Altered parvalbumin-positive neuron distribution in
basal ganglia of individuals with Tourette syndrome
Paul S. A. Kalanithi†‡, Wei Zheng†‡, Yuko Kataoka†, Marian DiFiglia§¶, Heidi Grantz†, Clifford B. Saper¶?,
Michael L. Schwartz††, James F. Leckman†, and Flora M. Vaccarino†‡‡
†Child Study Center and††Department of Neurobiology, Yale University, New Haven, CT 06520;§Department of Neurology, Massachusetts General Hospital,
Charlestown, MA 02129;?Department of Neurology, Beth Israel Deaconess Medical Center, Boston, MA 02215; and¶Department of Neurology, Harvard
Medical School, Boston, MA 02115
Edited by Rodolfo R. Llinas, New York University Medical Center, New York, NY, and approved July 20, 2005 (received for review March 30, 2005)
Tourette syndrome (TS) is a childhood neuropsychiatric disorder
characterized by motor and vocal tics. Imaging studies found
alterations in caudate (Cd) and putamen volumes. To investigate
possible alterations in cell populations, postmortem basal ganglia
tissue from individuals with TS and normal controls was analyzed
by using unbiased stereological techniques. A markedly higher
total neuron number was found in the globus pallidus pars interna
(GPi) of TS. In contrast, a lower neuron number and density was
observed in the globus pallidus pars externa and in the Cd. An
increased number and proportion of the GPi neurons were positive
for the calcium-binding protein parvalbumin in tissue from TS
subjects, whereas lower densities of parvalbumin-positive inter-
neurons were observed in both the Cd and putamen of TS subjects.
migration of some GABAergic neurons. The imbalance in striatal
and GPi inhibitory neuron distribution suggests that the functional
dynamics of cortico-striato-thalamic circuitry are fundamentally
altered in severe, persistent TS.
GABA ? human ? postmortem ? psychiatry ? tic
may or may not abate upon entering adulthood. Tics are sudden
stereotyped motor sequences of varying intensity and complex-
ity, often preceded by compulsions or sensory phenomena.
Neither the etiology nor the pathophysiology of TS is well
understood. Both genetic and environmental factors are thought
to be important, but the exact role of each has not yet been
identified (1). Epigenetic events that increase the risk of devel-
oping tic disorder or TS include perinatal hypoxic-ischemic
events that damage the periventricular germinal matrix and
adjacent deep regions of the brain (2). A considerable amount
of data implicates the cortico-striato-thalamo-cortical circuit in
TS pathophysiology, particularly basal ganglia (BG) abnormal-
ities. The BG, a richly interconnected set of nuclei, is essential
for the initiation and correct implementation of learned se-
quences of motor and cognitive segments that characterize
purposive behavior. The two major inputs into the BG, from the
cerebral cortex and the intralaminar nuclei of the thalamus,
enter into the striatum, which consists of the caudate (Cd) and
putamen (Pt). The firing of cortical inputs drives activity in both
medium spiny neurons (MSNs) (3) and several types of inter-
neurons, including parvalbumin (PV)-positive (PV?) GABAer-
electrical junctions and form a web of inhibitory synapses
throughout the striatum, coordinating the activities of MSNs,
and likely increasing their threshold of firing in response to
cortical inputs (5, 6). MSNs, which are the large majority of
neurons in the striatum, project to the globus pallidus pars
interna (GPi), either directly or indirectly via the subthalamic
nucleus and the globus pallidus pars externa (GPe). These two
pathways can be differentiated histologically. Substance P (SP)
is contained within striatal terminals of the direct pathway,
ourette syndrome (TS) is a childhood neuropsychiatric dis-
order characterized by persistent motor and vocal tics, which
whereas met-enkephalin (enk) is contained within terminals of
neurons: ?75% of GPi and 50% of GPe cells are positive for this
calcium-binding protein (7). However, unlike those in the stri-
atum, PV? cells in the GPi are projection neurons. The GPi
inhibitory projections to the thalamus have a major influence on
the firing rate and rhythmic activity of both ventrolateral and
intralaminar thalamic nuclei (8).
All major structures of the BG have been implicated in TS.
