Objective: The clinical diagnosis of primary lateral sclerosis can only be made after upper motor neuron symptoms have progressed for several years without developing lower motor neuron signs. The goal of the study was to identify neuroimaging changes that occur early in primary lateral sclerosis, prior to clinical diagnosis. Methods: MRI scans were obtained on 13 patients with adult-onset progressive spasticity for five years or less who were followed longitudinally to confirm a clinical diagnosis of primary lateral sclerosis. Resting state functional MRI, diffusion tensor imaging, and anatomical images were obtained. These "pre-PLS" patients were compared to 18 patients with longstanding, established primary lateral sclerosis and 28 controls. Results: Pre-PLS patients had a marked reduction in seed-based resting-state motor network connectivity compared to the controls and patients with longstanding disease. White matter regions with reduced fractional anisotropy were similar in the two patient groups compared to the controls. Patients with longstanding disease had cortical thinning of the precentral gyrus. A slight thinning of the right precentral gyrus was detected in initial pre-PLS patients' scans. Follow-up scans in eight pre-PLS patients 1-2 years later showed increasing motor connectivity, thinning of the precentral gyrus, and no change in diffusion measures of the corticospinal tract or callosal motor region. Conclusions: Loss of motor functional connectivity is an early imaging marker in primary lateral sclerosis. This differs from literature descriptions of amyotrophic lateral sclerosis, warranting further studies to test whether resting-state functional MRI can differentiate between amyotrophic lateral sclerosis and primary lateral sclerosis at early disease stages.

OBJECTIVES:The goal of this study was to better understand the changes in tissue microstructure that underlie white matter diffusion changes in ALS patients. METHODS:Diffusion tensor imaging was carried out in postmortem brains of 4 ALS patients and two subjects without neurological disease on a 7 T MRI scanner using steady-state free precession sequences. Fractional anisotropy (FA) was measured in the genu, body, and splenium of the corpus callosum in formalin-fixed hemispheres. FA of the body and genu was expressed as ratio to FA of the splenium, a region unaffected in ALS. After imaging, tissue sections of the same segments of the callosum were stained for markers of different tissue components. Coded image fields were rated for pathological changes by blinded raters. RESULTS:The FA body/FA splenium ratio was reduced in ALS patients compared to controls. Patchy areas of myelin pallor and cells immunostained for CD68, a microglial-macrophage marker, were only observed in the body of the callosum of ALS patients. Blinded ratings showed increased CD68 + microglial cells in the body of the corpus callosum in ALS patients, especially those with C9orf72 mutations, and increased reactive astrocytes throughout the callosum. CONCLUSION:Reduced FA of the corpus callosum in ALS results from complex changes in tissue microstructure. Callosal segments with reduced FA had large numbers of microglia-macrophages in addition to loss of myelinated axons and astrogliosis. Microglial inflammation contributed to reduced FA in ALS, and may contribute to a pro-inflammatory state, but further work is needed to determine their role.

Inhibitory repetitive transcranial magnetic stimulation (rTMS) of the primary motor area (M1) impairs motor sequence-learning, but not basic motor function. It is unknown if this is specific for motor forms of procedural learning or a more general effect. To investigate, we tested the effect of M1-inhibition on the weather prediction task (WPT), a learning task with minimal motor learning component. In the WPT, participants learn arbitrary, probabilistic, associations between sets of meaningless cues and fictional outcomes. In our "Feedback" (FB) condition, they received monetary rewards/punishments during learning. In the "paired associate" (PA) condition they learned the same information by passive observation of associations. The observational and feedback learning conditions were matched for their non-learning-specific motor demands. In each of two FB or PA sessions, we delivered Real (inhibitory) or Sham continuous theta-burst (cTBS) to the left-M1, before 150 training-trials. We then tested learning with 42 trials without feedback immediately after learning and again 1-h after cTBS. Compared to Sham, Real cTBS reduced performance during FB-learning, when learning was immediately reinforced, but not when knowledge was tested after PA learning. Furthermore, when FB-based memory was tested after learning without immediate incentive, there was no effect of TMS compared to post-PA test performance, showing the TMS effect operated only in the presence of incentive and feedback. We conclude that M1 is a node in a network underlying feedback-driven procedural learning and inhibitory rTMS there results in decreased network efficiency.

