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M o lecular Psychiatry (2015) 20, 2 0 1 -2 0 6 © 2015 Macmillan Publishers Limited All rights reserved 1359-4184/15

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ORIGINAL ARTICLE

Brain abnormalities in bipolar disorder detected by quantitative T ip mapping CP Johnson1, RL Follmer2, I Oguz3, LA Warren2, GE Christensen3, JG Fiedorowicz2'4'5, VA M agnotta1,2 and JA Wemmie2'6

Abnormal metabolism has been reported in bipolar disorder, however, these studies have been limited to specific regions of the brain. To investigate whole-brain changes potentially associated w ith these processes, we applied a magnetic resonance imaging technique novel to psychiatric research, quantitative mapping o f T1 relaxation in the rotating frame (Tip). This method is sensitive to proton chemical exchange, which is affected by pH, metabolite concentrations and cellular density w ith high spatial resolution relative to alternative techniques such as magnetic resonance spectroscopy and positron emission tomography. Study participants included 15 patients with bipolar I disorder in the euthymic state and 25 normal controls balanced for age and gender. T ip maps were generated and compared between the bipolar and control groups using voxel-wise and regional analyses. T ip values were found to be elevated in the cerebral white matter and cerebellum in the bipolar group. However, volumes o f these areas were normal as measured by high-resolution T1- and T2-weighted magnetic resonance imaging. Interestingly, the cerebellar T ip abnormalities were normalized in participants receiving lithium treatment. These findings are consistent with metabolic or microstructural abnormalities in bipolar disorder and draw attention to roles of the cerebral white matter and cerebellum. This study highlights the potential utility o f high-resolution T ip mapping in psychiatric research.

Molecular Psychiatry (2015) 20, 201-206; doi:10.1038/mp.2014.157; published online 6 January 2015

INTRODUCTION Although the pathophysiological mechanisms o f bipolar disorder are largely unknown, accumulating evidence is beginning to link the disease to abnormal metabolism. Magnetic resonance (MR) spectroscopy studies have found evidence for reduced intracel­ lular pH, N-acetyl aspartate, phosphocreatine and phosphomono- diesters and elevated lactate, glutamate, choline and myo-inositol in euthymic people with bipolar disorder compared with matched controls, particularly in the frontal lobes and basal ganglia.1 These factors suggest increased anaerobic metabolism as well as altered phospholipid metabolism.1 Mitochondrial dysfunction has been hypothesized to be a common thread between these various find­ ings.1,2 This view is buttressed by genetic evidence o f abnormal mitochondria gene expression in bipolar disorder3"5 as well as the presence o f w hite matter abnormalities that may have a metabolic source such as hyperintensities6"8 and dysconnectivity.9,10 Metabolic abnormalities may also result in an inflammatory response, which may lead to synaptic pruning,11 loss of oligodendrocytes,12,13 and ultimately disease progression14 and cognitive decline in bipolar disorder.15 These various observations suggest parenchymal brain abnormalities in bipolar disorder including acidosis, altered metabolite concentrations and loss o f cellular density.

New imaging tools are needed to further investigate potential abnormalities in bipolar disorder. MR spectroscopy has proven useful for detecting metabolically derived signals in the brain, with its primary advantage being that it can probe specific metabo­ lites.16 However, MR spectroscopy is limited by coarse spatial resolution (~8.0 cm3 per voxel), incomplete brain coverage (single

voxel or single slice) and long acquisition times ( > 5 m in for a single voxel). Positron emission tomography has also been used to study metabolism,17 but it is limited by dependence on radio- labelled markers as well as coarse spatial and temporal resolution. An alternative imaging modality that may be particularly useful to investigate bipolar disorder is quantitative MR mapping o f T1 relaxation in the rotating frame (Tip).18 The T ip signal is sensitive to chemical exchange between protons and other molecules largely via amide and hydroxyl groups.19 T1 p is increased by acidic pH, reduced cellular density and reduced concentrations of meta­ bolites such as glucose,20"24 all o f which have been suggested from studies of participants with bipolar disorder in the euthymic state. In contrast to MR spectroscopy, quantitative T ip mapping can be performed w ith an order o f magnitude higher spatial resolution and whole-brain three-dimensional coverage in less than 10 min, which provide the ability to efficiently probe for abnormalities throughout the brain and not just in targeted regions.

