''Hairy Baskets'' Associated with Degenerative Purkinje Cell

J Neuropathol Exp Neurol Vol. 69, No. 3 Copyright Ó 2010 by the American Association of Neuropathologists, Inc. March 2010 pp. 262Y271

ORIGINAL ARTICLE

‘‘Hairy Baskets’’ Associated With Degenerative Purkinje Cell Changes in Essential Tremor

Cordelia R. Erickson-Davis, BA, Phyllis L. Faust, MD, PhD, Jean-Paul G. Vonsattel, MD, Sachin Gupta, MD, Lawrence S. Honig, MD, PhD, and Elan D. Louis, MD, MSc Downloaded from https://academic.oup.com/jnen/article/69/3/262/2917229 by guest on 30 September 2021 (4). The tremor is usually progressive (5) and produces func- Abstract tional disabilities (6). Recent evidence also suggests an asso- Essential tremor (ET) is one of the most common neurologic dis- ciated increased risk of mortality (7). eases. Increased numbers of torpedoes and Purkinje cell (PC) loss A wealth of clinical data suggests that ET is a disorder have been documented in the brains of patients with ET. We re- of cerebellar dysfunction. Intention (i.e. Bcerebellar[) tremor cently observed a dense and tangled appearance (Bhairiness[) of the of the hands occurs in 44% of ET cases (8). Abnormalities basket cell axonal plexuses that surround PC soma in Bielschowsky in tandem gait and balance have been repeatedly described in preparations of cerebellar cortex in ET brains. Here, we assessed ET patients (9Y11), and ET patients with intention tremor basket cell Bhairiness[ in 37 ET (32 cerebellar ET; 5 Lewy body may also have other cerebellar signs such as dysdiado- variant ET), 21 nondisease control, and 48 disease control brains chokinesia (12). Eye movement abnormalities that indicate using a semiquantitative scale. In 8 cerebellar ET cases (25%), there cerebellar dysfunction have also been described in ET (13). were high basket scores (rating = 3), whereas no Lewy body variant Unilateral cerebellar stroke has been reported to abolish ip- ET, 1 nondisease control (4.8%), and 2 diseased controls (4.2%) had silateral arm tremor in ET (14), and cerebellar outflow path- high basket scores (p = 0.001). The hairy basket scores correlated ways are the targets of deep brain stimulation surgery, which with numbers of torpedoes (p G 0.001) and inversely with numbers is effective in treating ET (15, 16). Numerous neuroimaging of PCs (p = 0.06). Axonal plexus density obtained by image analysis studies using functional magnetic resonance imaging (17), of basket cell processes traced from digitized images was higher in positron emission tomography (18, 19), [1H] magnetic reso- ET than in nondiseased control cases (p = 0.016). Closely spaced nance spectroscopic imaging (20, 21), diffusion-tensor im- sites of synaptic contact between basket cell processes and PCs were aging (22), and voxel-based morphometry (23) have provided identified by electron microscopy in ET cases. These data indicate evidence that cerebellar structure and function are abnormal that structural changes are not restricted to PCs in ET, and that other in ET. neurons within their functional network may be involved in its Until recently, there had been few postmortem studies of pathogenesis. ET (24). Studies that explore the pathological anatomy and improve our understanding of the pathophysiology of ET are Key Words: Basket cells, Cerebellum, Essential tremor, Neuro- critically important as there is no cure for ET and first-line degenerative, Pathology, Pathophysiology, Purkinje cells. medications, of which there are only two, are estimated to be ineffective in as many as 50% of patients (25). In each of 2 INTRODUCTION recent large postmortem series, the presence of degenerative Essential tremor (ET) is a chronic brain disease, the changes in the cerebellum has become evident (24, 26Y28). To most recognizable feature of which is a 4- to 12-Hz action date, these changes consist of an increased number of tor- tremor (1, 2). It is one of the most common neurologic dis- pedoes (5- to 6-fold greater than seen in age-matched control orders present in 4.0% of individuals 40 years or older (3), and brains) and a mild, approximately 40%, loss of Purkinje cells in as many as 21.7% of individuals aged 95 years and older (PCs) (27, 29). Whether the structural changes in ET are restricted to PCs or whether they involve other neurons in their From the GH Sergievsky Center (CED, SG, LSH, EDL); Department of functional network has not been investigated. Pathology and Cell Biology (PLF, JPGV); Taub Institute for Research on Recently, we observed an unusual dense and tangled Alzheimer’s Disease and the Aging Brain (JPGV, LSH, EDL); Depart- (we now term Bhairy[) appearance of the basket cell axonal ment of Neurology, College of Physicians and Surgeons (LSH, EDL); plexus surrounding PC soma in Bielschowsky preparations and Department of Epidemiology, Mailman School of Public Health, of cerebellar cortical sections in ET cases. Basket cells are Columbia University, New York, New York (EDL). F Y Send correspondence and reprint requests to: Elan D. Louis, MD, MSc, Unit -aminobutyric acid ergic inhibitory interneurons found in 198, Neurological Institute, 710 West 168th Street, New York, NY 10032; the molecular layer; they send out axonal collaterals to form E-mail: [email protected] a pericellular basket around PC perikarya. To our knowl- Supported by Grant Nos. R01 NS42859, P50 AG08702, and P01 AG07232 edge, there are only 2 previous reports describing a dense from the National Institutes of Health (Bethesda, MD); the Parkinson’s Disease Foundation (New York, NY); and the Arlene Bronstein Essential and tangled appearance of basket cell axonal plexus pro- Tremor Research Fund (Columbia University). files occurring with cerebellar degeneration: a single case of Online-only figures are available at http://www.jneuropath.com. Creutzfeldt-Jakob disease (30) and an early study examining

