylaspartate.

^*p ≤.05;

^**p ≤ .01;

^***P ≤ .001

Riluzole treatment sub-analyses

There were 15 riluzole-treated ALS patients and 14 riluzole-naïve ALS patients. Subgroup characteristics are presented in Table 3. As seen in Figure 3, there were significantly higher levels of Cr, Glx, and NAA in the motor cortex of riluzole-naïve ALS patients compared to the riluzole-treated ALS patients. There were also significantly higher levels of Glx in the pons (P = .004) and higher levels of Cr in the SCWM (P = .05) in the riluzole-naïve patients compared to the riluzole-treated patients. There were no significant differences in the levels of GABA or other metabolites between the two subgroups.

Table 3.

Riluzole Subgroup Characteristics

	Riluzole-naive	Riluzole-treated
Numbers	14	15
Male:female	8:6	9:6
Age, yrs, mean ± SD (range)	60.4 ± 12.5 (32–78)	59.4 ± 7.4 (47–72)
Disease Duration, mo, mean ± SD (range)	25.4 ± 16.4 (4–64)	30.9 ± 12.6 (12–60)
ALSFRS-R, mean ± SD (range)	38.1 ± 6.1 (26–47)	30.4 ± 8.5 (18–25)*
UMN Score. mean ± SD (range)	14.7 ± 7.3 (1–24)	16.5 ± 7.2 (3–27)

Abbreviations: ALSFRS-R = revised Amyotrophic Lateral Sclerosis Functional Rating Scale, UMN = upper motor neuron.

^*p ≤0.05; no significant differences in other characteristics between subgroups.

Figure 3.

MRS PRESS results of left motor cortex (MC) metabolite levels for riluzole-naïve (⊖riluzole) and riluzole-treated (⊕riluzole) ALS patients. Circles represent respective brain metabolites for Cr (A), Glx (B) and NAA (C). Riluzole-naïve ALS patients have elevated levels of Cr, Glx and NAA in the left motor cortex compared to riluzole-treated ALS patients. Glx = glutamine + glutamate, Cr = Creatine, NAA = N-acetylaspartate, IU = institutional units.

Comment

This study demonstrates reductions of GABA levels in the motor cortex in ALS patients compared with HCs. In addition, it was found that Glx levels were reduced in ALS patients taking riluzole compared to those not on the medication. The inclusion of an additional 19 ALS patients augments the significance of our prior report that found lower levels of GABA in the motor cortex of 10 ALS patients. This is the first report of in vivo measurements of both GABA and Glx in ALS patients, thereby investigating both the GABAergic (inhibitory) and the glutamatergic (excitatory) neurotransmitter systems in the same patients.

GABA is the major inhibitory neurotransmitter in the central nervous system and plays an important role in regulating neuronal excitability. However, measurement of GABA has been challenging due to chemical shift overlap with other brain metabolites such as Cr, Glx, and NAA, which are present in brain tissue at higher concentrations than GABA.¹⁶ MRS editing techniques allow for improved differentiation of metabolites and quantification of GABA. Specifically, the MEGA-PRESS technique can edit out the overlapping Cr peak at 3.0 ppm, allowing for direct quantification of the underlying GABA peak, also at 3.0 ppm.⁸ A moderate correlation between GABA levels and disease duration was found, suggesting that GABA levels measured by MRS may reflect loss of cortical inhibition associated with disease progression. The findings are consistent with results from an animal model of ALS which demonstrated that cortical excitability is explained by reduced GABAergic inhibition.¹⁷ In addition, human histochemical and positron emission tomography studies have implicated GABA receptor alterations in the motor cortex of ALS patients.^{18, 19} The current result of decreased levels of GABA in the motor cortex provides further in vivo evidence of reduced inhibitory function in the pathophysiology of ALS.

Higher levels of Glx were also found in the motor cortex and pons of riluzole-naïve patients compared to riluzole-treated patients. Glutamate and glutamine are difficult to resolve independently at field strengths of 3T or lower, and are usually reported as a combined measure, ‘Glx’, however in normal brain glutamate is the larger contributor to this peak by about a 4-to-1 ratio.²⁰ Due to concerns of a potential relationship between NAA and Glx, we confirmed that there was not a significant correlation between the NAA levels and the Glx levels for the overall group or the treatment subgroups. Riluzole is thought to act on both the glutamatergic and GABAergic systems.^{6, 21} Riluzole has been shown to decrease glutamatergic neurotransmission by acting as an antagonist of presynaptic NMDA and AMDA glutamate receptors, as well as by increasing glutamate transporter uptake.^22–24 Riluzole has been shown to increase GABA levels in cell cultures, although the levels required for GABA modulation are thought to be higher than those required for glutamatergic inhibition, which may explain the lack of effect of riluzole on the GABA levels seen in the current study.^{25, 26} Reduced motor cortex excitability has been demonstrated after riluzole administration in healthy subjects, as well as partial restoration of increased cortical excitability in ALS patients, which is thought to be mediated through glutamatergic rather than GABAergic interactions.^{27, 28}