In light o f the previous suggestions o f abnormal metabolism and cellular damage in bipolar disorder and the potential sensitivity of T ip to these processes, we hypothesized that T ip would be elevated in bipolar I disorder. To test this hypothesis, we used whole-brain quantitative T ip mapping to compare people with bipolar I disorder in the euthymic state to healthy controls balanced for age and gender. Our results suggest T ip mapping may be a valuable strategy for investigating bipolar disorder and its underlying mechanisms.

'Department of Radiology, University of Iowa, Iowa City, IA, USA; de partm ent of Psychiatry, University of Iowa, Iowa City, IA, USA; departm ent o f Electrical and Computer Engineering, University of Iowa, Iowa City, IA, USA; de partm ent of Epidemiology, University of Iowa, Iowa City, IA, USA; de partm ent o f Internal Medicine, University of Iowa, Iowa City, IA, USA and 'Veterans Affairs Hospital Center, Iowa City, IA, USA. Correspondence: Dr JA Wemmie or Dr VA Magnotta, Carver College of Medicine, University of Iowa, 3139 Med Labs, Iowa City, 52242, IA, USA. E-mails: [email protected] or [email protected] Received 5 August 2014; revised 19 September 2014; accepted 9 October 2014; published online 6 January 2015

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T ip mapping o f bipolar disorder CP Johnson et at

MATERIALS AN D METHODS Participants Fifteen participants w ith a DSM-IV diagnosis o f bipolar I disorder in the euthymic state (Young Mania Rating Scale ^ 1 2 and Montgomery-Asberg Depression Rating Scale < 10) and a normal control group of 25 partici­ pants balanced for age and gender w ith no known psychiatric history were enrolled into this study after providing w ritten informed consent in accordance w ith the local Institutional Review Board. Euthymic participants consisted of individuals whose mood state had cycled from either depres­ sion or mania w ithin the past 2 years. All participants completed a medical and psychiatric history including current medications. Sample dem o­ graphic and clinical features are tabulated in Table 1.

Data collection Participants were imaged using a 3 T Siemens Tim Trio MRI system (Magnetom; Siemens Healthcare, Erlangen, Germany) and a vendor- provided 12-channel receiver head coil. Whole-brain T1- and T2-weighted anatomical acquisitions w ith 1.0 mm isotropic spatial resolution were acquired first, and the quantitative T ip acquisition followed. The T1- w eighted acquisition used a three-dimensional magnetization-prepared rapid gradient echo sequence w ith the follow ing parameters: coronal orientation: field-of-view = 2 5 .6x2 5 .6 x2 5 .6 cm3; sampling matrix = 256 x 25 6 x25 6 ; TR/TE/TI = 2530/2.8/909 ms; flip angle =10°; bandw idth = 180 Hz/pixel; and ft = 2 GRAPPA. The T2-weighted acquisition used a three- dimensional turbo spin echo w ith variable flip angle sequence w ith the following parameters: sagittal orientation; field-of-view = 26 x 22.8 x 17.6 cm3; sampling matrix = 2 5 6 x 2 3 0 x 1 7 6 ; TR/TE = 4000/406 ms; bandw idth = 592 Hz/pixei; and ft = 2 GRAPPA. The T ip acquisition used a segmented three- dimensional gradient echo sequence w ith the follow ing parameters: coronal orientation; field-of-view = 2 2 x 2 2 x 2 0 cm3; sampling matrix = 128 x 1 2 8x40; TR/TE = 5.6/2.5ms; segment block tim e = 1500 ms; views per segment = 24; flip angle = 10°; bandw idth = 260 Hz/pixel; ft = 2 GRAPPA; and 7/8 partial Fourier. The T ip spin-lock preparation used a self- compensating spin-lock cluster25 w ith spin-lock frequency = 330 Hz and spin-lock times (TSLs) = 10 and 55 ms.