Chemical and electrical stimulation of the Pt in both animals and
humans produces tic-like stereotypies (9). Structural imaging
studies have shown small, but significant, decreases in Cd and Pt
volumes in TS (10), whereas functional imaging has shown
decreased activity in the ventral striatum (11–13) and increases
in dopaminergic terminals in the ventral striatal region of TS
patients (14). Additionally, levels of neuronal activity as mea-
sured by functional MRI while patients are asked to actively
suppress their tics are related in a complex manner to tic severity
outside the magnet, such that neuronal activity in the right head
of the Cd is inversely correlated with tic severity, whereas
neuronal activity in the GP and Pt is directly correlated with tic
In an effort to better understand the cellular abnormalities
that may be present in TS, we undertook a quantitative post-
mortem study of the BG of individuals with TS as compared with
age- and sex-matched normal controls (NCs), staining with
cresyl violet, PV, met-enk, and SP. We report here that in a small
number of TS patients thus far examined there is a profound and
consistent imbalance in the number of PV? neurons among
structures of the BG. The number of PV? neurons is increased
in the GPi and decreased in the striatum of TS brains compared
with NC. Although there may be additional abnormalities in TS
yet to be discovered, these data point to a strong imbalance in
PV? inhibitory neurons in TS, which may provide explanations
for TS symptomatology and pathophysiology and additional
avenues for research.
Subjects. A cohort of human brains obtained from the Harvard
Brain Tissue Resource Center at McLean Hospital and the
Yale Department of Critical Technologies was used in this
study and included five NCs (mean age, 60 ? 9.7) and three
subjects with severe, persistent TS (mean age, 42 ? 11.9)
(Table 2, which is published as supporting information on the
PNAS web site). NC subjects were collected after routine
autopsy at Yale University and Massachusetts General Hos-
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: TS, Tourette syndrome; Cd, caudate; Pt, putamen; GP, globus pallidus; GPi,
GP pars interna; GPe, GP pars externa; BG, basal ganglia; MSN, medium spiny neuron; PV,
parvalbumin; NC, normal control; SP, substance P; enk, enkephalin.
‡P.S.A.K. and W.Z. contributed equally to this work.
‡‡To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
September 13, 2005 ?
vol. 102 ?
no. 37 ?
pital. The TS specimens were obtained after informed consent
from the next of kin, and donated brain tissue was collected
under the sponsorship of the TS Association tissue resource.
This group of TS subjects was selected from a larger group of
donated specimens (n ? 41). Reasons for exclusion included
the presence of a neurological condition that might limit the
interpretation of the findings, e.g., Alzheimer’s disease, brain
tumors, problematic agonal events (such as a prolonged in-
terval on a respirator before death), or an excessive postmor-
tem interval (n ? 17); an inability to locate the next of kin (n ?
5); the presence of a significant, severe comorbid psychiatric
disorder, e.g., schizophrenia, bipolar disorder (n ? 8); or
insufficient or improperly processed tissue (n ? 8). Tissue
sections from one hemisphere were examined by routine
neuropathological tests, and all TS or NC brains with any
evidence of gross pathological changes or CNS cytological
abnormalities, including cellular ischemic changes, were ex-
cluded from analyses.
Each of the TS subjects was matched with one or more NC
subjects on the basis of age, sex, postmortem interval, and,
whenever possible, hemisphere (i.e., right vs. left). All of the
subjects in this study were male. Psychiatric diagnoses were
established by using a retrospective review of medical records
and an extensive family questionnaire that included the medical,
psychiatric, and social history of the subjects. For the diagnosis
of TS, attention-deficit hyperactivity disorder, obsessive-
compulsive disorder, major depressive disorder, and alcohol or
drug dependence DSM-IV criteria were used. A best-estimate
procedure was used once all available data had been compiled
(by J.F.L. and H.G.) (16). The TS diagnostic confidence index
was also estimated (17). All TS subjects had a ‘‘definite’’
Diagnostic and Statistical Manual-IV diagnosis of TS. They also
had TS diagnostic confidence index scores of ?90 and a history
of severe tic symptom severity, scoring 49–50 of 50 points on the
Yale Global Tic Severity Scale at the ‘‘worst ever’’ point in their
lives (18) (see Table 2). Two cases were associated with a positive
family history for TS or chronic tics, and the third case was
associated with a history of perinatal hypoxia. One of the three
TS subjects (case 4454) was not taking antipsychotic medications
at the time of death. The other two subjects were on a variety of
medications (Table 2). No subject in the NC group was receiving
any psychotropic agents at the time of death.
