Download Early Microglial Activation Precedes Neuronal Loss in Mouse Model of Progressive

J Neuropathol Exp Neurol
Copyright Ó 2012 by the American Association of Neuropathologists, Inc.
Vol. 71, No. 1
January 2012
pp. 40Y53
ORIGINAL ARTICLE
Early Microglial Activation Precedes Neuronal Loss in
the Brain of the Cstbj/j Mouse Model of Progressive
Myoclonus Epilepsy, EPM1
Saara Tegelberg, MSc, Outi Kopra, PhD, Tarja Joensuu, PhD, Jonathan D. Cooper, PhD,
and Anna-Elina Lehesjoki, MD, PhD
Abstract
Progressive myoclonus epilepsy of Unverricht-Lundborg type
(EPM1) is a hereditary neurodegenerative disorder caused by mutations in the cystatin B (CSTB) gene encoding an inhibitor of cysteine
proteases. Here, we provide the first detailed description of the onset
and progression of pathologic changes in the CNS of Cstb-deficient
(Cstbj/j) mice. Our data reveal early and localized glial activation in
brain regions where neuron loss subsequently occurs. These changes
are most pronounced in the thalamocortical system, with neuron loss
occurring first within the cortex and only subsequently in the corresponding thalamic relay nucleus. Microglial activation precedes the
emergence of myoclonia and is followed by successive astrocytosis
and selective neuron loss. Neuron loss was not detected in thalamic
relay nuclei that displayed no glial activation. Microglia showed
morphologic changes during disease progression from that of phagocytic brain macrophages in young animals to having thickened
branched processes in older animals. These novel data on the timing
of pathologic events in the CSTB-deficient brain highlight the potential role of glial activation at the initial stages of the disease. Determining the precise sequence of the neurodegenerative events in Cstbj/j
mouse brains will lay the basis for understanding the pathophysiology
of EPM1.
Key Words: Astrocyte activation, Cerebellum, Cerebral cortex,
Cerebrum, Microglia, Myoclonus epilepsy, Neurodegeneration, Posterior thalamic nuclei, Somatosensory cortex.
From the Folkhälsan Institute of Genetics (ST, OK, TJ, A-EL); Haartman
Institute, Department of Medical Genetics and Research Program’s Unit,
Molecular Medicine, and Neuroscience Center (ST, OK, TJ, A-EL), University of Helsinki, Helsinki, Finland; and Pediatric Storage Disorders
Laboratory (JDC), Department of Neuroscience, MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King’s College, London,
United Kingdom.
Send correspondence and reprint requests to: Anna-Elina Lehesjoki, MD, PhD,
Folkhälsan Institute of Genetics and Neuroscience Center, University of
Helsinki, Helsinki, Finland; E-mail: [email protected]
This study was funded by the Folkhälsan Research Foundation, the Sigrid
Jusélius Foundation, and the Academy of Finland, as well as grants to
Saara Tegelberg from the European Molecular Biology Organization, the
Finnish Cultural Foundation, and the University of Helsinki Funds, and
grants to Jonathan D. Cooper from the Batten Disease Support and Research
Association (United States), the Batten Disease Family Association (United
Kingdom), and The Natalie Fund.
40
INTRODUCTION
The progressive myoclonus epilepsies (PMEs) are a
heterogeneous group of inherited disorders characterized by
myoclonus, epilepsy, and progressive neuronal degeneration
(1, 2). The pathogenesis of the PMEs is poorly understood,
and these disorders are generally refractory to treatment. The
most common single cause of PME is Unverricht-Lundborg
disease (EPM1, OMIM no. 254800), an autosomal recessively
inherited disorder with an onset at 6 to 16 years, usually with
stimulus-sensitive myoclonus or tonic-clonic seizures. Other
neurologic symptoms, including ataxia and dysarthria, appear
as the disease progresses but cognition is essentially preserved
(3Y5). Symptomatic pharmacologic and rehabilitative management including psychosocial support are the mainstay of EPM1
patient care (5).
EPM1 is caused by loss-of-function mutations in the
cystatin B gene (CSTB; OMIM no. 601145) encoding an
inhibitor of cysteine proteases (6Y8). These proteases include
lysosomal cysteine cathepsins, and based on studies on EPM1
patient lymphoblastoid cells, it has been suggested that their
increased activity is related to EPM1 pathogenesis (9). However,
the precise role of CSTB and the mechanisms by which its loss
leads to EPM1 remain poorly understood.