Additional data were acquired to investigate factors that may affect T1 p. Respiratory rate and heart rate were recorded using a physiological m onitoring system (Biopac Systems, Inc; Goieta, CA, USA) either during or follow ing the T ip acquisition. Additionally, for all participants except four controls, blood samples were collected on the same day as the MRI exam (with the exception o f four participants w ith bipolar disorder whose blood was collected during a visit 10-16 months prior to the exam) to measure the levels of 14 peripheral inflammatory markers (C-reactive protein, interleukin (IL)-ip, IL-1RA, IL-4, IL-6, IL-10, IL-17, IL-18, IL-18BP, interferon y, TNF-a, TNF-R1, TNF-R2 and monocyte chemotactic protein 1).

Data analysis BRAINS (Brain Research: Analysis of Images, Networks, and Systems) AutoW orkup26 was used to align the T1- and T2-weighted anatomical images for each participant to a common brain atlas (NAC HNCMA Atlas 20 1 3).27 During this process, a deformable transformation was calculated using Advanced Normalization Tools28 to warp each participant's

Table 1. Euthymic bipolar and normal control g ro u p dem ographic and clinical features

Euthymic bipolar group Normal control group

Participants N (male/female) 15 (7/8) 25 (13/12) Age m ean±s.d. 40.5 ± 1 4 .9 41.5 ± 1 2 .7 Age range 21-66 21-62

Medications (NJ Lithium 6 0 Anticonvulsants 6 0 Antidepressants 6 0 Antipsychotics 4 0 Sedative hypnotics 7 0 None 2 25

anatomical images to the common brain atlas, which includes a set of manually defined tissue classification labels for cerebral and cerebellar gray and white matter and subcortical structures.27 Another atlas w ith labels for 48 w hite matter tracts (ICBM-DTI-81 White Matter Labels Atlas)29 was also registered to the common brain atlas space to provide a more detailed segmentation o f the w hite matter. These transformations and atlas labels were subsequently used to regionally measure T ip values. Additionally, using segmentation voxel counts generated from BRAINS AutoWorkup, volumes of brain regions o f interest were calculated relative to intracranial volume.

For each participant, a T ip map was calculated by fittin g the 10 and 55 ms spin-lock tim e (TSL) image signals (S) according to the relationship:

S(TSL) = S(0)e“ TSL/Tlp. [1]

Using the TSL = 55 ms image as a reference, the T ip map was aligned to the anatomical T1-weighted image using Analysis o f Functional Neurol- mages.30 The T ip map was then interpolated to 1.0mm isotropic resolu­ tion to match that o f the anatomical T1-weighted image, and the deformable transformation calculated during the BRAINS AutoWorkup processing was used to subsequently warp the T1-aligned T1 p map to the common brain atlas. For visualization and group-wise comparisons, the warped T ip map was masked to only include voxels corresponding to brain tissue in the atlas.

Statistical analysis A voxel-wise comparison was performed to investigate brain tissue T ip differences between the bipolar and control groups and identify regions of interest for further evaluation. For this analysis, the groups' mean T ip maps were calculated and compared voxel-wise using an independent samples two-tailed f-test (ft <0 .05 ) corrected for m ultiple comparisons by cluster thresholding (a = 0.05). Calculations were performed using Analysis o f Functional Neuroimages.

Two approaches were then used to investigate regional T1 p differences between the euthymic bipolar and normal control groups in specific regions o f interest identified using the voxel-wise comparison. First, label-specific histograms were generated for each group's mean T ip map. Twelve common brain atlas labels were available for investigation: cerebral and cerebellar gray and white matter; putamen; hippocampus; globus pallidus; amygdala; nucleus accumbens; caudate; thalamus; and pons. Only voxels w ith a T ip value between an expected range of 50 and 100 ms31 in both maps were included in the analysis to reduce the influence o f outliers (e.g., because o f cerebral spinal fluid partial volume artifacts). The Pearson's correlation r between the tw o groups' voxel-paired mean T ip values in each label was also calculated. Second, the median T ip relaxation times in the common brain atlas labels of interest and all 48 white matter tract labels were calculated for each individual participant T ip map. The median T ip values for each label were then compared between the bipolar and control groups using a one-way analysis o f variance (ANOVA) with a significance threshold of ft <0.05. Sub-threshold differences with significance level P < 0.1 were also recorded to determine whether regions were trending toward significance. Calculations were performed using R statistical software (R Foundation for Statistical Computing, Vienna, Austria).32

