Midbrain dopamine function in schizophrenia and major depression- a post-mortem
and PET imaging study
Supplementary methods and results
Study 1
All the brains come from the Corsellis Brain Collection, consisting of over 6500 brains collected
during the period 1952 to 1997. The collection consists of brains of individuals whose death was
reported to the coroner's court reported by Professor Jan Corsellis and retained for further research.
The majority of samples come from the county of Essex but a smaller number came from national
referrals. The collection procedures and methods have been previously described1.
Histochemical staining
The first mounted 10µm section of each specimen was stained with haematoxylin and eosin (H&E)
for anatomical identification of the SN. Sections were submerged in xylene for 30 minutes and
serially placed in troughs of 2 x 100%, 90%, 70% ethanol (EtOH) and pure distilled water, each for 2
minutes, before 5 minute immersion in Mayer’s haematoxylin stain and washing, followed by
differentiation in acid-alcohol (1% HCl/70% EtOH) for 10 seconds and into distilled water for 2
minutes. Sections were immersed in 1%w/v eosin for 5 minutes and washed before dehydration in
70%, 90% and 100% EtOH before placed in xylene and mounted with DPX.
Immunohistochemical staining
Three slides per hemisphere, a total of six per case, were dewaxed in xylene, hydrated in 100%, 90%,
70% ethanol and dH20 for 2 minutes each and incubated in methanol/3% hydrogen peroxide (H202)
for 10 minutes. Sections were washed phosphate buffered saline (PBS) and incubated for one hour
at room temperature with primary antibody (1:200 TH; Abcam ab51191) diluted in PBS/10% normal
horse serum. After washing and incubation with HRP-conjugated secondary antibody (Dako K4002)
for 30 minutes at room temperature sections were then washed in phosphate buffered saline (PBS).
Visualisation was done by 10 minute incubation in 1% Vector Red (Vector SK-5100)/1% H2O2. After
washing in PBS and rinsing in dH20 sections were immersed in cresyl-violet for 5 minutes, rinsed,
differentiated in 95% EtOH/1% ethanoic acid, rinsed and counterstained in Mayers haematoxylin,
rinsed again and dehydrated through serial alcohols, immersed in xylene and mounted using DPX.
Following staining slides were randomised and blinded by an independent investigator.
Substantia nigra identification
H&E stained specimens were viewed under x40 magnification to identify structures anatomically
related to the SN, such as the cerebral aqueduct, red nucleus, superior colliculi and cerebral
pedunculi. This gave an indication of the rostrocaudal level of the section in anatomical variations.
Dopaminergic neurons of the SN were identified by their large size, oval shape and according to the
presence of neuromelanin within the cell soma.
Image capture and Quantification
Images were captured using an Olympus Vanox AHBT 3 micropscope with Q Imaging Micropublisher
RTV 3.3 camera and analysed using Image-Pro Plus 5.1 software (Media Cybernetics, US).
Sample cells showing no staining, moderate staining and heavy staining were captured at x400
magnification and used as standards.
No staining visible in the cytoplasm with clear cresyl
counterstain was rated as zero, moderate staining visible in the cytoplasm distinct from
neuromelanin as one and heavy staining forming a clear halo around the nucleus and intense
staining observed elsewhere in the cytoplasm as two (supplementary figure 1). The medial region of
each TH-stained nigra section was imaged at x40 magnification and all dopaminergic cells tagged.
These were defined as large, oval stained neurons containing neuromelanin and a counterstained
nucleus. A random number generator (www.graphpad.com, US) was used to select eight unique
neurons, which were imaged at x400 magnification and semi-quantitatively assessed for TH
expression as described above.
Study 2
The thirteen patients who were taking medication were taking the following treatment: aripiprazole
(n=1), chlorpromazine (n=2), clozapine (n=2), olanzapine (n=6), quetiapine (n=1), risperidone (n=3),
sulpiride (n=1). The striatal but not the nigral data from fourteen antipsychotic treated patients and
twelve controls has been included in a previous publication2.