A mouse model for EPM1 has been generated by targeted
disruption of the mouse Cstb gene (Cstbj/j mice) (10). These
mice recapitulate the key clinical features of EPM1, including
appearance of myoclonic seizures at 1 month of age and progressive ataxia (10). Their major neuropathologic phenotype is
a severe loss of cerebellar granule neurons due to apoptotic
death. Widespread gliosis accompanied by increased expression of apoptosis- and glial activation-related genes have been
also reported in aged Cstbj/j mice (11Y13). Deficiency of
Cstb on either seizure-prone or seizure-resistant backgrounds
displays similar neuropathologic changes, indicating that these
events are independent of seizures (10, 12). Our recent findings from cultured cerebellar granule neurons from Cstbj/j
mice point to an elevated susceptibility to oxidative stress that
is at least partially mediated through elevated cathepsin B
activity (14). Furthermore, mice deficient for both CSTB and
cathepsin B show significantly less cerebellar apoptosis but no
changes in their seizure or ataxia phenotypes (15). These results suggest that, although cathepsin B seems to contribute to
the pathogenesis of EPM1, cathepsin inhibition is not the sole
J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
Glial Activation and Neuron Loss in Mouse EPM1
MATERIALS AND METHODS
Mice
FIGURE 1. Schematic diagram of the cortical regions and thalamic nuclei analyzed in Cstbj/j mice. Measurements of cortical thickness were made in the primary somatosensory (S1),
somatosensory barrel field (S1BF), primary visual (V1), primary
motor (M1), and lateral entorhinal (LEnt) cortices, together
with optical fractionator estimates of the number of neurons in
laminae IV, V, and VI of S1 and V1. The same unbiased stereological method was used to investigate the survival of thalamic relay neurons in the posterior thalamic nuclear group
(Po) and dorsal lateral geniculate nucleus (LGNd) at different
stages of disease progression.
function of CSTB and that CSTB has a wider role in maintaining normal neuronal function and architecture.
In this study, we have systematically investigated the
onset and progression of neuron loss and glial activation in the
Cstbj/j mouse brain at different stages of the disease. Our
results show early microglial activation followed by astrocytosis and neuron loss, suggesting a central role for microglia in
the pathogenesis of EPM1.
The cystatin BYdeficient (Cstbj/j) mice used in this
study were obtained from The Jackson Laboratory (Bar Harbor,
ME; 129-Cstbtm1Rm/J; stock no. 003486) (10). Wild-type offspring from heterozygous matings were used as controls.
Animal research protocols were approved by the Animal Ethics
Committee of the State Provincial Office of Southern Finland
(decision no. STU376A, no. STH660A and no. STH524A). All
animals used were males.
Histologic Processing
For histologic analysis, Cstbj/j and control mice (n = 6
per genotype and age, except n = 4 for P14) were killed at
2 weeks (P14) and at 1, 2, 4, and 6 months of age. Brains
were removed, bisected along the midline, and immersion
fixed in 4% paraformaldehyde in 0.1 mol/L sodium phosphate buffer pH 7.4 for at least 1 week before cryoprotection
in 30% sucrose in 50 mmol/L Tris-buffered saline (TBS) containing 0.05% sodium azide. Forty-micrometer frozen coronal
sections were cut through the cerebrum with Microm HM430
Sliding Microtome (Microm International, Walldorf, Denmark),
and cerebella were cut sagittally. Sections were collected, 1 per
well, into 96-well plates containing cryoprotectant solution
(TBS/30% ethylene glycol/15% sucrose/0.05% sodium azide),
as described (16, 17), and stored at j80-C before histologic
processing. All histologic processing and subsequent analyses
were done using all these samples unless otherwise stated and
were performed with no prior knowledge of genotype.
FIGURE 2. Progressive cerebellar atrophy in Cstbj/j mice. (A) Nissl-stained sections reveal the overall atrophy of cerebellum during
disease progression. There was no difference detected between Cstbj/j and control mice at 1 month of age, but the cerebellum
was markedly atrophied in 6-month-old Cstbj/j mice. Scale bar = 1 mm. (B) Cavalieri estimates of total cerebellar volume of
Cstbj/j mice compared with control mice. Data are expressed as mean regional volume in cubic micrometers T SEM. *, p e 0.05;
**, p e 0.01; ***, p e 0.001.
Ó 2012 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
41
Tegelberg et al
42
J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
Ó 2012 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
Nissl Staining
To provide direct visualization of neuronal morphology,
every sixth section was Nissl stained as previously described
(16, 17). Sections were mounted onto gelatin-chrome alumY
coated slides, air-dried overnight, and incubated for 45 minutes
at 60-C in a solution of 0.05% Cresyl fast violet (VWR, Dorset,
UK)/0.05% acetic acid, rinsed in distilled water, and differentiated through a graded series of alcohols before clearing in
xylene (VWR) and coverslipped with DPX (VWR).
Regional Volume Measurements
Unbiased Cavalieri estimates of the volume of cerebral
cortex, cerebellum, and cerebellar layers (molecular and granular cell layer, white matter) were made in Nissl-stained sections using StereoInvestigator software (Microbrightfield, Inc,
Williston, VT), as previously described (16, 17). A sampling
grid with appropriate spacing (200 Km for cerebral cortex and
150 Km for cerebellar layers) was superimposed over Nisslstained sections, and the number of points covering the relevant
areas was counted using a 1.25 objective. Regional volumes
were expressed in cubic millimeters, and mean volume was
calculated for control and Cstbj/j mice at each age.