Potential covariates o f the group T ip differences include respiratory rate, heart rate, medication use, regional brain volumes and inflammatory marker levels. To assess w hether respiratory rate, heart rate and regional brain volumes were potential covariates o f group T ip differences, euthymic bipolar and normal control group mean values of each were compared using a tw o-tailed f-test (P < 0.05). Each o f the 14 inflammatory markers were similarly assessed using a Wilcoxon rank sum test (P < 0.05). The relationship between T ip values and respiratory rate and heart rate were also considered using a one-way ANOVA as above for each common brain atlas region of interest w ith significance level P < 0.05. A similar ANOVA was performed to evaluate the influence o f broad medication classes (lithium, anticonvulsants, antidepressants, antipsychotics and sedative hypnotics) on T ip values w ithin the euthymic bipolar group. Although there may be a relationship between T ip , age and gender,31 these factors were not evaluated as potential covariates because they were balanced in this study. Calculations were performed using R.

RESULTS To explore the potential utility of whole-brain high-spatial-resolu­ tion T ip mapping in bipolar disorder, we quantified and

Molecular Psychiatry (2015), 201 -2 0 6 © 2015 Macmillan Publishers Limited

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b

Mean T ip (ms) Mean T ip (ms) Mean T ip (ms)

----- Normal Control Group ------Euthymic Bipolar Group

Figure 1. T ip values are elevated in the euthymic bipolar group in the cerebral white matter and cerebellum, (a) Statistical maps of the voxel- wise differences for the right (R) and left (L) brain hemispheres. Z-scores are thresholded at a significance level o f P < 0.05 with correction for multiple comparisons. A positive Z-score indicates the mean T1 p value for the bipolar group was greater than that of the control group at that voxel. The statistical map is overlaid on the average common-atlas-aligned Tl-weighted image for all participants, (b) Histograms of each group's T ip values in these regions o f interest. Pearson's correlation coefficient r between the groups' T ip values is indicated for each label. The bipolar group histograms are shifted to the right, which indicates a general increase in T ip compared with the controls.

compared T ip values between a euthymic group o f participants w ith bipolar I disorder and a normal control group balanced for age and gender (Table 1). First, we generated and averaged T ip maps for the euthymic bipolar and normal control groups. Voxel- wise comparison between these maps revealed significantly (P < 0.05, corrected) greater T ip values in the cerebral white matter and cerebellum o f the euthymic bipolar group (Figure 1a). This finding was supported by histogram analyses for which we plotted the number of voxels versus T1 p values in these regions of interest as defined by the common brain atlas labels27 (Figure 1 b). Although the patterns o f these histograms were very similar and showed a strong correlation between groups (/•> 0.90), the curves were shifted to the right in the bipolar group, suggesting a general increase in T ip across each region. A between-group ANOVA o f T1 p values in the cerebral white matter and cerebellar regions o f interest also revealed an increase in the bipolar group, although these findings were not statistically significant after correction for multiple comparisons (Table 2). Sub-dividing the cerebral white matter into regions defined by the white matter tract atlas labels29 consistently revealed increased T ip values in the bipolar group, although these differences were also not significant after correction for multiple comparisons.

One potential source of elevated T ip values in the euthymic bipolar group is inflammation. To investigate this possibility, we compared serum levels o f 14 inflammatory markers between the bipolar and control groups. None o f the inflammatory marker levels were significantly different between the tw o groups. Although the extent to which these markers in peripheral blood reflect inflammation in the brain is unknown,33 this finding suggests that the T ip differences are not due to active systemic inflammatory processes.

Another potential source of elevated T ip is cellular loss, perhaps due to prior inflammation or injury. To explore this potential explanation for the observed T ip abnormalities, we compared regional brain volumes o f the cerebral and cerebellar

gray and white matter as a percentage o f intracranial volume between the bipolar and control groups. No regional volume differences were found (cerebral white matter: P = 0.68, 35.7 ± 1.3 vs 35.8±1.7%; cerebellar white matter: P = 0.15, 4.55±0.40 vs 4.74±0.38%; cerebellar gray matter: P = 0.39, 4.95 ±0.52 vs 4.80 ±0.53%), suggesting an absence of extensive cellular loss.