262 J Neuropathol Exp Neurol Volume 69, Number 3, March 2010

Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol Volume 69, Number 3, March 2010 Hairy Baskets Associated With Essential Tremor brains of individuals diagnosed with Bsenile dementia[ (31). of New York, or were outside referrals to the brain bank. We Because this pathologic finding may provide important mech- also used the brains of 12 PSP cases and of 1 case of clinically anistic insights into the pathogenesis of ET, we assessed bas- diagnosed adult-onset focal dystonia (torticollis) even if they ket cell changes in ET, other neurodegenerative diseases, and were younger than the cerebellar ET cases because tissue nondiseased control cases. availability was limited. Nondiseased control brains (n = 21) were matched to the age distribution of cerebellar ET brains and were from MATERIALS AND METHODS individuals who had been enrolled as normal elderly control Brain Repository subjects in the Alzheimer’s Disease Research Center or the Washington Heights Inwood Columbia Aging Project; they This study was conducted at the Essential Tremor were followed prospectively with serial neurologic assess- Centralized Brain Repository at the New York Brain Bank, ments and were free of the diagnosis of AD, ET, PD, DLBD, Columbia University (32). The 106 brains included 37 from or PSP, and had normal neuropathologic examinations. Downloaded from https://academic.oup.com/jnen/article/69/3/262/2917229 by guest on 30 September 2021 ET cases; 31 (83.8%) of these were collected prospectively beginning in 2003, and 6 were archival. All 37 had been Tissue Processing diagnosed by their treating neurologist, and all diagnoses All brains underwent complete neuropathologic assess- were confirmed using Essential Tremor Centralized Brain ment at the New York Brain Bank (26, 27, 32, 33, 36). Repository criteria (27). Essential tremor cases were divided Brains received ratings of neurofibrillary tangles using Braak into 2 groups based on the presence or absence of brainstem and Braak staging (37, 38) and Consortium to Establish a Lewy bodies on postmortem examination (27, 29, 33, 34). Registry for Alzheimer’s Disease (CERAD) ratings (39) for Essential tremor brains without Lewy bodies have an array of neuritic plaques. National Institute on AgingYReagan Insti- cerebellar abnormalities that include increased numbers of tute criteria for AD (40) were also assigned. Postmortem in- Y torpedoes and PC loss (27, 29). The Lewy body free brains terval (PMI) was the number of hours between death and have been referred to as cerebellar ET and brains with Lewy placement of the brain in a cold room or on ice. bodies as Lewy body variant of ET in previous publications A standard 3 Â 20 Â 25Ymm parasagittal tissue block (24, 32). Demographic and clinical information were col- from the neocerebellum was harvested from each brain as lected on each brain; the severity of tremor on Archimedes described (26, 27, 29, 33). Seven-micrometer-thick paraf- Y spiral drawings was rated using a validated rating scale (0 3) fin sections, impregnated using a modified Bielschowsky by a neurologist (E.D.L.) who was blinded to postmortem silver technique, were used for semiquantitative and quantita- data (35). tive analyses of basket cell processes and quantification of Diseased control brains (n = 49) included individuals torpedoes. Sections were also stained with Luxol fast blue- diagnosed with Alzheimer disease (AD; n = 20), Parkinson hematoxylin and eosin (LH&E) for quantification of torpedoes disease (PD) or diffuse Lewy body disease (DLBD; n = 15) that and PCs (26, 27, 33). For immunohistochemical analyses, were selected for matching to the age distribution of available sections were incubated with mouse monoclonal SMI-31 cerebellar ET brains. Alzheimer disease brains were from indi- (Covance, Princeton, NJ), as previously described (41). In viduals who had been enrolled as AD cases in the Alzheimer each brain, torpedoes in an entire LH&E section and an entire Disease Research Center or the Washington Heights Inwood Bielschowsky preparation were counted, and PCs in five 100Â Columbia Aging Project, where they were followed prospec- LH&E fields were counted and averaged, as previously tively with serial neurologic assessments, including assess- described (27). ment of action tremor, and were free of ET, PD, DLBD, or progressive supranuclear palsy (PSP). Parkinson disease or Semiquantitative Rating of Hairy Baskets DLBD brains at the New York Brain Bank had been patients A semiquantitative rating of the appearance of the basket at the Movement Disorder Division, Neurological Institute cell plexus surrounding PC bodies throughout Bielschowsky