Immunocytochemistry. Intact half brains were stored in 10%
formalin. The telencephalon and brainstem were cut coronally
into 2.5-cm blocks. Blocks were rinsed in PBS?0.1% NaN3
(PBS?azide) and cryoprotected in 15% sucrose. Tissue was
serially sectioned at 50 ?m with a cryostat or a freezing
microtome, and 24 series of sections were collected.
Sections were rinsed three times in PBS, and endogenous
peroxidase was quenched by incubation in 3% H2O2for 30 min
at room temperature. Sections were blocked in PBS containing
5% normal horse or goat serum and 0.1% Triton X-100 (PBS
serum) for 30 min at room temperature and then incubated with
the first antibody for 48 h at 4°C in PBS serum. Primary
antibodies were anti-PV (1:2,500; Sigma), anti-SP (1:1,000;
Immunostar, Hudson, WI), and anti-met-enk (1:1,000; Immu-
nostar). After three washes in PBS, sections were incubated with
biotinylated secondary antibodies (Vectastain Elite kit, Vector
Laboratories) in PBS serum and processed for immunoperoxi-
dase staining according to the manufacturer’s instructions. Tis-
sue from control and TS was processed concurrently and ex-
posed to the substrate for exactly equal amounts of time.
Stereological Analyses. One series of sections, 1.2 mm apart, was
stained with cresyl violet and subjected to unbiased stereo-
logical analysis with a Zeiss Axioskop 2 mot microscope
equipped with an automatic stage and coupled to a computer
running STEREOINVESTIGATOR and NEUROLUCIDA software.
The Cd, Pt, GPe, and GPi regions were drawn in each section
in the series based on cytoarchitectonic landmarks. When
applicable, the volume of these nuclei was estimated by
planimetry, which is computed by adding up the cross-
sectional areas of the nucleus of interest in each section and
multiplying this number by the section interval and the average
section thickness (measured at the time of counting).
method with a ?40 oil-immersion objective, by randomly placing
a sampling grid over each contour. Sampling grids measured
1,800 ? 1,800 ?m for GPi, 1,500 ? 1,500 ?m for GPe, and
3,300 ? 3,300 ?m for Cd and Pt. Tridimensional counting frames
regions) with three of six exclusion borders (19, 20) were
automatically placed by STEREOINVESTIGATOR at each grid in-
tersection point. Approximately 115 frames per brain were
sampled in the Cd, 135 in the Pt, 412 in the GPe, and 124 in the
GPi. On average, 1,202 neurons per brain were counted in the
Cd, 1,264 in the Pt, 280 in the GPe, and 81 in the GPi. Neurons
and glia were separately counted in each frame, distinguished on
the basis of their cytological appearance. The total number of
cells per region was calculated by STEREOINVESTIGATOR using
where ?Q was the total number of nuclei counted, t the mean
section thickness, h the height of the optical dissector, asf the
The density for each cell type was calculated by dividing the total
number of cells by the total counting volume.
For the PV-stained sections, the protocol was identical to the
above, except for the sizes of the sampling grids and counting
frames. Sampling grid in the GP measured 1,000 ? 1,000 ?m,
and counting frames measured 130 ? 130 ? 15 ?m. In the
striatum the sampling grid measured 2,500 ? 2,500 ?m, and the
counting frame measured 700 ? 500 ? 15 ?m (magnification:
?10). The counting frames were placed in the most superficial
portion of the sections (1 ?m from the surface), to avoid
variability caused by differential penetration of antibodies. Cell
density within tissue was calculated by dividing the total number
of cells by the total volume of tissue sampled. Approximately 356
frames per brain were sampled in the Cd, 417 in the Pt, 368 in
per brain were counted in the Cd, 348 in the Pt, 172 in the GPe,
and 244 in the GPi.