We have previously shown that respiratory rate and C02 inhalation can alter T ip throughout the brain.22 However, we monitored respiratory rate in these studies and did not find a difference between the groups (P = 0.9: 15.5 ±2.8 vs 15.4±3.0 breaths per minute). We did detect a general increase in heart rate in the bipolar group (P = 0.03: 70.0 ± 11.1 vs 62.7 ±8.6 beats minute), suggesting cardiovascular deconditioning. Nevertheless, general physiological parameters such as these are unlikely to cause the focal T ip changes observed here, and the heart rate increase did not correlate w ith the region o f interest median T1 p values.

Because medications m ight also potentially influence T ip values, we assessed whether broad medication classes (Table 1) were associated w ith T ip abnormalities in the euthymic bipolar group. ANOVA did not reveal any significant relationships between T ip and use o f anticonvulsants, antidepressants, antipsychotics or sedative hypnotics in the key regions o f interest, the cerebral white matter and cerebellum. Note that the statistical power of this analysis was limited by small sample sizes. Interestingly, however, we did detect an effect of lithium in the cerebellum. T1 p values in the cerebellum were elevated in participants in the bipolar group not taking lithium ( L i- ) , whereas T ip values in the cerebellum were normal in their counterparts who were taking lithium (Li+) (Figure 2). Similar results were observed in voxel-wise comparisons between the average T1 p maps for the Li - , Li+ and control groups (Figure 3). The Li - and Li+ groups were of similar age (mean = 40.9 ±16.6 and 40.0 ±13.4 years, respectively) and gender (male/female = 4/5 and 3/3, respectively). These findings suggest that medications were unlikely to cause the elevated T ip values in the bipolar group. Moreover, they suggest that lithium

2015 Macmillan Publishers Limited Molecular Psychiatry (2015), 201 -206

T1 p mapping of bipolar disorder CP Johnson ef al

Table 2. AIMOVA of euthymic bipolar vs normal control group T1 p values in the cerebral white matter and cerebellar regions o f interest as defined by the common brain and white matter tract atlas labels

Euthymic bipolar group: Tip meanis.d. (ms) Normal control group: Tip mean±s.d. (ms) P F

Common brain atlas labels Cerebellar white matter 70.6 ±1.6 69.6 ±1 .4 0.039 4.6 Cerebellar gray matter 74.2 ±1.5 73.1 ±1.6 0.032 4.9 Cerebral white matter 72.4 ±1.9 71.5 ± 1.1 0.061 3.7

White matter tract labels Body o f corpus callosum 73.7 ±2.0 72.5 ±1.1 0.022 5.7 Left superior corona radiata 74.9 ±2.5 73.5 ±1.3 0.024 5.5 Right superior longitudinal fasciculus 72.7 ±2.1 71.5 ±1.4 0.025 5.4 Left cingulum 76.1 ±2.4 74.5 ±1.9 0.034 4.9 Left superior longitudinal fasciculus 72.3 ±2.5 71.0 ± 1.3 0.037 4.7 Middle cerebellar peduncle 74.5 ± 2.3 72.9 ±2.2 0.038 4.6 Right sagittal striatum 71.6 ±2.2 70.0 ±2.4 0.046 4.3 Right posterior thalamic radiation 75.2 ±2.4 73.8 ±1.9 0.046 4.3 Left posterior limb o f internal capsule 72.7 ±2.6 71.4 ±1.4 0.046 4.2 Splenium o f corpus callosum 74.6 ±1.9 73.6 ±1.6 0.062 3.7 Left superior cerebellar peduncle 77.4 ±2.8 76.0 ±2.0 0.063 3.7 Right inferior cerebellar peduncle 76.5 ±2.1 75.0 ±2.8 0.070 3.5 Left posterior thalamic radiation 74.6 ±2.5 73.3 ±1.8 0.071 3.4 Right medial lemniscus 69.0 ± 2.0 67.4 ± 2.9 0.073 3.4 Right fornix and stria terminalis 72.7 ±2.0 71.7 ± 1.4 0.073 3.4 Left tapetum 86.0 ±2.5 84.2 ± 3.1 0.077 3.3 Genu o f corpus callosum 73.1 ±2.9 71.8 ± 1.6 0.078 3.3 Right superior cerebellar peduncle 81.2 ±2.5 79.5 ±3.1 0.080 3.2 Left posterior corona radiata 76.2 ±2.2 75.2 ±1.6 0.090 3.0 Left sagittal stratum 72.0 ±2.7 70.8 ±1.7 0.098 2.9 Right superior corona radiata 74.5 ±2.2 73.6 ±1.3 0.099 2.9

Only those white matter tract labels with P < 0.1 (uncorrected) are shown.