TABLE 1. Demographic and Clinical Characteristics of Cases and Controls Nondiseased Diseased All Cerebellar ET LBVET AD PD/DLBD PSP Dystonia Controls Controls Controls N 32 5 20 15 12 1 21 48 69 Age (years) 83.6 T 7.6 86.8 T 4.7 82.9 T 5.2 78.1 T 6.7 73.4 T 7.2* 68 77.3 T 13.6 78.7 T 7.3* 78.3 T 9.6* Female sex 20 (62.5%) 2 (40.0%) 12 (60.0%) 6 (40.0%) 5 (41.7%) 1 (100%) 8 (38.1%) 24 (50.0%) 32 (46.4%) PMI (hours) 4.7 T 4.1 7.7 T 2.8 7.7 T 7.3 7.7 T 4.8 8.0 T 7.3 7.1 8.8 T 8.4 7.8 T 6.5† 8.1 T 7.0* Brain weight (g) 1,211 T 138 1,184 T 131 1,097 T 119 1,128 T 272 1,164 T 110 1,632 1,284 T 182 1,135 T 192 1,180 T 200 CERAD score 0.9 T 1.1 1.2 T 1.3 3.0 T 0.0‡ 1.5 T 1.1 0.4 T 0.7 0 0.2 T 0.4 1.8 T 1.3* 1.3 T 1.3 Braak AD score 2.0 T 1.2 2.2 T 0.8 5.8 T 0.4‡ 1.3 T 1.0 1.2 T 0.9 1 0.6 T 0.9‡ 3.2 T 2.4* 2.4 T 0.4

Values are either mean T SD or number (percentage). Diseased controls = AD + PD/DLBD + PSP + dystonia. All controls = diseased controls and nondiseased controls. *, p G 0.01; †,pG 0.05; ‡,pG 0.001 compared with cerebellar ET (analysis of variance with Tukey post hoc comparison). AD, Alzheimer disease; DLBD, diffuse Lewy body disease; ET, essential tremor; LBVET, Lewy body variant of ET; PD, Parkinson disease; PMI, postmortem interval; PSP, progressive supranuclear palsy.

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TABLE 2. Hairy Basket Categories by Diagnosis Cerebellar ET LBVET AD PD/DLBD PSP Dystonia Diseased Nondiseased All Controls (n = 32) (n = 5) (n = 20) (n = 15) (n = 12) (n = 1) Controls (n = 48) Controls (n = 21) (69) Hairy basket category 1 (low) 13 (40.6%) 2 (40.0%) 15 (75.0%) 10 (66.7%) 9 (75.0%) 1 (100%) 35 (72.9%) 14 (66.7%) 49 (71.0%) 2 (intermediate) 11 (34.4%) 3 (60.0%) 4 (20.0%) 4 (26.7%) 3 (25.0%) 0 (0.0%) 11 (22.9%) 6 (28.6%) 17 (24.6%) 3 (high) 8 (25.0%) 0 (0.0%) 1 (5.0%) 1 (6.7%) 0 (0.0%) 0 (0.0%) 2 (4.2%) 1 (4.8%) 3 (4.3%) P* Not applicable 0.51 0.01 0.068 0.02 † 0.001 0.03 0.001