It is also interesting to note that the riluzole-treated ALS patients had lower levels of Cr than the riluzole-naïve subjects. Cr is a marker of brain energy metabolism.²⁹ It has been proposed that an energy-depleted state may be an inciting factor in ALS⁶ and that riluzole may reduce neuronal cellular energy demand.³⁰ A potential confound to the above findings is that the riluzole-treated patients had lower levels of NAA than the riluzole-naïve group. There was a moderate correlation between NAA and Cr levels (r = 0.49, P = 0.01) in the MC for the overall 29 ALS patients. Two prior studies have reported a longitudinal increase in the NAA/Cr ratio of ALS patients after short periods (≤ 3 weeks) of riluzole treatment, however the studies did not report absolute concentrations of the NAA and Cr metabolites, making direct comparison difficult.^{10, 11} However, overall, these results do suggest the potential of MRS measurements of GABA and Glx as surrogate markers of disease progression and treatment response, which might be useful in future pharmacological trials.

The cause of ALS remains elusive, although excitotoxic neuronal injury is thought to play an important role, primarily mediated through glutamate toxicity.^{31, 32} There is increasing evidence that an ‘interneuronopathy’ may be a central player in the pathophysiology of ALS.³³ Interneuronopathy is the hypothesis that inhibitory or GABAergic dysfunction results in relatively unopposed excitotoxic neuronal damage. Neuroimaging, animal, histochemical, genetic, and clinical studies support the role of an interneuronopathy in ALS.³⁴ For example, an ALS functional MRI study demonstrated increased functional connectivity in ALS, suggesting a loss of inhibitory neuronal tone.³⁵ A significant barrier to further support this hypothesis has been the challenge of directly measuring in vivo GABA concentrations. The current results support this notion, and suggest the possibility of the development of new ALS disease-modifying treatments aimed at increasing inhibitory neuronal tone, thus normalizing the excitatory to inhibitory balance in the central nervous system.

The current results are also in agreement with the findings of a number of prior MRS studies of ALS. NAA is generally considered a marker for neuronal integrity. As in our study, almost all prior MRS studies reported decreases in levels of NAA or ratios of NAA/Cr, particularly in the motor cortex. The finding of elevated mI in the MC and SCWM is thought to represent increased numbers of glial cells.²⁹ Although mI has not been investigated to the same extent as NAA, prior studies have reported mI (or mI/Cr) elevations in the motor cortex.^36–38 Interestingly, only a few studies have measured Glx, with the only positive finding being higher levels of Glx in the pons in one study.³⁹ Of the prior studies that measured Glx, it is important to note that riluzole status was not mentioned^{36, 39, 40} or unclear³⁷ which is a potential mediating factor as the current study would indicate.

As reported before, limitations of MRS (including the MEGA-PRESS technique) include inability to differentiate between the intracellular and extracellular contribution of the metabolites, potential macromolecular component contributions, and relatively large voxel sizes required. We were not able to implement the MEGA-PRESS sequence to measure GABA in the pons given the relatively small volume of the brain stem. The riluzole-treated group had on average a lower ALSFRS-R score compared to the riluzole-naïve group, indicating greater disability from both UMN and LMN impairment, although the UMN disease burden of disease was not significantly different between groups. Direct causality between riluzole treatment and effect on brain metabolites cannot be established given the cross-sectional nature of the study. A longitudinal imaging trial would be required to establish better the response of brain metabolites to treatment as well as probe ALS central nervous system changes over time.

Conclusion

Reductions in GABA levels in the motor cortex of ALS patients were observed, as well as elevations of Glx in riluzole-naïve compared to riluzole-treated ALS patients. The results support the hypothesis that imbalance of excitatory and inhibitory neurotransmitters is an important contributing factor in the pathogenesis of ALS. These findings also support the potential of MRS to establish Glx and GABA measurements as clinically relevant markers of disease, although additional research efforts are needed to better understand the findings reported here.