Three researchers (P.S.A.K., Y.K., and W.Z.) performed
the majority of the stereological analyses, and the PV-
immunostained sections of GPi and Pt were counted by two of
them. Neither researcher was aware of the disease state of the
tissue during counting. Interperson count variability was ?3%.
All brains from the TS cases showed a ?60% increase in both
density and total neuron number in the GPi as compared with
NC. An opposite 40% decrease in neuron number and density
was observed in the GPe of the same subjects (Table 1). A
3.2 in TS and NC, respectively. These changes were remarkably
consistent in our sample, such that both the increase in neuron
number in the GPi and the decrease in neuron number and
density in the GPe and Cd detected in TS versus NC brains were
statistically significant despite the small number of subjects (P ?
0.025, Mann–Whitney U test). In contrast, no significant change
control (data not shown).
www.pnas.org?cgi?doi?10.1073?pnas.0502624102 Kalanithi et al.
No change in size or shape of the neuronal cell bodies was
detected. The total number, density, and morphology of glial
cells did not differ among TS or control brains in either the GPe
or GPi. No differences in the volume of the GPe or the GPi were
detected among the subjects (Table 1).
To ascertain the phenotype of neuronal cells in both the GP
and the striatum, we immunostained sections for PV, which is
normally expressed by the majority of GP neurons as well as
in a subset of striatal interneurons, and assessed the density of
these cells in various regions of the BG by unbiased sampling
methods. The density of PV? cells was higher by 122% in the
GPi of the subjects with TS as compared with NC. In contrast,
the brains from TS subjects exhibited a lower density of PV?
neurons in all other BG regions assessed. This difference was
strongest in the Cd (51% lower) and smallest in the Pt (37%),
whereas the difference in the GPe (23%) was not statistically
significant (Fig. 1).
PV? cells in both pallidal regions are large, multipolar
projection neurons. This morphology did not appear to differ in
the brains of TS subjects (Fig. 2). In the striatum, PV? cells are
much smaller and display an extensive dendritic?axonal tree.
Again, no apparent difference in PV cell morphology was
present in the Cd and Pt of brains of TS cases as compared with
NCs (see Fig. 3 and data not shown). However, the density of PV
immunoreactivity in the neuropil (presumably reflecting the
dendrites and axons of these cells) was clearly decreased in the
Cd and Pt of TS cases as compared with corresponding regions
of NCs (Fig. 3 and data not shown).
To estimate the total number of PV? cells, we multiplied the
PV? cell density in the GPi and GPe of each subject by the
corresponding volumes (Table 3, which is published as support-
ing information on the PNAS web site). The results suggest that
the number of PV? neurons was 31% smaller in the GPe of
patients with TS, whereas it was 129% greater in the GPi of
patients with TS in comparison with NC. These changes are
virtually identical to those noted for PV? neuron density in
these regions, as total volumes were not significantly different
between the groups. For the Pt and the Cd, we could not obtain
total volumes by stereological analyses with the available tissue.
To have an estimate of total number of PV? neurons in these
regions, we used an extensive neuroimaging data set of 130 NCs
and 154 TS subjects (10) and used their average Cd and Pt
volumes after correcting for shrinkage caused by tissue process-
ing as explained in the Table 3 legend. The results suggest that
the numbers of PV? neurons were 54% and 40% fewer in the
Cd and Pt, respectively, of patients with TS in comparison with
NC (Table 3). These differences in total PV? cell number
between NC and TS individuals are very similar to the difference
in PV? cell density reported in Fig. 1. Interestingly, the total
number of PV? neurons in the BG was decreased in TS by only
14.0%, indicating that this group of TS patients differs from the
NC with respect to the distribution and not the overall number
of PV neurons in the BG.
By subtracting the total PV neuron number from the total
neuron number, we determined changes in PV-negative (PV?)
fewer PV? cells: the GPi of control brains had 1.7 ? 105PV?