Hairy basket category: 1(low; hairy basket rating = 0Y1.5), 2 (intermediate; hairy basket rating = 2Y2.5), 3 (high; hairy basket rating = 3). Values are number of cases (percentage). *Compared with cerebellar ET (Mantel-Haenszel W2 test). †Only 1 case; p value not computed. AD, Alzheimer disease; DLBD, diffuse Lewy body disease; ET, essential tremor; LBVET, Lewy body variant of ET; PD, Parkinson disease; PSP, progressive supranuclear palsy. Downloaded from https://academic.oup.com/jnen/article/69/3/262/2917229 by guest on 30 September 2021 preparations was performed by a senior neuropathologist the program whereby each pixel of the basket tracing was (P.L.F.) who was blinded to all clinical information. The fol- assigned a gray value using a variable gray scale and then lowing scale was used: 0 (few or no discernible processes); 1 summed). (sparse number of processes); 2 (moderate number of processes); and 3 (dense tangle of processes). In some instances, Ultrastructural Analysis the rater used intermediate values (0.5, 1.5, and 2.5). Hence, the Flat Eponate 12 (Ted Pella, Redding, CA) embedded hairy basket rating ranged included the values 0, 0.5, 1, 1.5, 2, vibratome sections of cerebellar cortical tissue were dissected 2.5, and 3. into 3 Â 4Ymm sections and re-embedded in the same resin. Semithin sections were then stained with toluidine blue and Quantitative Analysis of Basket Cell Plexus examined by light microscopy for PCs. Once PCs were Quantification of the density of the basket cell axonal identified on semithin section, the block was further trimmed plexus formations was performed on a subsample of 10 brains around the PCs to the size required for ultrathin sectioning. (including 5 randomly selected cerebellar ET brains and 5 Ultrathin sections were stained with uranyl acetate and lead randomly selected nondiseased control brains) by a single citrate and then examined by transmission electron micro- trained physician who was blinded to clinical information. scope (JEOL 1200EXII, Peabody, MA), as described (41). Digital images of the Bielschowsky neocerebellum preparations were obtained using a Zeiss Axioplan 2 microscope fit Statistical Analyses with an Axiocam HR digital camera (20Â objective lens). For All analyses were performed in SPSS (version 16.0). As image acquisition, slides were oriented with the dentate previously noted, a priori, the focus of our interest was cer- nucleus situated on the right and the folia branches extending ebellar ET cases rather than Lewy body variant of ET cases. leftward. Folia were labeled numerically, and the 2 folia Demographic and clinical features of diagnostic groups (e.g. located most centrally in the slide were selected. Within each cerebellar ET vs controls) were compared using W2 tests and of these folia, 3 smaller subregions consisting of 1 to 2 dis- analysis of variance (ANOVA) with Tukey post hoc compar- tal peaks (gyri) and 1 to 2 distal troughs (sulci) were chosen isons (Table 1). such that there were 6 subregions photographed per brain. In The hairy basket rating included the values 0, 0.5, 1, each photograph, the basket cell processes surrounding each 1.5, 2, 2.5, and 3. In nondiseased controls, the correlates of visible PC perikaryon were traced using Adobe Photoshop the hairy basket rating were assessed using Pearson correla- 5.0 (approximately 40 PCs/brain). Images of the basket cell tion coefficients (e.g. hairy basket rating by age). To facilitate tracings were then inverted and imported into Image J (NIH), further analyses, hairy basket ratings were then collapsed into where the integrated density was assessed (i.e. a function of a smaller number of rating categories as follows: low (0 or 1,

FIGURE 1. Semiquantitative rating of basket cell processes. (AYD) A rating scale (from 0 to 3) of the appearance of the basket cell plexus surrounding extant Purkinje cell bodies was determined in a blinded analysis of Bielschowsky preparations of neocerebellum sections. (A) Score = 0 (few, or no discernible processes); (B) Score = 1 (sparse number of processes); (C) score = 2 (moderate number of processes); (D) score = 3 (dense tangle of processes, i.e. hairy basket).

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Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol Volume 69, Number 3, March 2010 Hairy Baskets Associated With Essential Tremor Downloaded from https://academic.oup.com/jnen/article/69/3/262/2917229 by guest on 30 September 2021

FIGURE 2. Immunohistochemical analysis of basket cell processes. (A, B) Vibratome sections (100-Km-thick) immunostained with monoclonal antibody SMI-31 demonstrate prominence of phosphorylated neuroﬁlaments in basket cell processes (long arrows). (A) A dense and tangled, or ‘‘hairy,’’ appearance of basket cell axonal plexus formation in a section from a cerebellar essential tremor case with a hairy basket rating of 3. (B) A control case with a rating of 0. Torpedoes (short arrows) are also seen. and also including any ratings of 0.5 and 1.5); intermediate ket rating category (Mantel-Haenszel W2 test; Table 2). For (2 and also including any ratings of 2.5); and high (3). Di- several analyses, diagnostic groups were combined (AD + agnostic groups were compared with regard to hairy bas- PD/DLBD + PSP + dystonia = diseased controls; diseased

FIGURE 3. Quantiﬁcation of basket cell axonal plexus density. (AYD) Examples of tracings scored according to a semiquantitative scale are shown: (A) 0 (few, or no discernible processes); (B) 1 (sparse processes); (C) 2 (moderate numbers of processes); and (D) 3 (dense tangle of processes). (E) Basket cell plexus densities were higher in cerebellar essential tremor (ET) cases (closed circles; median value = 15.10) than in controls (open squares; median value = 6.98; p = 0.016).

