Electronic Supplementary Material Online Resource

Electronic Supplementary Material – Online Resource article title: Involvement of the human ventrolateral thalamus in olfaction. submitted: Journal of Neurology authors: S. Zobel, T. Hummel, M.D., J. Ilgner, M.D., A. Finkelmeyer, PhD.,

U. Habel, PhD., D. Timmann, M.D., J.B. Schulz, M.D., M. Kronenbuerger, M.D. corresponding author: Martin Kronenbuerger, M.D., Dept. of Neurology, RWTH Aachen University,

e-mail: [email protected]

Details of the olfactory testing. The “Sniffin’ Sticks” [23, 24] are a well established and reliable method to assess olfactory functioning. For odor presentation, the cap of a selected Sniffin’ Stick was removed by the experimenter and the pen’s tip was held approximately 1 cm under the examined nostril for approximately 3 seconds. The participants were tested blindfolded to prevent visual identification of the odorant-containing pens.

The olfactory subtest odor discrimination was assessed first followed by testing odor threshold, and lastly odor identification. The three different olfactory functions were assessed separately for each nostril (lateralized olfactory testing). For instance, odor discrimination was tested at one nostril and then at the other nostril before testing continued with the assessment of odor threshold and odor identification in the same fashion. Odorless soft tape (Microfoam Surgical Tape; 3M, Neuss, Germany) was used to temporarily seal one nostril for the purpose of lateralized olfactory testing. There was a five minute break between testing of the three olfactory subtests.

Odor thresholds for n-butanol were assessed using a staircase, three alternative forced choice procedure (3-AFC procedure). Sixteen dilutions were prepared in a geometric series starting from a 4% n-butanol solution (dilution ratio 1:2 in deionized aqua conservans as solvent). Three pens were presented in randomized order, with two containing the solvent and the third containing the odorant. Patients were asked to identify the odor-containing pen. Reversal of the staircase (i.e., the presentation of the triplet with the next lower odor concentration) was initiated when the odor-containing pen was correctly identified in two successive trials. When patients gave an incorrect answer, the triplet with the next higher odor concentration was presented and thus, the staircase was reversed. Testing was completed after 7 reversals of the staircase. Odor threshold was defined as the mean of the last four out of the seven staircase reversals.

In the odor discrimination task, using a 3-AFC procedure, 16 triplets of pens were presented in randomized order, with two containing the same and one a different odorant. Within the presentation of a given triplet, each pen of the triplet was presented only once. The participant always had to choose one of the three pens as the one that (probably) contained the odor even when s/he eventually felt not to have perceived an odor. For odor threshold and odor discrimination, the presentation of triplets was separated by 30 seconds with the interval between presentations of individual pens being approximately 3 seconds.

Odor identification was assessed for 16 common odors. Half of these were used for testing one nostril, the second half were used to test the other nostril. The odors were presented in a randomised sequence. A multiple choice forced task was used for testing odor identification. Immediately before the odor was presented, the examiner read a list of four response options from which the subject had to choose the correct answer. The participant was asked to choose one answer option that she / he felt to be correct after the odor had been presented. The interval between odor presentations was approximately 30 seconds.

The patients’ scores ranged between 1 and 16 per nostril for the odor threshold task and the odor discrimination task, where lower scores indicated poorer performance. For the odor identification task the score for each nostril ranged from 0 to 8. Superimposed brain images in the two patient groups. The axial images of the computer tomography or the magnetic resonance tomography made during the routine care were used to localize the brain lesion. The location and extent of the lesions were entered manually into MRIcroN software (Lazarus INC, USA, Version 15) and superimposed. a The superimposed lesions in the group of all patients with a thalamic lesion involving the ventrolateral thalamus examined at the level of the inter-commissural line are shown in the left image. Right hemispheric lesions were flipped to the left hemisphere. The image on the right shows the region of maximum overlap in the group of patients with an odor threshold deficit at the ipsilesional nostril subtracted by the area of the lesions in the group of patients with no or a contralesional odor threshold deficit. b The upper part shows the superimposed brain lesions in the group of all patients with a focal cerebellar lesion examined. Left hemispheric lesions were flipped to the right hemisphere. The images below show the region of maximum overlap in the group of patients with a cerebellar lesion and an odor threshold deficit at the contralesional nostril subtracted by the area of the lesions in the group of patients with no or an ipsilesional odor threshold deficit. Table 3 Airflow through the nostrils as assessed with rhinomanometry – Online Resource

patients with a patients with a le- cerebellar lesion of the ventro- controls sion lateral thalamus

contralesional nostril* 261.5 (126.2) 220.5 (97.7) 218.8 (127.0)

ipsilesional nostril** 232.5 (186.8) 231.0 (125.0) 238.4 (169.9)

Values are means (standard deviation) in cm³/s. Airflow was assessed at a pressure difference of 150 Pa between nostril and nasopharynx. *refers to the left nostril in controls; **refers to the right nostril in controls.

Side difference between the three groups: P=0.84 as assessed with the Kruskal-Wallis-Test Table 4 Performance on the neuropsychological tests in the three groups – Online Resource

patients with a lesion of patients with the ventro- a cerebellar lateral thal- controls lesion amus

logical memory tests [20] immediate recall 12.4 (4.4) 12.8 (4.5) 11.5 (5.3) delay recall 11.9 (5.7) 11.7 (4.8) 10.0 (5.5)

digit span [20] forward 7.4 (1.4) 5.5 (1.9) 6.7 (1.9) backward 6.2 (1.7) 4.1 (2.0) 5.3 (1.9)

Multiple Choice Vocabu- lary Intelligence Test [21] 29.8 (5.1) 29.6 (3.9) 29.4 (5.8)

Values are means (standard deviation).