Expansion mutations in the C9orf72 gene may cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), or mixtures of the two clinical phenotypes. Different imaging findings have been described for C9orf72-associated diseases in comparison with sporadic patients with the same phenotypes, but it is uncertain whether different phenotypes have a common genotype-associated imaging signature. To address this question, 27 unrelated C9orf72 expansion mutation carriers (C9 +) with varied phenotypes, 28 age-matched healthy controls and 22 patients with sporadic ALS (sALS) underwent 3T MRI scanning and clinical phenotyping. Measures of brain volumes and cortical thickness were extracted from T1 images. Compared to healthy controls and sALS patients, symptomatic C9 + subjects had greater ventricular volume loss and thalamic atrophy for age, with diffuse, patchy cortical thinning. Asymptomatic carriers did not differ from controls. C9 + ALS and ALS-FTD patients had less thinning of the motor cortex than sALS patients, but more thinning in extramotor regions, particularly in frontal and temporal lobes. C9 + ALS patients differed from sporadic ALS patients in the thickness of the superior frontal gyrus and lateral orbitofrontal cortex. Thickness of the precentral gyrus was weakly correlated with the revised ALS functional rating scale. Thickness of many cortical regions, including several frontal and temporal regions, was moderately correlated with letter fluency scores. Letter fluency scores were weakly correlated with ventricular and thalamic volume. To better understand how imaging findings are related to disease progression, nineteen C9 + subjects and 23 healthy controls were scanned approximately 6 months later. Ventricular volume increased in C9 + patients with FTD and ALS-FTD phenotypes and remained stable in asymptomatic C9 + subjects. We conclude that diffuse atrophy is a common underlying feature of disease associated with C9orf72 mutations across its clinical phenotypes. Ventricular enlargement can be measured over a 6-month time frame, and appears to be faster in patients with cognitive impairment.

Introduction: Patients with mutations in C9orf72 can have amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), or ALS-FTD. The goals were to establish whether cortical hyperexcitability occurs in C9orf72 patients with different clinical presentations. Methods: Cortical thresholds and silent periods were measured in thenar muscles in 19 participants with C9orf72 expansions and 21 healthy controls using transcranial magnetic stimulation (TMS). El Escorial and Rascovsky criteria were used to diagnose ALS and FTD. Fourteen participants with C9orf72 expansions were re-tested 6 months later. Correlations with finger-tapping speed, timed peg test, the ALS functional rating scale, and Dementia Rating Scale were examined. Results: Most participants with C9orf72 expansions had normal or low cortical thresholds. Among them, ALS patients had the lowest thresholds and significantly shorter silent periods. Thresholds correlated with timed peg-test scores. TMS did not correlate with the Dementia Rating Scale. Conclusions: TMS measures of cortical excitability may serve as non-invasive biomarkers of ALS disease activity.

OBJECTIVE: To assess the relationship between volumes of particular brain regions and clinical measures in persons with a repeat expansion in the C9ORF72 gene. BACKGROUND: Persons with a repeat expansion in the C9ORF72 gene may present with varied degrees of motor and cognitive dysfunction. Quantitative MRI measures may help provide biomarkers that correlate with clinical symptoms. DESIGN/METHODS: Patients and asymptomatic carriers with a confirmed mutation were enrolled in an ongoing natural history study collecting clinical and brain MRI measures at baseline and at a 6-month follow-up. Clinical measures included the ALSFRS-R, the Frontobehavioral Inventory (FBI), and cognitive measures included the Dementia Rating Scale (DRS2) and a verbal fluency index. T1 weighted brain MRI scans with slice thickness 1 mm were acquired with a GE 3T scanner. The semi-automated software program FreeSurfer (surfer.nmr.mgh.harvard.edu) was used to segment brain CSF, gray matter and white matter, and parcellate cortical regions. FreeSurfer was used to obtain measures of volume and thickness for selected brain and intracranial regions. RESULTS: 15 persons were enrolled in the first year, with varying amounts of motor and cognitive dysfunction. Preliminary analysis showed the ratio of total brain gray and white matter to intracranial volume significantly correlated (p<0.05) with DRS2 (r=0.61) and FBI scores (apathy r=-0.82; disinhibition r=-0.68). The DRS2 score was also correlated with the ratio of brain matter to ventricular volume (r=-0.82). There was a trend (p=0.07) towards a positive correlation between the thickness of the precentral gyrus and ALSFRS-R. CONCLUSION: Measures of atrophy correlated with cognitive dysfunction at baseline. Follow up studies will be performed to see if these measures can be used as a marker of disease progression. Supported by the intramural programs NINDS and NIA, NIH Disclosure: Dr. Floeter has nothing to disclose. Dr. Schanz has nothing to disclose. Dr. Danielian has nothing to disclose. Dr. Braun has nothing to disclose. controls funded by Merck Inc., $1,300,000. Dr. Bageac has nothing to disclose.