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FIGURE 5. Ultrastructural morphology of basket cellYPurkinje cell (P) contacts in essential tremor cases. (AYC) Multiple, closely spaced sites of synaptic contact (arrows) between a neurofilament-rich basket fiber and a Purkinje cell soma. Post-synaptic Downloaded from https://academic.oup.com/jnen/article/69/3/262/2917229 by guest on 30 September 2021 densities are apparent in (A) and (B) (arrows). The basket fiber in (C) shows several vesicle-lined contacts with the Purkinje cell (arrows). (D) Numerous processes converging on the initial segment (axon hillock) of a Purkinje cell (P-axh); at least 1 of these is a neurofilament-rich basket cell axon (large arrow). (d¶) In a higher magnification of (D), the Purkinje cell contact shows 2 distinct clusters of vesicles (arrows). Magnifications: (A, B, d¶), 40,000Â; (C) 50,000Â; (D) 20,000Â. controls + nondiseased controls = all controls; Table 2). Be- diseased controls), cerebellar ET cases were older by an cause of the modest sample size, basket cell plexus density average approximately 5 years, and mean PMI was shorter was compared across diagnostic groups using the Mann- (Table 1). All groups were similar with respect to gender and Whitney test. brain weight (Table 1). Consortium to Establish a Registry In an unadjusted logistic regression model, diagno- for Alzheimer’s Disease scores and Braak AD scores were sis (cerebellar ET vs all controls) was the outcome variable. higher in AD cases than cerebellar ET cases (Table 1). Braak In this model, the hairy basket rating category was the in- AD scores were higher in cerebellar ET cases than non- dependent categorical variable (high, intermediate, and low, diseased controls (Table 1). as previously described). In adjusted regression models, covariates that were associated with either the outcome or Basket Cell Plexus Alterations in Cerebellar ET independent variable or for which a priori evidence was Bielschowsky sections of cerebellar neocortex revealed considerable that the variable may be a confounder were variable appearances of basket cell axon collaterals that sur- included. round the PC perikarya. Figure 1 illustrates examples of the The correlates of the hairy basket rating in cerebellar ET semiquantitative scores of the basket cell plexus morphol- B [ cases and nondiseased controls and in cerebellar ET cases alone ogies. Hairy baskets (rating = 3, the severe end of the were determined using Pearson correlation coefficient when spectrum) were defined as a dense and tangled axonal plexus both variables were normally distributed; otherwise, Spearman (Fig. 1D). These hairy baskets generally surrounded viable- correlation coefficient was used. The degeneration index was appearing PCs, rather than PCs that had previously died B [ defined as the number of torpedoes on Bielschowsky section (i.e. empty baskets ). In each of the 8 ET cases with hairy divided by the mean number of PCs per 100Â LH&E-stained basket scores of 3, 20 hairy baskets were assessed (160 hairy high-power field. High values indicated large numbers of baskets). Purkinje cell somata were visible in 141 of these torpedoes and/or low PC counts (i.e. PC loss) (42). (88.1%). Immunohistochemistry highlighted the neurofilament- rich basket cell axonal processes that comprise the hairy baskets and PC axonal torpedoes (Fig. 2). RESULTS We also assessed whether the hairy basket rating was similar in different neocerebellar regions within the same brain. ET, Nondiseased Controls, and Diseased Controls For this analysis, 1 Bielschowsky section was examined from Cerebellar ET cases were similar to Lewy body variant 3 separate neocerebellar blocks in 7 brains with hairy basket of ET, AD, and PD/DLBLD cases and nondiseased controls ratings ranging from 0 to 3. The agreement between ratings was with respect to age and PMI. However, when diseased high (Block 1 vs Block 2, Pearson r = 0.91, p = 0.005; Block 1 controls (AD + PD/DLBD + PSP + dystonia) were combined vs Block 3, Pearson r = 0.95, p = 0.001; and Block 2 vs Block and all controls were combined (diseased controls + non- 3, Pearson r =0.96,pG 0.001). This indicated that the data

FIGURE 4. Ultrastructure of neurofilament-rich basket cell processes in cerebellar essential tremor (ET) and control cases. (A) The dense plexus of basket cell processes (arrows) adjacent to the Purkinje cell soma (P) in a cerebellar ET case contains neurofilaments and mitochondria. (B) Only 1 to 2 layer(s) of basket cell processes (arrows) are seen adjacent to a Purkinje cell soma (P) in a control case. (C) Higher magnification of basket cell axonal plexus in (A) shows many layers of the basket cell formation (arrows). (D) A dense nest of basket cell processes (arrows) surrounds another Purkinje cell in an ET case. (E) Intertwining of basket cell processes around one another in an ET case. Some processes are longitudinal to the plane of section (long arrows); others have been cut transversely (short arrows). Magnifications: (A, B) 10,000Â; (C) 20,000Â; (D) 25,000Â; (E) 30,000Â.