Cerebral Cortex Thickness Measurements
To explore the extent of cortical atrophy, cortical
thickness measurements were made in Nissl-stained sections
through cortical regions that serve different functional roles,
including the primary somatosensory (S1), primary somatosensory barrel field (S1BF), primary visual (V1), primary motor
(M1), and lateral entorhinal (LEnt) cortices defined according to
the landmarks described by Paxinos and Franklin (18) (Fig. 1).
Ten perpendicular lines extending from white matter to pial
surface were placed on 4 consecutive sections using a 5
objective. Lines were measured using either StereoInvestigator
or Fiji (ImageJ 1.44a) software. Results were expressed as the
mean cortical thickness in micrometer per region for Cstbj/j
and control mice.
Counts of Neuronal Number and Volume
To examine neuronal survival within individual thalamic nuclei and their target cortical regions, we used StereoInvestigator software to obtain unbiased optical fractionator
estimates of neuronal numbers and nucleator estimates of neuronal volumes from Nissl-stained sections. Estimates of neuronal number were obtained in posterior thalamic nucleus (Po)
and dorsal lateral geniculate nucleus (LGNd), and their target
areas S1 and V1 (laminae IV, V, and VI), respectively. Estimates for neuronal volume were obtained only for neurons
in Po and lamina V of S1 of P14 and 6-month-old animals.
Glial Activation and Neuron Loss in Mouse EPM1
Measurements were performed as previously described (19),
with random starting section followed by every 6th (Po, LGNd,
and V1) or every 12th (S1) section thereafter. The boundaries
of selected areas were defined by reference to landmarks in
Paxinos and Franklin (18). Only neurons with clearly identifiable nucleolus were sampled, and all counts were carried out
using a 100 oil objective (NA 1.4). The following sampling
scheme was applied to the regions of interest: grid area for
Po 30,625 Km2, frame area 2400 Km2; grid area for LGNd
15,625 Km2, frame area 2400 Km2; grid area for S1 and
laminae IV and V 50,625 Km2, frame area 2400 Km2; grid area
for S1 and lamina VI 50,625 Km2, frame area 1000 Km2; grid
area for V1 and laminae IV, V, and VI 30,625 Km2, frame area
1000 Km2.
Immunohistochemistry
To assess the extent of glial activation, adjacent 1-in-6
series of free-floating sections were immunostained, as described
previously (17) for detection of astrocytic (rabbit antiYglial fibrillary acidic protein [GFAP], 1:5000; DAKO, Cambridge, UK)
and microglial (rat anti-F4/80, 1:100; Serotec, Oxford, UK)
markers. Briefly, sections were incubated for 15 minutes in 1%
H2O2 in TBS to quench endogenous peroxidase activity, rinsed
in TBS, and incubated for 40 minutes in TBS/0.3% Triton
X-100 (TBS-T)/15% normal serum. Sections were incubated
overnight at 4-C in primary antibody diluted with 10% normal
serum in TBS-T and subsequently rinsed in TBS and incubated for 2 hours in biotinylated secondary antiserum (Vector
Laboratories, Peterborough, UK) diluted with 10% normal
serum in TBS-T. After rinsing with TBS, sections were incubated for 2 hours in Vectastain avidin-biotin-peroxidase complex (Vectastain Elite APC kit; Vector Laboratories), diluted
with 10% normal serum in TBS-T, and rinsed again in TBS. Immunoreactivity was visualized by a standard diaminobenzidineY
hydrogen peroxide reaction (Sigma, Dorset, UK), after which
the sections were transferred to excess ice-cold TBS, mounted
onto gelatine-chrome alumYcoated SuperFrost microscope
slides (VWR), air-dried overnight, and passed through a graded
series of alcohols before clearing in xylene and coverslipping
with DPX mounting medium (VWR).
Statistical Analysis
The statistical significance of differences between genotypes of all quantitative data was assessed using 1-way analysis
of variance with Bonferroni correction (GraphPad Prism 5.0c;
GraphPad Software, La Jolla, CA) with p e 0.05 considered
as statistically significant. The mean coefficient of error for
all individual optical fractionator and Cavalieri estimates was
calculated according to the method of Gundersen and Jensen
(20) and was less than 0.08 in all of the analyses.
FIGURE 3. Progressive atrophy of cerebral cortex in Cstbj/j mice. (A) Cavalieri estimates of the volume of cerebral cortex in Cstbj/j
mice compared with control (ctrl) mice. Progressive cortical atrophy was seen in Cstbj/j mice from 2 months of age onward. (B)
Primary somatosensory cortex (S1) in Nissl-stained sections from Cstbj/j and ctrl mice showed progressive atrophy in Cstbj/j mice,
which occurred to a similar extent across all cortical laminae. Scale bars = 100 Km. (CYG) Cortical thickness measurements reveal
progressive thinning of the cortex, at 2 months in S1, S1BF, and V1; at 4 months in M1; and at 6 months in LEnt. Data are expressed
as mean regional volume in cubic micrometers T SEM (A) or mean thickness in micrometers T SEM (CYG). One-way analysis of
variance: *, p e 0.05; **, p e 0.01, ***, p e 0.001.