Table 1. Differences in total cell number in the BG of NC and TS
GPGroup Volume, mm3
No. of neurons,
103cells per mm3
No. of glial
Glial density, 103
cells per mm3
491.4 ? 41.1
442.4 ? 10.5
223.2 ? 32.8
230.0 ? 25.7
1,695 ? 304
910 ? 105*
540 ? 49
905 ? 108*
3.41 ? 0.36
2.05 ? 0.20*
2.54 ? 0.46
4.00 ? 0.54
177 ? 21.4
158 ? 6.2
63.5 ? 2.1
72.4 ? 9.1
372 ? 74.6
358 ? 17.6
297 ? 42.5
319 ? 39.4
Morphometric analysis of BG subregions after cresyl violet staining in NC and TS patients. Values represent
group means ? SEM. The volume, total cell number, and density were assessed in serial sections by stereological
analyses using STEREOINVESTIGATOR, as described in the text.
*TS versus NC statistically different; P ? 0.025, Mann–Whitney U test.
using stereological analyses. Each small square represents a single subject.
Differences in PV neuronal density in the Cd, Pt, and GPi of TS patients were
statistically significant (P ? 0.025, Mann–Whitney U test).
Unbiased estimates of PV cell density in three TS and three NC brains
Kalanithi et al.PNAS ?
September 13, 2005 ?
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neurons, whereas the GPi from subjects with TS had less than
half that number, 5.8 ? 104. In the GPe of the subjects with TS
there were also fewer PV? neurons, 2.0 ? 105compared with
6.7 ? 105. The differences were roughly similar, 59% in the GPi
and 69% in the GPe.
To assess whether functional neurotransmitter abnormalities
may be present in TS, we performed immunostaining for SP and
met-enk, neuropeptides contained within striatal MSN of the
direct and indirect pathways, respectively. Consistent with pre-
vious data (21), SP-positive ‘‘woolly fibers’’ were concentrated in
the GPi, suggesting that they represented terminals of the direct
pathway; in contrast, enk-positive woolly fibers were greatly
enriched in the GPe although a subset reached the GPi, consis-
tent with being terminals of the indirect pathway. No difference
and controls was apparent. A close comparison of sections for
the TS and NC cases under examination showed no differences
in the apparent density or distribution of fibers immunoreactive
for these neuropeptides between NC and cases of TS (data not
No correlation was found between changes in PV- or cresyl
violet-stained cell densities and neuroleptic intake or postmor-
tem interval. Although TS patients had comorbid diagnoses, TS
was the only diagnosis in common among them. Although two
patients had positive family histories of TS, and one had a
perinatal hypoxic event, no correlation existed between mor-
TS brain were at risk for secondary complications because of the
agonal history (Table 2); however, no correlations were found
between this history and PV? neuron number or density.
The data presented above provide quantitative cellular charac-
terization of specific cell types within the BG of subjects with
severe and persistent TS. Our data demonstrate a consistent and
profound PV? neuronal imbalance in the BG of the three
patients examined. The striatum and the GPe have fewer PV?
neurons, whereas the GPi has substantially more. There are
parallel changes in total neuron number in the GPe and GPi
(46% decrease in GPe and 68% increase in GPi of TS as
compared with NC) and the changes in the PV? neuron
distribution partially account for these changes in total neuron
number. Confirming earlier studies in TS (22), no changes were
SP-immunoreactive direct or the enk-positive indirect pathways.
Taking the BG as a whole, there is a small (14%) nonsignif-
icant decrease in PV? neurons in TS (Table 3), whereas the
distribution of PV cells is strikingly altered in TS, with far more
PV? cells in the GPi and substantially fewer in other regions of
the BG, particularly the Cd (Fig. 4). With unbiased sampling
methods, 51% and 37% statistically significant decreases in PV
neuron density were detected in the Cd and Pt, respectively, of
TS patients. In contrast, in the GPi in the same subjects, both
density and number of PV? neurons were more than twice as
large in TS as compared with NC (Fig. 4). We think that the
above changes in PV? neuron distribution represent altered
number of cells and that it is unlikely that they reflect changes
in PV expression for two reasons. First, the PV? cell changes in
the GPi are paralleled by changes in total neuron number, as
assessed by cresyl violet staining; and second, it is improbable
that variables that might affect the expression or the immuno-
detection of PV would manifest in opposite ways among closely
spaced BG subregions. For example, the increase in cresyl
violet-stained neurons in the GPi from subjects with TS is fully
accounted for by the increase in PV? cells in this nucleus.