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Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. Erickson-Davis et al J Neuropathol Exp Neurol Volume 69, Number 3, March 2010 from a single block were likely to be broadly representative of ally inversely with the number of PCs (Spearman r = j0.25; the entire neocerebellum. p = 0.49), and with the degeneration index (Spearman r = The hairy basket rating was not correlated with age 0.76; p = 0.01). (Pearson r = 0.24, p = 0.31), PMI (Pearson r = j0.04, p = 0.87), brain weight (Pearson r = j0.20, p = 0.41), CERAD Ultrastructural Analysis of the Basket Cell Plexus score (Pearson r = j0.09, p = 0.71), or Braak AD score We performed ultrastructural studies to compare bas- (Pearson r = 0.05, p = 0.83) in nondiseased controls. It also ket cell processes in ET and control cases. In an ET case did not differ by gender in nondiseased controls (1.5 T 0.9 in with a hairy basket rating of 3, there were several layers of males vs 1.1 T 0.8 in females, p = 0.34). neurofilament-rich basket cell processes adjacent to the PC Hairy basket rating category was higher in cerebellar soma that intertwined at various angles (Fig. 4A, CYE). In ET cases than in AD, PSP, dystonia, PD/DLBD (marginal contrast, in a nondiseased control with a hairy basket rating of difference), and nondiseased controls (Table 2). When con- 0, only 1 or 2 basket cell processes having a somewhat thinner trol groups were combined, the differences with cerebellar diameter were found adjacent to the PC soma (Fig. 4B). Downloaded from https://academic.oup.com/jnen/article/69/3/262/2917229 by guest on 30 September 2021 ET were more marked. In 8 of 32 ET cases (25.0%), there The identity of these neurofilament-rich processes as was a hairy basket rating of 3, whereas only 4.2% of diseased basket cell fibers was further confirmed in the ET case by the controls and 4.8% of nondiseased controls had a score of identification of synapses with the PC soma and its initial axon 3 (Table 2). In an unadjusted logistic regression model in segment. In several instances, contacts with the PC somata which diagnosis (cerebellar ET vs all controls) was the were marked by multiple, closely spaced synaptic sites within outcome variable, brains in the high (rating = 3) hairy basket 1 basket cell process (Fig. 5AYC; arrows). A basket fiber axon rating category were 10 times more likely to be cerebellar was also identified making contact with the axon hillock of ET than controls (odds ratio [OR] = 10.05; 95% confidence the PC. In the basket axon, there were multiple synaptic sites interval [CI] = 2.33Y43.32; p = 0.002). Brains in the inter- marked by vesicle accumulations beneath the axonal mem- mediate (rating = 2) basket rating category were 2 to 3 times brane (Fig. 5D, d¶). These findings further corroborate and more likely to be cerebellar ET than controls (OR = 2.44; extend our light microscopic findings of basket cell plexus 95% CI = 0.92Y6.46; p = 0.07). In a logistic regression model morphology. that adjusted for age, gender, PMI, brain weight, CERAD score, and Braak AD score, these values were as follows: Correlates of the Hairy Basket Rating OR = 18.95 (95% CI = 1.88Y191.56; p = 0.01) and OR = 3.52 In cerebellar ET cases and nondiseased controls, the (95% CI = 0.82Y15.02; p = 0.089). hairy basket rating was robustly correlated with number of To provide a quantitative measure of these basket cell torpedoes (Spearman r =0.49,pG 0.001; Fig. 6A), inversely plexus alterations, the axonal plexuses surrounding extant with the number of PCs (Spearman r = j0.27; p = 0.06; PCs in 5 cerebellar ET cases and 5 nondiseased controls were Fig. 6B), and inversely with the degeneration index (Spearman traced from Bielschowsky preparation images (Fig. 3AYD). r = 0.48; p = 0.001; Fig. 6C), indicating that these basket cell When the densities of the tracings were determined, the av- changes were associated with torpedo formation and PC loss. erage basket cell plexus in cerebellar ET cases was approx- Within cerebellar ET cases, the hairy basket rating was imately twice the density of the average plexus in nondiseased associated with age (Pearson r = 0.35; p = 0.048) and controls (mean = 13.97 T 3.56 [median = 15.10] vs 6.98 T marginally with disease duration (disease duration was known 2.20 [median = 6.68]; Mann-Whitney z = 2.40; p = 0.016; in 19 cases and Pearson r = 0.35; p = 0.19). Hairy basket Fig. 3E). Basket cell plexus density was highly correlated with rating was associated with number of torpedoes (Spearman r = the hairy basket rating (Pearson r = 0.73; p = 0.018). Basket 0.49; p = 0.005) but not with number of PCs (Pearson r = cell plexus density was also strongly associated with the 0.01; p = 0.98); however, it was associated with the number of torpedoes (Spearman r = 0.79; p = 0.007), margin- degeneration index (Spearman r = 0.45; p = 0.01). Hairy

FIGURE 6. Correlations of hairy basket scores in cerebellar essential tremor (ET) and nondiseased control cases. (AYC) Hairy basket score correlated with number of torpedoes (A) (Spearman r = 0.49; p G 0.001), inversely with the number of Purkinje cells (B) (Spearman r = j0.27; p = 0.06), and degeneration index (C) (Spearman r = 0.48; p = 0.001).