Ó 2012 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
43
Tegelberg et al
44
J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
Ó 2012 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
Image Analysis
All microphotographs were obtained as color images
with AxioCam HRc (Zeiss, Oberkochen, Germany) and converted to black-and-white images using Adobe Photoshop
CS4 software (Adobe Systems, Inc, San Jose, CA). Images of
F4/80 staining were converted using blue filter to enhance the
visualization of the staining.
RESULTS
Progressive Atrophy and Cortical Thinning in
Cstbj/j Mouse Brain
Previous studies have reported a progressive loss of
cerebellar granule cells in Cstbj/j mice (10). Our analyses
also show marked progressive loss of cerebellar volume in
Cstbj/j versus control mice (Fig. 2A). Cavalieri estimates of
regional volumes revealed significant and progressive atrophy
of the cerebellum of Cstbj/j mice from 2 months of age
onward, reaching almost 50% reduction in cerebellar volume
by the age of 6 months (Fig. 2B). No significant differences
in the rate of volume loss between molecular layer, cerebellar
granule cell layer, and white matter were observed (data not
shown).
The occurrence of pathologic changes in the cerebrum
of Cstbj/j mice, particularly in young animals, has not yet
been systematically analyzed. Our data reveal progressive
atrophy of the cerebral cortex of Cstbj/j mice from 2 months
of age onward (Figs. 3A, B). To assess whether any subregions of the cortex may be preferentially affected, we carried out thickness measurements of different cortical regions
as representative areas that receive information of different
sensory modalities (Figs. 3CYG). Significant and progressive
thinning was observed in S1, S1BF, and V1 from 2 months
of age onward in Cstbj/j compared to control mice. In M1,
the difference was significant at 4 months, and in LEnt, the
difference was significant only at 6 months of age. Cortical
thinning at 2 months of age coincided with the onset of atrophy in the Cstbj/j brains, but no differences were seen in
rate of thinning between individual cortical laminae (Fig. 3B,
numerical data not shown).
Microglial Activation
To examine glial activation in Cstbj/j mice, we surveyed the distribution of cells immunoreactive for the microglial marker F4/80 in the brains of Cstbj/j and control mice
at different ages (Fig. 4). Intensely stained F4/80 immunoreactive microglia were already apparent in restricted regions
Glial Activation and Neuron Loss in Mouse EPM1
of the Cstbj/j CNS at P14 and became more widespread and
intense with disease progression (Table). The most pronounced
microglial activation in Cstbj/j brains was seen in midline
and posterior thalamic nuclei, substantia nigra, claustrum, amygdaloid nuclei, and cingulate cortex (Figs. 4AYE). The hippocampal formation of Cstbj/j mice contained considerable
amounts of activated microglia at P14, but at later stages
(with the exception of the hippocampal dentate gyrus, which
displayed activated microglia throughout disease progression),
it was almost completely devoid of activated microglia (Fig. 4F).
Throughout the cerebral cortex, many more intensely stained
microglia were present in the most dorsal (I, II) and ventral
laminae (VI) than in intervening laminae III-V (Fig. 4G). In the
cerebellum of Cstbj/j mice, activated microglia were evenly
distributed with no consistent differences between individual
layers (white matter, granule cell layer, or molecular layer) or
individual lobules (data not shown). In contrast to gray matter,
white matter of Cstbj/j mice only displayed microglial activation at 6 months of age.
A striking feature of Cstbj/j mice was the change in
morphology of microglia during disease progression (Fig. 4H).
At P14, microglial cells in Cstbj/j brain show the classic,
activated phenotype with thickened processes and round soma,
whereas in control brains, microglia were much less darkly
stained with many thin ramified processes. In 1-month-old
Cstbj/j mice, most microglia displayed intense F4/80 immunoreactivity with a dramatically enlarged cell soma and
numerous short tufted processes, with a subset exhibiting an
amoeboid brain macrophage-like morphology. The occurrence
of darkly stained F4/80Yimmunoreactive microglia in Cstbj/j
mice declined from 2 to 4 months of age, with many cells
displaying more branched processes. At 6 months, microglia
in Cstbj/j mice displayed much less intense F4/80 staining
but frequently displayed a characteristic morphology with a
large cell soma and thick processes that branched several times.
In contrast to the Cstbj/j mice, in the control brains,
F4/80 staining was barely detectable above background, with
a network of faintly stained ramified processes evident in the
neuropil. This was most evident at P14, in the same regions as
the most intensive F4/80 immunoreactivity in P14 Cstbj/j
brains. Control brains displayed only sporadic activated microglia from 1 month onward (Fig. 4G).
Astrocytosis
In contrast to microglial staining, GFAP immunoreactivity revealed no clear differences between Cstbj/j and control
mice at P14 (Fig. 5A). However, at 1 month of age, there was a
FIGURE 4. Progressive microglial activation in Cstbj/j brain. (AYH) Patchy immunohistochemical staining for the microglial
marker F4/80 revealed the progressive activation of microglia in selected brain regions of Cstbj/j mice but not in age-matched
controls (also see the Table). (A) Midline and posterior thalamic nuclei, P14. (B) Substantia nigra, P14. (C) Claustrum, 1 month.