Exclusion criteria for all subjects were history of neurological or medical illness or head injury, drug
or alcohol abuse or dependency, pregnancy, or contraindication to scanning. In addition, all controls
were required to have no history of psychiatric illness.
PET methods
The ECAT/EXACT3D PET scanner (Siemens/CTI, Knoxville, USA) used in the study has a sensitivity of
69 cps/Bq/ml and a spatial resolution of 4.8±0.2 mm. High resolution whole brain images were
reconstructed from 95 planes with a slice spacing of 2.425 mm. One hour prior to scanning subjects
were given a one off dose of 150mg carbidopa and 400mg entacapone orally. This greatly reduces
the formation of radiolabeled metabolites which may cross the blood-brain-barrier, and so improves
the specificity of the signal.3
Data acquired in list-mode were rebinned into 26 time frames of increasing duration (1 x 30s, 4 x
60s, 3 x120s, 3 x 180s, 15 x 300s). Data were reconstructed using a 3D re-projection algorithm. A
structural MR scan was also conducted to exclude intracranial abnormalities. A mutual information
algorithm was used to correct for movement using the images that had not been attenuation
corrected.4 The resulting transformation parameters were applied to the corresponding attenuationcorrected frames, and the realigned frames combined to create a movement corrected dynamic
image for analysis.
Voxel-wise analysis
To confirm the results of the ROI analysis at the voxel level, individual parametric (18F-DOPA Kicer)
images were constructed from movement corrected images using a wavelet approach.5 The
individual parametric images were normalised to MNI space using a decay-weighted 18F-DOPA add
image and an 18F-DOPA template in SPM5. To examine sub-regional group contrasts the search
volume was restricted to the substantia nigra via application of a nigral mask derived from the
HamNet probabilistic atlas.6 Fully corrected results (p<0.05 adjusted for family wise error rate with
no extent threshold) are presented for this analysis. Additionally we conducted a whole brain voxel-
based analysis to determine the specificity of the differences to the regions-of-interest using a liberal
threshold of p<0.01 to ensure high sensitivity, although it should be noted that this increases the risk
of type-II error. The results of the whole brain contrast schizophrenia>controls identified two
clusters, one in the striatum and one including the right nigra (figure 5a). The contrast
controls>schizophrenia identified no significant elevation in controls (figure 5b).
Supplementary table 1
Clinical and demographic data for post-mortem samples used (M – male, F – female, NK – not known, SZ –
schizophrenia, MDD – major depressive disorder, CoD- cause of death, PMI- post-mortem interval (hours)).
Case
Age
Sex
PMI
Diagnosis
CoD
1
39
M
24
Control
Acute Myocardial Infarction
2
40
M
NK
Control
Acute Myocardial Infarction
3
62
M
21
Control
Acute Myocardial Infarction
4
68
M
66
Control
Multiple Myeloma
5
63
M
28
Control
Ischaemic Heart disease
6
52
M
NK
Control
Pneumonia
7
61
M
NK
Control
Ischaemic Heart disease
8
54
F
27
Control
Ischaemic Heart disease
9
59
M
68
Control
Pulmonary Heart disease
10
46
F
23
Control
Acute Myocardial Infarction
11
33
M
NK
Control
Acute myocarditis
12
48
M
25
Control
Acute ischaemic heart diseases
13
45
F
66
Control
Pneumonia
14
55
M
94
SZ
Cachexia
15
40
M
76
SZ
Inhalation of gastric contents
16
59
F
119
SZ
Congestive heart failure
17
63
F
87
SZ
Ischaemic Heart diseases
18
60
M
24
SZ
Acute Myocardial Infarction
19
66
M
60
SZ
Pneumonia
20
69
M
27
SZ
Acute Myocardial Infarction
21
55
M
NK
SZ
Intentional self-poisoning by gases
22
56
F
126
SZ
Acute Myocardial Infarction
23
76
F
NK
SZ
Pneumonia
24
66
F
42
SZ
Intentional self-poisoning by gases
25
66
M
NK
SZ
Ischaemic Heart diseases
26
60
F
NK
MDD
Intentional self-poisoning by drugs
27
49
F
57
MDD
Intentional self-poisoning by alcohol
28
41
M
24
MDD
Intentional self-poisoning by gases
29
70
F
48
MDD
Pneumonia
30
68
F
NK
MDD
Pneumonia
31
47
F
NK
MDD
Intentional self-harm by suffocation
32
63
M
99
MDD
Ischaemic Heart disease
33
54
M
27
MDD
Intentional self-harm by fire
34
53
F
NK
MDD
Intentional self-harm by suffocation
35
45
F
66
MDD
Intentional self-poisoning by drugs
36
58
F
NK
MDD
Intentional self-harm by suffocation
37
60
F
23
MDD
Ischaemic Heart disease
38
65
M
39
MDD
Intentional self-poisoning by drugs
Supplementary Table 2.