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Copyright @ 2010 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol Volume 69, Number 3, March 2010 Hairy Baskets Associated With Essential Tremor basket rating did not differ by family history (presence vs creased profiles observed in our study represent an accumu- absence of Q1 first-degree relative with ET), age of tremor lation of converging basket cell processes recruited from onset (Pearson r = j0.21; p = 0.44), severity (0Y3) of tremor neighboring PCs that have been damaged or died. Although on Archimedes spiral (Pearson r = j0.08; p = 0.79), or pres- there has been little investigation of such a phenomenon in ence versus absence of head tremor (data not shown). the human cerebellum, selective preservation and reorganization of basket cell axonal processes has been demonstrated in basket cells in the CA1 and CA3 region of the hippocam- DISCUSSION pus (49, 50). These cells form baskets around hippocam- Recent postmortem studies have documented degener- pal pyramidal cells and function as local circuit inhibitory ative changes in the cerebellum of ET cases, as manifested by F-aminobutyric acidYergic interneurons, analogous to the increased numbers of torpedoes and loss of PCs (24, 27Y29, 42). relationship between cerebellar basket cells and PCs. These These postmortem data complement a rich history of clinical, disease-resistant hippocampal basket cells undergo extensive physiologic, and imaging studies that have long implicated reorganization in the setting of pyramidal cell death (51). In a Downloaded from https://academic.oup.com/jnen/article/69/3/262/2917229 by guest on 30 September 2021 cerebellar and cerebellothalamacortical circuitry abnormalities few cases, basket cell plexuses displayed increased density in ET (8Y15, 19Y21, 43). Although previous pathologic studies adjacent to areas with severe pyramidal cell loss and no bas- focused on degenerative changes in PCs, the relationship be- ket formations, suggesting that these basket cell processes tween ET and cerebellar interneurons has remained unexplored. might be converging on and reorganizing around remaining We examined archival and prospectively acquired cerebellar pyramidal cells (51). tissue of ET cases and both diseased and nondiseased controls The molecular mechanisms that underlie the degener- and found that the axonal plexuses of basket cells surrounding ative changes observed in the ET cerebellum are not clear. PCs in cerebellar ET brains are significantly Bhairier[ as com- The array of cellular changes within the complex neuronal pared with controls. network in cerebellar cortex have yet to be fully catalogued Basket cells are F-aminobutyric acidYergic inhibitory in ET; thus, further work with human postmortem tissue is interneurons that receive input from parallel fibers and, to a needed. In addition, genes have not been identified for ET, limited extent, from climbing and mossy fibers. In humans, and no transgenic mice currently exist that provide an animal up to 50 axon collaterals from neighboring basket cells de- model for ET. The most widely used animal model for ET is scend from the molecular layer and combine to form a com- based on the administration of neurotoxins (e.g. harmaline plex basket structure around the PC soma to which the basket and ibogaine) to rodents and other mammals. These toxins cells’ entire axonal output is devoted (44). Reductions in induce synchronous firing of inferior olivary neurons at an 8- cerebellar basket cell axonal plexuses have been reported in to 12-Hz frequency and sustained glutamatergic stimulation several disease states, including hypothyroidism (44), ataxia of PCs via their climbing fiber afferents (52Y54), triggering a telangiectasia (45), and Menkes kinky hair disease (46). Pre- massive increase in intracellular calcium and an excitotoxic- servation of basket cell plexuses in the setting of PC death mediated PC degeneration and cell death (55Y57). However, (i.e. Bempty baskets[) has been reported in Creutzfeldt-Jakob this toxin-based animal model produces an acute, reversible disease (47) and spinocerebellar ataxia Type 1 (48), but a action tremor. Most animals shake for a matter of hours, and dense and tangled appearance of the axonal plexus profile there is more severe destruction of cerebellar cortex in readily has not, to our knowledge, been documented in the ataxia identifiable linear bands. Although these latter clinical and literature. Curiously, an early 20th century study describing pathologic features clearly differ from those in ET, this model PC abnormalities in 16 brains of individuals diagnosed with demonstrates the functional significance of the olivocerebel- Bsenile dementia[ noted increased basket cell fiber Btangles lar system and a potential role for excitotoxic-mediated PC and masses[ alongside PC soma showing signs of degener- death in the generation of this type of tremor. It is intriguing ation in 3 cases (31). Another more recent study qualitatively that harmane (a tremor-producing, indole-alkaloid structurally examined basket cells in Creutzfeldt-Jakob disease and similar to harmaline) has been found in the blood of some ET noted empty baskets with abnormally dense phosphorylated patients at levels up to 50% higher than age-matched controls neurofilament positivity in a single case (31). Here, we docu- (58). Although additional studies are needed to establish mented that individuals with a diagnosis of cerebellar ET whether this exposure is of etiologic importance in ET, we were more than 10 times as likely as controls (both diseased postulate that the PC loss documented in ET (whether exci- and nondiseased) to have a hairy basket rating of 3 (the totoxic or not) leads to a reorganization of the cerebellar in- marked or severe end of the spectrum). In addition, the me- terneuron network, with sprouting and/or accumulation of dian axonal plexus density in cerebellar ET was approxi- neighboring basket cell processes on surviving PC bodies. mately 2 to 3 times higher than in controls. BHairiness[ of Ultrastructural analysis revealed the presence of multiple, basket cell axonal plexuses in these independent semiquanti- closely spaced sites of synaptic contact between the dense, tative and quantitative measures was positively associated multilayered basket cell processes and PC soma in ET. The with number of torpedoes and the degeneration index, and in- neurophysiologic abnormality in ET is not clear, but is thought versely with number of PCs, suggesting that hairy baskets and to be the result of decreased cerebellar PC inhibitory output. torpedoes may be concomitant features of cerebellar degener- Our observations raise the possibility that the increased density ation in ET. of basket cell processes identified in a significant number of The mechanism by which this increased Bhairiness[ ET cases contribute to this inhibitory effect on PC output. Re- occurs is unknown. One possible explanation is that the in- cent studies have demonstrated that climbing fiber responses in