(D) Nuclei of the amygdala, 4 months. (E) Cingulate cortex, 6 months. (F) The hippocampal formation displayed no microgliosis
even at 6 months of age, except for the dentate gyrus (arrowhead). (G) Laminar-specific microglial activation in primary somatosensory cortex (S1) of Cstbj/j, but not control (ctrl) mice. (H) Progressive changes in microglial morphology in Cstbj/j during
disease progression. Darkly stained activated microglia with a brain macrophage-like morphology could be seen in Cstbj/j mice at
P14 and were more pronounced at 1 month of age, whereas age-matched controls show much paler-stained microglia with many
thin ramified processes. By 6 months of age, microglia in Cstbj/j mice lose their activated phenotype with a pronounced decline in
the intensity of F4/80 staining, but displaying distinctive thickened and branched processes. Cells are from the midline thalamus.
Arrowheads indicate the regions of interest. Scale bars = (AYF) 200 Km; (G, H) 100 Km.
Ó 2012 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
45
J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
Tegelberg et al
TABLE. Distribution of F4/80-Immunoreactive Microglia
Cstbj/j
ctrl
Brain Region
Cortex
Prelimbic
Primary motor
Secondary motor
Infralimbic
Dorsal peduncular
Lateral orbital
Primary sensory
Secondary sensory
Somatosensory barrel field
Agranular insular
Granular insular
Piriform
Cingulate
Auditory
Primary visual
Secondary visual
Retrosplenal agranular
Retrosplenal granular
Lateral entorhinal
Ectorhinal
Perirhinal
Septal region
Medial septum
Lateral septum
Diagonal band vertical
Diagonal band horizontal
Basal ganglia
Caudate putamen
Substantia innominata
Interstitial nucleus of posterior limb of anterior commissure
Bed nucleusVstria terminalis
Nucleus accumbens
Ventral pallidum
Claustrum
Dorsal endopiriform claustrum
Basal nucleus of Meynert
Lateral accumbens shell
Anterior olfactory area
Globus pallidus
Olfactory tubercle
Amygdala
Central nucleus
Medial nucleus
Basomedial nucleus
Basolateral nucleus
Posteromedial cortical amygdaloid nucleus
Anterior cortical amygdaloid area
Hippocampus
CA1
CA2
CA3
Dentate gyrus
Lacunosum moleculare layer
Subiculum
Hypothalamus
Lateral nucleus
Posterior nucleus
Suprachiasmatic nucleus
Ventromedial nucleus
46
P14
P14
1 mo
2 mo
4 mo
6 mo
PrL
M1
M2
IL
DP
LO
S1
S2
S1BF
AI
GI
Pir
Cg
Au
V1
V2
RSA
RSG
LEnt
Ect
PRh
+
j
++
+
+
+
j
j
j
j
j
+
+
j
j
j
+
j
+
j
+
++
+
+++
++
+++
+++
+
+
+
+
+
++
++
++
++
++
+++
++
+++
+++
+++
++
+
++
++
j
+
++
+
++
++
++
+
++
+
+
+
+
++
+
++
++
++
++
+++
++
j
++
++
++
++
++
++
+
+++
++
++
++
+
++
++
+++
+++
++
++
+++
+++
++
++
++
++
+++
++
++
++
+++
++
+
++
+++
++
+
++
++
++
++
+++
+
+
+++
++
++
++
++
+++
++
++
j
+
++
+++
++
j
+
+
MS
LS
VDB
HDB
j
+++
j
j
++
+++
+
+
+
+
+
+
+
+
j
j
+
+
j
j
j
+++
++
j
CPu
SI
IPAC
BST
Acb
VP
Cl
DEn
B
LAcbSh
AO
GP
Tu
+
j
++
+
+
++
+
++
++
++
++
++
+
+++
++
+++
++
++
+++
+++
+++
+++
+++
+++
+++
+
++
j
++
j
+
++
+++
+
j
++
++
++
++
++
++
+++
j
+
++
+++
++
+
++
+
++
+
++
+++
+++
j
+
+++
+
++
++
++
+
+++
+
+
++
++
j
j
+++
++
++
+++
++
+
+++
j
Ce
Me
BM
BL
PMCo
ACo
+
+
+
j
j
+
++
++
+++
+
+++
+++
j
j
j
+
++
+
+
+
j
++
+
++
+
+
j
++
+
j
j
j
j
j
+
j
DG
LMol
S
++
++
++
++
++
+
+++
+++
+++
+++
+++
+++
j
j
+
++
+
j
j
j
+
+++
+
j
j
j
j
+++
j
j
j
j
j
+++
j
j
LH
PH
SCh
VMH
+
j
j
j
++
+++
j
+
j
++
j
j
j
++
j
j
++
++
j
j
+++
+++
++
j
Ó 2012 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
Glial Activation and Neuron Loss in Mouse EPM1
TABLE. Continued
Cstbj/j
ctrl
Brain Region
Hypothalamus
Arcuate nucleus
Optic tract
Thalamic nuclei
Anteromedial
Anteroventral
Anterodorsal
Habenular
Paraventricular
Ventromedial
Ventrolateral
Ventral posteromedial
Lateral posterior
Mediodorsal
Reticular
Reunions
Subthalamic
Zona incerta
Intermediodorsal thalamic
Pretectal
Parafascicular
Centromedial
Posterior thalamic
Rhomboid
Laterodorsal thalamic
Lateral geniculate (dorsal part)
Subparafascicular thalamic
Lateral geniculate (ventral part)
Medial geniculate (dorsal)
Medial geniculate (ventral)
Midbrain
Precommisural nucleus
Prerubral field
Deep mesencephalic nuclei
Substantia nigra reticular
Substantia nigra compacta
Ventral tegmental area
Interpeduncular nucleus
Deep gray layer of SC
Deep white layer of SC
Optic nerve layer of SC
Ventral periaqueductal gray
Cerebellum
Granule cell layer