Cerebellar White Matter

76 ■ p=0.016

p=0.0027 i----------------

I I I ♦

74 •

72 ■ •♦ r

70 - ♦

*

68 - ♦•

♦ ♦

••

Control Bipolar: Li- Bipolar: Li+ (N=25) (N=9) (N=6)

Cerebellar Gray Matter

Figure 2. Cerebellar T1 p values appear normalized w ith lith ium use. Median T1 p relaxation tim es fo r each participant in th e cerebellar w h ite and gray m atter regions o f interest are grouped by normal control participants, euthym ic bipolar participants w ho were n o t using lith ium ( L i - ) and euthym ic bipolar participants w h o were using lith iu m (Li+). The average median value fo r each group is indicated by the red dot, and th e red bars show the standard error. The L i - participants have significantly higher T ip values than the normal control and Li+ participants, whereas th e normal control and Li+ participants have similar values.

may have a normalizing effect on T ip values in the cerebellum. If so, then T ip may be sensitive to biological processes that contribute to bipolar disorder and that are corrected by lithium.

DISCUSSION To our knowledge, this is the first study to apply quantitative Tip mapping to a psychiatric disorder. By mapping T ip relaxation times throughout the brain with high spatial resolution, we found cerebral white matter and cerebellum abnormalities in a group of euthymic participants with bipolar I disorder compared with a

normal control group, suggesting a potential role for these regions in the pathophysiology of the disease.

Our findings provide new evidence for widespread white matter alterations in bipolar disorder. Whereas prior imaging studies of white matter in bipolar disorder have largely focused on the frontal-limbic networks associated with emotion processing,9,34-36 our findings include abnormalities in the corpus callosum, sagittal striatum, superior longitudinal fasciculus and cerebellar peduncle. There is evidence for white matter inflammation, tissue loss and altered metabolism in bipolar disorder,13,14,34,37 all of which may have a broad effect and could be reflected in the T ip findings.

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T ip mapping of bipolar disorder CP Johnson et al

205

-1.96

-3.00

Li- vs. Control Li+ vs. Control Li- vs. Li+ Z-Score: +3.00

+1.96

Figure 3. The effect o f lith ium on T ip values is unique to the cerebellum. Mean T ip map differences between bipolar participants n o t using lith ium ( L i- ) , those using it (Li+) and all controls indicate tha t lith iu m use is associated w ith normalized T ip values in the cerebellum b u t not the cerebral w h ite matter. Statistical maps o f the voxel-wise differences are shown fo r one slice, b u t the effect is seen th ro u g h o u t th e brain. Z-scores were thresholded at a significance level o f P < 0.05 w ith correction fo r m ultiple comparisons.

However, w e did n o t fin d evidence fo r peripheral in flam m atio n or w h ite m a tte r volum e loss in th e bipo lar group, w h ich lends some sup po rt th a t o u r w h ite m a tte r fin d in g s may reflect abnorm al m etabolism . If th e elevation o f T ip is in fact due to m etabolic factors, th e n ou r fin d in g is consistent w ith reduced pH and reduced glucose con cen tra tion .20,22' 24 Others have fo u n d evi­ dence fo r bo th reduced pH and abnorm al glucose m etabolism in th e w h ite m a tte r o f bip o la r disorder,6,38' 40 w h ich fu rth e r supports a m etab olic in te rp re ta tio n o f o u r results. O ur T ip fin d in g s suggest a need to focus on these w h ite m a tte r regions and on th e cerebellum in fu tu re studies, fo r example, by ta rg e tin g MR spectroscopy specifically to these areas.