Patient data by diagnostic group (M – Male, F – Female, PMI – post-mortem interval (hours), NK – not
known, SZ – schizophrenia, MDD – major depressive disorder, CoD=cause of death).
Group
Total n (n where
Mean (SEM) Age/
Sex ratio (M/F)
Mean (SEM) PMI/
CoD was suicide)
years
Control
13 (0)
51.9(2.8)
9/4
39.6(5.4)
SZ
12 (2)
60.1(2.3)
7/5
68.5(9.4)
MDD
13 (9)
56.5(2.3)
4/9
51.3(6.3)
hours
Supplementary figure 1. Illustrating the grades of cytoplasmic TH staining used in Study 1.
1a. Illustrating grade 0 staining (indicating low staining for tyrosine hydroxylase). Low/ absent
cytoplasmic TH staining with visible cresyl counterstain (pale violet color) is indicated by the arrows
labeled A. Staining of neuromelanin, characteristic of dopamine neurons in the substantia nigra, is
indicated by the arrow labeled Nm.
1b Illustrating grade 1 staining (indicating moderate staining for tyrosine hydroxylase). Moderate
levels of TH staining in the cytoplasm is indicated by the arrows labeled B. Staining of neuromelanin,
characteristic of dopamine neurons in the substantia nigra, is indicated by the arrow labeled Nm.
1c Illustrating grade 2 staining (indicating high staining for tyrosine hydroxylase). High levels of TH
staining in the cytoplasm with a clear stained halo around cell nucleus is indicated by the arrows
labeled C. Staining of neuromelanin, characteristic of dopamine neurons in the substantia nigra, is
indicated by the arrow labeled Nm.
Supplementary figure 2
2a The nigral region of interest used in study 2 is shown in red on a transverse view of a T1 weighted
structural MRI scan in Montreal Neurological Imaging Space.
2b The nigral region of interest is shown (in red within the red circle) following normalisation to
native space on the transverse dynamic PET summation images from three patients.
Patient 1.
Patient 2
Patient 3
Supplementary figure 3
The relationship between nigral [18F]-DOPA uptake and striatal [18F]-DOPA uptake in
patients (in red) and controls (in blue).
Supplementary figure 4
a. Showing a significant positive relationship between total symptom severity score and dopamine
synthesis capacity (Kicer /min) in the substantia nigra (r=0.39, p=0.035)
b. Showing a significant positive relationship between psychotic symptom severity score and
dopamine synthesis capacity (Kicer /min) in the substantia nigra (r=0.37, p=0.048)
Supplementary figure 5
5a. Statistical parametric map for the whole brain voxel-wise contrast schizophrenia>controls
showing two clusters where the Kicer values were significantly elevated in schizophrenia (one
cluster in the striatum and the other cluster with a peak within the right substantia nigra
(indicated by the arrow, MNI coordinates: 12, -18 and -10)).
5b. Statistical parametric map for the whole brain voxel-wise contrast controls>schizophrenia
showing no voxels where there is a significant elevation in the controls relative to
schizophrenia.
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