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PCs are modulated through cerebellar interneuron networks 10. Stolze H, Petersen G, Raethjen J, et al. The gait disorder of advanced that include the basket cell (59). Basket cell output is also in- essential tremor. Brain 2001;124:2278Y86 11. Parisi SL, Heroux ME, Culham EG, et al. Functional mobility and pos- hibited by the axon collaterals of PCs and excited by both tural control in essential tremor. Arch Phys Med Rehabil 2006;87: parallel fibers and collaterals of climbing fibers (60). Cerebel- 1357Y64 lar interneurons are also coupled by dendro-somatic, dendro- 12. Koster B, Deuschl G, Lauk M, et al. Essential tremor and cerebellar dendritic, and somato-somatic gap junctions, which may lead dysfunction: Abnormal ballistic movements. J Neurol Neurosurg Psy- Y to synchronization of firing in groups of organized neural net- chiatry 2002;73:400 5 13. Helmchen C, Hagenow A, Miesner J, et al. Eye movement abnormalities works (61). Specialized axo-axonic septate junctions electri- in essential tremor may indicate cerebellar dysfunction. Brain 2003;126: cally couple basket fiber processes in the axon hillock region 1319Y32 (62). Thus, the disease process in ET may cause an imbalance 14. Dupuis MJ, Delwaide PJ, Boucquey D, et al. Homolateral disappearance in the interactions between cerebellar afferents, PCs and their of essential tremor after cerebellar stroke. Mov Disord 1989;4:183Y87 axon collaterals, and the cerebellar interneuron network that 15. Schuurman PR, Bosch DA, Bossuyt PM, et al. A comparison of con- tinuous thalamic stimulation and thalamotomy for suppression of severe includes basket cells. Additional studies on other cerebellar tremor. N Engl J Med 2000;342:461Y68 Downloaded from https://academic.oup.com/jnen/article/69/3/262/2917229 by guest on 30 September 2021 neurons and their interactions in ET are clearly needed to 16. Benabid AL, Pollak P, Seigneuret E, et al. Chronic VIM thalamic stim- understand the basis for tremor generation in this disorder. ulation in Parkinson’s disease, essential tremor and extra-pyramidal dys- Despite being the largest series of ET brains reported to kinesias. Acta Neurochir Suppl (Wien) 1993;58:39Y44 date, the sample size was modest. Despite this limitation, we 17. Bucher SF, Seelos KC, Dodel RC, et al. Activation mapping in essential tremor with functional magnetic resonance imaging. Ann Neurol 1997; were able to detect significant case-control differences, indi- 41:32Y40 cating that the sample was sufficient. This study had several 18. Colebatch JG, Findley LJ, Frackowiak RS, et al. Preliminary report: strengths. This was the first investigation of a relatively novel Activation of the cerebellum in essential tremor. Lancet 1990;336: pathologic finding in a large series of ET brains. We examined 1028Y30 basket cell profiles both semiquantitatively and quantitatively 19. Jenkins IH, Bain PG, Colebatch JG, et al. 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