OBJECTIVE: To determine utility of physiological measures of upper or lower motor neuron dysfunction as markers of disease status in persons with the C9ORF72 gene repeat expansion. BACKGROUND: Patients with the C9ORF72 mutation have progressive upper and lower motor neuron dysfunction. Physiological measures have the potential to serve as sensitive, non-invasive indicators of treatment effectiveness, which will be needed during clinical trials. DESIGN/METHODS: Confirmed patients and asymptomatic carriers were enrolled in an ongoing natural history study collecting clinical and physiological data at baseline and 6-month follow-up. All patients had a diagnostic EMG. LMN physiology measures included electrical impedance myography (EIM) on 8 muscles of the upper (EIM-UE) and lower (EIM-LE) limb, the motor unit number index (MUNIX), thenar CMAP amplitudes. UMN physiology consisted of transcranial magnetic stimulation (TMS) measures of cortical thresholds, MEP amplitudes, and central motor conduction times from bilateral thenar and extensor digitum brevis or tibialis anterior muscles. Clinical measures included the ALSFRS-R and measures of movement speed: finger/foot tapping rates, 9-hole peg test times (9HPT), timed gait. The ALSFRS-R was broken into a lower extremity score (ALSFRS-R-LE, questions 8-9) and an upper extremity score (ALSFRS-R-UE, questions 4-6). RESULTS: 15 persons were enrolled in the first year. Preliminary analysis of LMN baseline data showed correlation between CMAP amplitude and ALSFRS-R-UE and 9HPT (|r| values ≍ 0.7, p<0.01). MUNIX correlated with 9PHT (|r| values ≍ 0.7). EIM-LE and EIM-UE 50kHz phase were not correlated with clinical measures. TMS thenar MEP amplitudes correlated with the ALSFRS-R-UE (|r| values ≍ 0.6, p<0.05). Thresholds for lower extremity MEPs were correlated with ALSFRS-R-LE (|r| values ≍ 0.6). CONCLUSION: Some baseline physiological measures exhibit modest correlations with clinical indicators of upper or lower motor neuron dysfunction. Follow-up studies will be performed to correlate with clinical progression. Supported by intramural programs NINDS and NIA, NIH Disclosure: Dr. Floeter has nothing to disclose. Dr. Braun has nothing to disclose. Dr. Bageac has nothing to disclose. “ALS gene discovery using exome sequencing” Dr. Lehky has nothing to disclose. Dr. Schanz has nothing to disclose.

Inhibitory transcranial magnetic stimulation (TMS), of which continuous theta burst stimulation (cTBS) is a common form, has been used to inhibit cortical areas during investigations of their function. cTBS applied to the primary motor area (M1) depresses motor output excitability via a local effect and impairs procedural motor learning. This could be due to an effect on M1 itself and/or to changes in its connectivity with other nodes in the learning network. To investigate this issue, we used functional magnetic resonance imaging to measure changes in brain activation and connectivity during implicit procedural learning after real and sham cTBS of M1. Compared to sham, real cTBS impaired motor sequence learning, but caused no local or distant changes in brain activation. Rather, it reduced functional connectivity between motor (M1, dorsal premotor & supplementary motor areas) and visual (superior & inferior occipital gyri) areas. It also increased connectivity between frontal associative (superior & inferior frontal gyri), cingulate (dorsal & middle cingulate), and temporal areas. This potentially compensatory shift in coupling, from a motor-based learning network to an associative learning network accounts for the behavioral effects of cTBS of M1. The findings suggest that the inhibitory TMS affects behavior via relatively subtle and distributed effects on connectivity within networks, rather than by taking the stimulated area “offline”.