Molecular layer
Deep cerebellar nuclei
White matter
White matter tracts
Fimbria
Corpus callosum
Internal capsule
Cerebral peduncle
Anterior commissure (anterior part)
Periventricular fiber system
Rostral interstitial nucleus of medial longitudinal fasciculus
P14
P14
1 mo
2 mo
4 mo
6 mo
Arc
opt
j
j
+
j
j
j
j
j
j
j
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j
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The distribution of F4/80-immunoreactive microglia in different brain regions of Cstbj/j mice from P14 to 6 months of age and control (ctrl) mice at P14.
j , no significant F4/80 immunoreactivity; +, few scattered F4/80-immunopositive cells; ++, considerable F4/80 immunoreactivity; +++, region full of densely packed
F4/80-immunopositive cells.
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47
Tegelberg et al
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J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
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J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
Glial Activation and Neuron Loss in Mouse EPM1
FIGURE 6. Activation of microglia and astrocytes in the posterior thalamic nuclei (Po) and the dorsal lateral geniculate nucleus
(LGNd). (A) Po of a Cstbj/j, but not a control (ctrl) mouse, shows microglial and astrocyte activation at 1 month of age. (B) LGNd
remained devoid of activated microglia and astrocytes even in a 6-month-old Cstbj/j mouse, as well as in control mouse. F4/80 was
used as a marker for microglia, and glial fibrillary acidic protein (GFAP) was used as a marker for astrocytes. Scale bars = 200 Km.
marked increase in intensely GFAP-immunopositive cells in
Cstbj/j brains. These activated astrocytes displayed hypertrophy with enlarged soma and thickened, branched processes
compared to the sporadic GFAP-positive astrocytes in controls
(Fig. 5B).
Glial fibrillary acidic protein immunoreactivity in
Cstbj/j brains became more pronounced and widespread
with increased age. The distribution of the astrocytes overlapped with that of microglia, that is, staining for GFAP
again was more intense in the most dorsal and ventral cortical laminae but was less evident in the intervening laminae
(Fig. 5A). Prominent GFAP staining was present within
midline and posterior thalamic nuclei, the substantia nigra,
claustrum, and amygdaloid nuclei (Figs. 5CYF) in the Cstbj/j
but not the control brains. However, the extent of GFAP immunoreactivity in hippocampal formation was similar in both
Cstbj/j and control mice (Figs. 5G, H).
Neuronal Loss
To determine whether there was any spatiotemporal
correlation between gliosis and neuron survival, we used
unbiased stereology to document the onset and progression of
neuronal loss in Nissl-stained sections through the thalamocortical system. On the basis of the pattern of glial activation,
we focused this analysis on the Po and the LGNd, together
with their corresponding cortical projection areas, S1 and V1,
respectively. Po was one of the first areas to display microglial
activation at P14 (Fig. 4A) and also displayed pronounced
astrocytosis from 1 month onward (Fig. 6A). In contrast, LGNd
remained devoid of activated microglia and astrocytes even in
6-month-old Cstbj/j mice (Fig. 6B).
Significant neuron loss was first evident within lamina
V of S1 in 1-month-old Cstbj/j mice (Figs. 7AYC) and
progressed to all laminae analyzed (Lam IV, V, and VI) by
2 months. In contrast, neuron loss within Po (the corresponding thalamic relay nucleus) did not become significant
in Cstbj/j mice until 2 months of age (Fig. 7D), occurring
after the onset of neuron loss within S1. Neuron loss within
these somatosensory nuclei of Cstbj/j mice occurred after
the onset of microglial activation and coincided with the
beginning of astrocyte activation within these pathways. In
contrast, neuron loss in the primary visual cortex (V1) of
Cstbj/j mice only became apparent at 6 months of age
(Figs. 7EYG), with no differences in the degree of neuronal
loss between individual laminae in V1. The visual relay
nucleus LGNd did not show significant neuron loss, even in
6-month-old Cstbj/j mice (Fig. 7H). No differences were
detected in neuronal volumes between Cstbj/j and control
mice at P14 or 6-month-old animals at Po and lamina V of S1
(data not shown).