Our T ip data also p o in t to th e cerebellum as a key region o f interest. A lth o u g h prior studies have n o t w id e ly appreciated th a t th e cerebellum may be critical in bipo lar disorder, th e cerebellum has been suggested to play an im p o rta n t role in e m o tio n pro­ cessing.41,42 Indeed, some prior studies have detected cerebellar abnorm alities in bip o la r disorder, in clud in g structural deficits,43 reduced cerebellar bloo d vo lu m e 44,45 and dysfunction as assessed by eyeblink, posture and fin g e r ta p p in g tests.46' 48 Finding th a t lith iu m alters T ip in th e cerebellum adds to this evidence and is consistent w ith o u r in te rp re ta tio n th a t abnorm al m etabolism underlies th e observed T ip abnorm alities. Lithium is unlikely to reverse cellular loss b u t it has been suggested to change glucose consum p tion in th e cerebellum ,49,50 perhaps th ro u g h its effects on glycogen synthase kinase-3 or calcium signaling.51

Primary advantages o f T ip m ap ping include sensitivity, spatial resolution, w hole -bra in coverage and q u an tificatio n, b u t specifi­ c ity remains a challenge. We a tte m p te d to address some o f th e p o tentia l factors th a t may have influenced T ip to b e tte r under­ stand th e source o f th e ab norm alities observed here. The T ip abnorm alities may very w ell reflect abnorm al m etabolism rather than in fla m m a tio n or cellular loss. However, at present, w e cannot rule o u t local changes in tissue structure or w a te r con tent. These possibilities could be investigated in fu tu re studies by using co m p le m e ntary m ap ping o f T2,52 adiabatic T ip and T2p,53 and T ip dispersion.54

O ther lim itation s o f this study can also be addressed in fu tu re work. First, alth o u g h o u r sample size was sufficie nt to de tect statistically sig nifica nt differences across groups, we were pow ered to d e tect o n ly relatively large effects. In particular, increasing th e sample size could im prove th e po w e r to estim ate effects o f m edications. Second, alth ou gh w e exam ined patients in th e e u th ym ic state, w e can no t presently rule o u t effects o f linge rin g m oo d states or th e ir consequences. Future studies exam ining T ip in d iffe re n t m ood states w o u ld pro vid e im p o rta n t ad d itio n a l insight. Third, given previously detected im aging abnorm alities in th e fronta l, subcortical and cerebral gray m atter regions in bip o la r disorder,55 it was som ew hat surprising th a t T ip d id n o t d e tect changes in these areas. T1 p may n o t be sensitive to these same abnorm alities o r technical factors may have prevented th e ir detection, in cluding: (i) d isto rtio n fro m fro n ta l sinus

susceptibility artifacts; (ii) partial vo lu m e averaging o f cerebral gray m a tte r w ith cerebral spinal flu id and increased v a ria b ility in gray m a tte r due to lo w er spatial resolution (5.0 m m ) in th e slice- encode direction; o r (iii) lim ite d T ip fittin g precision due to use o f o n ly tw o TSLs to reduce acquisition tim e. In th e future , pulse sequence advances such as T ip preparation w ith reduced sensitivity to field in h o m o g e n e ity,56,57 variable flip angle schemes coupled w ith corrective m ethods fo r T1 co n ta m in a tio n to enable hig h e r spatial resolution and faster acquisitions,58 and m ore o p tim a l selection o f TSLs fo r im p roved T ip m ap precision59 w o u ld be beneficial. Finally, w e d id n o t assess w h e th e r th e w h ite m a tte r and cerebellar abnorm alities are specific to bip o la r disorder, alth ou gh it w ill be im p o rta n t in fu tu re studies to determ ine w h e th e r these abnorm alities are shared by patients suffering from o th e r diseases such as schizophrenia and depression.

The findings presented in this paper potentially represent an im portant advance in the study o f bipolar disorder. Using an im aging strategy novel to psychiatric illness, we identified abnor­ malities in cerebral w h ite m atter and cerebellum th a t have n o t been previously observed and may reflect locally altered metabolism and consequences o f lithium action. This w o rk points to T1 p m apping as an im p ortan t new too l fo r studying psychiatric disorders, which com bined w ith existing techniques may lead to im proved insight in to mechanisms and treatm ent o f severe mental illnesses.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGMENTS This project was partially supported by a gift from Roger Koch. JGF was supported by the National Institutes of Health (1K23MH083695-01A210). JAW was supported by the Department of Veterans Affairs (Merit Award), National Institute of Mental Health (5R01MH085724), National Heart Lung and Blood Institute (R01HL113863) and a NARSAD Independent Investigator Award. Preliminary data for this study were reported in a conference abstract.60

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