However, PV expression has been shown to be sensitive to
neurotransmitter changes, such as those elicited by cocaine
exposure (23), and we cannot completely rule out that complex
changes in both the number of neurons and PV expression occur
Our data also suggest that other neuronal populations besides
PV neurons may be affected in TS. In the Cd, the observed 27%
sections from the GPi of NC (A and C) and TS (B and D) are shown. Note the corresponding increase in the density of total neurons and PV-containing neurons
in the GPi of TS brains. V indicates blood vessels; arrows indicate neuronal cell bodies. (Scale bar: 100 ?m.)
www.pnas.org?cgi?doi?10.1073?pnas.0502624102 Kalanithi et al.
decrease in total neuron density cannot be accounted for by the
decrease in density of PV? interneurons, because PV? cells
represent only ?0.5% of the total neuron number in this region.
We hypothesize that the Cd of TS individuals could have a
decrease in other types of interneurons, as MSN projections in
the GP appear to be qualitatively unchanged. However, we must
await unbiased estimations of the total number of MSNs by using
appropriate markers to definitively settle the question of
whether MSNs are altered in TS. The decrease in PV? and PV?
neurons in the Cd is consistent with the small (?5%) decreases
in Cd volume that is present in both children and adults with TS
later in life (25). In NC, PV? neurons are the majority of
neurons in the GP, representing 60% and 68% of the total
neurons in GPe and GPi, respectively. In TS, a 46% statistically
significant decrease in total neuron number was observed in the
GPe, whereas the decrease in number of PV? cells was not
statistically significant in this region, suggesting a change in PV?
cells. Similarly, there appears to be a decrease in non-PV?
neurons in the GPi from subjects with TS, because the propor-
tion of PV? is much higher in the GPi of TS patients. These data
suggest that parallel changes may be occurring in a PV?
population of cells in this disorder. Future studies will have to
clarify the nature of these PV? neurons that in the striatum and
the pallidum are changed in TS. Although PV’s calcium binding
ability has been hypothesized to affect refractory hyperpolar-
ization, and therefore, firing frequency (26), whether there are
any functional differences between PV? and PV? neurons in
the GP is currently unclear.
circuit-based models of BG pathophysiology (27) and emerging
oscillation models (28, 29). In the traditional circuit, cortical
input excites PV? interneurons (30), which, in turn, powerfully
inhibit MSNs (5), maintaining the characteristic low activity of
the striatum. The loss of PV? interneurons (and, possibly, other
interneurons) in this circuit should lead to MSN hyperactivity
and elicit hyperkinesis. Recent recordings from monkeys indi-
cate that, at rest, striatal activity is synchronized at a beta-band
oscillatory frequency (15–30 Hz); during movement, certain
localized striatal sites desynchronize, resynchronizing after the
movement ceases (31). Other in vivo recordings suggest that
high-amplitude spindle and theta-band oscillatory frequencies
are prominent in the striatum of awake rats (32). Because PV?
striatal interneurons form an electrically coupled inhibitory
network that can entrain large assemblies of neurons (4–6),
PV? interneurons may play a critical role in the synchrony of
striatal oscillations and their modulation by the cerebral cortex
and thalamus (32). Hence a deficit in striatal PV? interneurons
may lead to inappropriate desynchrony of small populations of
striatal MSNs, which presumably results in tic-like behavior.
The potential role of PV? interneuron deficits in promoting
tic-like behavior finds confirmation in a hamster model of
idiopathic paroxysmal dystonia. The dtszhamster phenotype
shares many features with TS, including facial contortions,
co-contractions of opposing muscle groups (33), and a similar
age-dependent time course (34). Remarkably, the dtszhamster
also has a strikingly similar reduction in striatal PV? interneu-
ron number (41% decrease, compared with 54% decrease in Cd
of TS) (35). These emerging lines of data strongly support a role
for striatal PV? interneuron deficit in TS and other hyperkinetic
Intriguingly, the changes we observed in the GP do not easily
fit with classic models of the BG. Microinjection studies in
monkeys provide evidence for the involvement of projections
from the ventral Cd to the GPe in tic-like stereotypies (36, 37).