Taken together, these data reveal that cortical neurons
are consistently lost in Cstbj/j mice before any loss of thalamic
FIGURE 5. Extensive and progressive astrocytosis in Cstbj/j mice. Immunostaining for the astrocyte marker glial fibrillary acidic
protein (GFAP) reveals progressive astrocytosis in selected brain regions of Cstbj/j mice but not in age-matched control (ctrl) mice.
(A) Laminar-specific astrocyte activation in primary somatosensory cortex (S1) of Cstbj/j but not ctrl mice. (B) Astrocytes show an
activated phenotype in 1-month-old Cstbj/j mouse midline thalamus but not in the ctrl. (C) Midline and posterior thalamic nuclei,
1 month. (D) Substantia nigra, 1 month. (E) Claustrum, 1 month. (F) Nuclei of amygdala, 4 months. (G, H) The hippocampal
formation displayed similar level of GFAP immunoreactivity in both Cstbj/j (G) and control mice (H) at 6 months of age.
Arrowheads indicate the regions of interest. Scale bars = (A, B) 100 Km; (CYH) 200 Km.
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49
Tegelberg et al
J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
FIGURE 7. Progressive region-specific neuron loss in Cstbj/j mice. (AYH) Histograms show unbiased optical fractionator estimates
of numbers of Nissl stained neurons in Cstbj/j and control (ctrl) mice during disease progression. Loss of neurons is first seen in
the lamina V of the primary somatosensory cortex (S1) (B), followed by the laminae IV and VI (A, C) and the corresponding
thalamic relay nuclei, the posterior thalamic nuclear group (Po) (D). In contrast, the primary visual cortex (EYG) showed later
neuron loss at 6 months of age and the corresponding thalamic relay nuclei, the dorsal lateral geniculate nucleus (LGNd) (H)
retained neurons even at this time. Data are expressed as mean neuron number T SEM. One-way analysis of variance: *, p e 0.05;
**, p e 0.01; ***, p e 0.001.
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J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
relay neurons occurs and suggest a correlation between the
extent of glial activation and neuron loss within the thalamocortical system.
DISCUSSION
We here provide the first detailed description of the
extent and sequence of pathologic changes in the CNS of
Cstbj/j mice, from young presymptomatic mice, to the stage
when ataxia starts to occur at 6 months of age. Previous studies of these mice have largely focused on cerebellum (10) or
have studied older mice with more advanced disease (12, 13).
Our results agree with the suggestion that EPM1 is a neurodegenerative disorder (10). However, we show that the disease pathogenesis is associated with a remarkable and early glial
involvement. Indeed, our data reveal localized microglial activation as early as P14 in Cstbj/j mice, when these mice are still
clinically normal. This microglial activation is followed successively by intense astrocytosis, but the first signs of neuron
loss in the cerebral cortex only become evident at 1 month, coinciding with the appearance of myoclonic seizures (10) (Fig. 8).
Our data also reveal that atrophy of the cerebral cortex
parallels that previously described in the cerebellum (10).
This atrophy and glial activation within the cerebral cortex is
widespread, although not uniform, but neuron counts reveal
substantial region-specific effects, suggesting no direct rela-
Glial Activation and Neuron Loss in Mouse EPM1
tionship between glial activation and neuron survival within
the cerebral cortex.
The region-specific neuron loss in Cstbj/j mice also
extends to subcortical structures, with loss of afferent relay
neurons in the corresponding thalamic relay nucleus, which
was only observed after the onset of cortical neuron loss.
In contrast to the cortex, the distribution of thalamic neuron
loss corresponded closely with the degree of glial activation
within these nuclei. For example, neuron loss was pronounced
within the somatosensory relay nucleus Po, which displayed
prominent early microglial activation, whereas in those without evident glial activation (eg, visual relay system), neurons
were not significantly lost.
Our data for selective effects on the somatosensory thalamocortical system are consistent with magnetoencephalography studies on EPM1 patients, which suggest that EPM1 is
characterized by hyperreactivity of the sensorimotor thalamocortical loop but not of the cortex in general (21). Neuropathologic studies on EPM1 patients are sparse, with neuron
loss reported in medial thalamic nucleus and the striatum, but
not in the hippocampal formation, in 3 patients with EPM1
(22). The interpretation of such pathologic findings in EPM1
patients have been complicated by the effects of antiepileptic
drug treatment, especially the association of phenytoin treatment with loss of Purkinje cells (23). However, our histopathologic findings in Cstbj/j mice are remarkably similar to
FIGURE 8. Schematic representation of the pathologic changes in the Cstbj/j mouse brain during disease progression. Extensive
microglial activation is evident at P14 in the Cstbj/j brain. This is followed by astrocytosis at 1 month of age, when the first signs of
myoclonus appear. Region-specific neuron loss in the primary somatosensory cortex (S1) also begins at this time and is followed by
neuron loss in the corresponding thalamic relay nucleus, the posterior thalamic nuclear group (Po). Evidence of regional atrophy
and cortical thinning can be seen from 2 months of age onward. Ataxia appears by 6 months of age, at which time the Cstbj/j
brain is severely shrunken and gliotic.
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51
Tegelberg et al
the pathology reported in human patients, suggesting that this
neuron loss may be the direct result of deficient CSTB function.
Localized glial activation and selective neuron loss has
also been documented in mouse models of neuronal ceroid
lipofuscinosis (NCLs), another group of disorders that display PME (19, 24Y26). These disorders also consistently display early glial activation before neuron loss, but the precise
relationship between astrocytosis, microglial activation, and
whether neuron loss begins in the thalamus or cortex varies
markedly among the different forms of NCL (27). However,
the characteristic pattern of microglial activation, its early
intense phase and later decline in Cstbj/j mice, has not been
seen in any form of NCL. Although present in many widespread brain regions, glial activation in Cstbj/j mice is in
several instances remarkably localized to individual brain structures. For example, in 1-month-old Cstbj/j mice, the claustrum
shows extremely intense and sharply demarcated activation
of both microglia and astrocytes. The claustrum is a telencephalic subcortical structure with largely unknown physiological function, but it has been suggested to be critical to
coordinating information within and across sensory and motor
modalities (28), as well as epileptiform activity and spreading and generalization of the seizures (29). Interestingly, the
claustrum connects several brain areas that display pronounced
gliosis and neuron loss in Cstbj/j mice, including somatosensory and motor cortex (30), anterior cingulate cortex (31)
and substantia innominata (32), amygdala (33), striatum (34),
substantia nigra (34), and thalamus, particularly the posterior
thalamic nuclei and midline thalamus (35).
A characteristic feature of our data is the activation of
both microglia and astrocytes before the onset of regional
atrophy or significant neuron loss in Cstbj/j mice. Although
gliosis has long been considered a hallmark of neurodegenerative changes, it is now apparent that glial responses often
precede neuron loss and may indicate neuronal dysfunction
(36Y39). Reactive changes in microglia and astrocyte populations in older Cstbj/j mice have been reported previously
(12, 13), as well as hyperexcitability possibly caused by loss
of GABAergic inhibition (13). We did not detect changes in
neuron volumes at P14 when there is intense microgliosis but
no neuron loss or in severely affected 6-month-old Cstbj/j
mice in the areas with the greatest neuron loss and most intense
gliosis. In line with our results, Shannon et al (12) reported
no significant difference in cross-sectional areas of superficial
granular cell neurons of the parasubiculum and prosubiculum
between 3- to 6-month-old control and Cstbj/j mice. Significant change in neuron size was seen only in substantially
older animals of 16 to 20 months of age (12). They also
reported region-specific neuron loss despite difficulties in
detecting apoptosis. However, we cannot exclude the role of
early neuronal changes, such as abnormal synaptic activity
in inducing early glial activation in Cstbj/j mice. Notably,
microglia and astrocytes are central regulators of both synaptogenesis and normal synaptic function (40). Therefore, further
studies are needed to unravel the neurodegenerative mechanisms in Cstbj/j mice.
Our results from Cstbj/j mice suggest a key role for
microglia in the pathogenesis of EPM1. Several lines of evidence show that cathepsins, secreted by activated microglia,
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J Neuropathol Exp Neurol Volume 71, Number 1, January 2012
directly induce neuron loss (41, 42). Because CSTB is an
inhibitor of cysteine cathepsins (9), it is plausible that an
imbalance in the cathepsin homeostasis in CSTB-deficient
microglia may provoke neuronal death in Cstbj/j mice, although this remains to be proven. Although microglia are
traditionally thought to be negative players in neurodegeneration by causing neuroinflammation, this view has been
challenged recently by the suggestion that a loss of microglialmediated neuroprotection may be an important contributor to
neuropathology. Indeed, there is a wealth of new evidence for
a compromised protective role of microglia in several neurodegenerative disorders, including Alzheimer disease (43) and
amyotrophic lateral sclerosis (44). The progression of microglia from an activated brain macrophage-like morphology to
a more ramified appearance with increased age in Cstbj/j
mice may hint at microglial dysfunction. Alternatively, it may
simply reflect that the molecular cues that caused their activation (presumably released from diseased neurons) are no
longer present because these neurons have died. It will be
important to distinguish between these possibilities, but both cellautonomous changes and alterations in the extracellular milieu
probably have a role in this distinct microglial phenotype.
The contribution of early microglia activation to subsequent pathologic changes in Cstbj/j mouse remains to be
elucidated. Moreover, whether such microglial activation plays
a role in the pathogenesis of human EPM1 remains unknown.
Our study provides the first evidence for the early involvement of microglial activation in this disorder. Further studies
should be directed at discovering disease-related changes in
microglia to understand the mechanistic basis for the morphologic alterations. Indeed, a very topical question raised from
this study is whether modulation of microglial function could
be used as a therapeutic tool for preventing or combating neurodegeneration in EPM1.
ACKNOWLEDGMENT
The authors thank Dr Jaana Tyynelä for critical reading
of the article.
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