Further, the GPe sends direct projections to all other nuclei of
the BG and the thalamus and appears to serve as a pacemaker
of the BG (38). Hence the overall 46% decrease in total neuron
number in the GPe in our cohort of severe cases of TS may
implicate a profound change on overall BG oscillatory activity.
However, beyond our data, support for a GPe defect in tic
production remains limited.
Because the GPi is a major output of the BG and prominently
involved in processing sensorimotor information, it is likely to
figure centrally in kinetic disorder pathophysiology. Recent
models of BG function have suggested that altered patterns of
GPi discharge might be important in movement disorders (39).
Recordings from the GPi analogue in the dtszhamster reveal
patients. PV immunostaining in the dorsolateral portion of the Cd nucleus at
the level of the GP of NC (A and C) or TS (B and D). C and D are magnifications
?m, A and B; 50 ?m, C and D.)
Decrease in the density of PV-containing neurons in the Cd of TS
uals. Differences in total PV cell number in the entire BG was not statistically
significant between NC and TS, whereas distribution among different BG
regions was significantly altered. Note that although NC subjects had a
relatively even PV neuron distribution between striatum and GP, in TS there
is an increased proportion of PV neurons in the GP, caused by a decrease in
Cd?Pt and an increase in the GPi.
Altered distribution of PV? neurons in the BG of NC and TS individ-
Kalanithi et al.PNAS ?
September 13, 2005 ?
vol. 102 ?
no. 37 ?
symptoms disappear (40). Similarly irregular firing patterns have Download full-text
also been recorded in the GPi of humans either with severe
dystonia or severe TS (41).§§Although it is possible that these
firing patterns may be a downstream effect of striatal abnor-
malities in these conditions, they highlight the possible signifi-
cance of an intrinsic GPi defect. An increase of ?100% of PV?
GPi neurons in TS, increasing by 68% the overall GPi neuronal
population, may very well generate aberrant firing patterns,
which in turn may elicit secondary effects at the thalamic level.
For example, excessive inhibitory input may provoke rebound
excitation in thalamic relay nuclei or alter the pace of their
oscillatory activity (42).
Although other cell populations may have also been af-
fected, these major changes in PV? neuron distribution in the
BG can be expected to have a dramatic effect in cortico-
striato-thalamo-cortical functioning and may suggest possible
etiologies for TS. A hypothesis consistent with an altered
distribution in the absence of major changes in number of PV
cells would be a defect in PV? cell migration. During embry-
ogenesis, PV? interneurons tangentially migrate from the
medial ganglionic eminence (MGE), the precursor of the GP,
to the lateral ganglionic eminence, the precursor to the
striatum, as well as to the cortex and hippocampus. Future
studies should investigate whether other PV? neuron popu-
lations are similarly affected or whether these changes are
specific to the BG. The Nkx2.1 gene, which is necessary for the
specification of the MGE, is also required for the specification
of these interneurons of the striatum, whereas the Dlx1?Dlx2
and Lhx6 genes are required for their migration (43, 44). Thus,
a possible explanation for our data is that abnormalities in
these or related genes during development cause altered
migration of neurons arising from the MGE in TS patients. It
is interesting to note that haploinsufficiency for Nkx2.1 has
been associated with a mild form of dyskinesia and chorea in
humans (45, 46). In addition to genetic factors, environmental
factors may play an important role. For example, one possible
environmental culprit might be perinatal hypoxia, as these
PV? striatal interneurons appear to be sensitive to ischemic
insults (47). Our data suggest that future gene–environmental
interaction studies focused on the development of PV?
GABAergic cells may be a worthwhile line of inquiry for TS.
We thank Dr. Francine Benes and the personnel at the Harvard Brain
Tissue Resource Center for help in tissue preservation and storage and
suggestions in tissue processing techniques; and Drs. Brian Ciliax and
Neal Swerdlow and other members of the Tourette Syndrome Associ-
ation Scientific Advisory Board and Tissue Committee for organizing
tissue collection and providing essential suggestions and encouragement.
This work was supported by National Institutes of Health Grant P01
MH49351, the Tourette Syndrome Association, and Yale University
School of Medicine.
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