J. Biomedical Science and Engineering, 2013, 6, 1129-1136 JBiSE
http://dx.doi.org/10.4236/jbise.2013.612141 Published Online December 2013 (http://www.scirp.org/journal/jbise/)
A mutation in protein kinase C-gamma alters SNC neuron
morphology and decreases synaptic vesicles in
dopaminergic striatal terminals in the AS/AGU rat
Abdullah Glil Al-Kushi1, David Russell2, Anthony Philip Payne2*
1College of Medicine, Umm Alqura University, Makkah, Kingdom of Saudi Arabia
2School of Life Sciences, Glasgow University, UK
Received 2 October 2013; revised 5 November 2013; accepted 21 November 2013
Copyright © 2013 Abdullah Glil Al-Kushi et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In accordance of the Creative Commons Attribution License all Copyrights © 2013 are reserved for SCIRP and the owner of the
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ABSTRACT
A spontaneous mutation in the Albino Swiss (AS) rat
has been shown to be a single point mutation (agu) in
the gene coding for the gamma isoform of protein
kinase C (PKC-γ). The characteristics of the mutant
include movement disorders, a failure to release do-
pamine in the striatum and elevations of molecules
such as parkin and ubiquitin in the substantia nigra
pars compacta (SNC). This present study examined
SNC cell bodies and dopaminergic synaptic terminals
within the caudate-putamen. Cell volume and nuclear
volume were reduced in the AS/AGU mutant com-
pared to the AS control, but the volume fractions of
mitochondria and rough endoplasmic reticulum were
significantly higher. No Lewy bodies were present in
the mutant, although microglia were found adjacent
to some SNC cells. Dopaminergic terminals were
identified in the caudate-putamen by electron mi-
croscopy with low-glutaraldehyde fixation and im-
munohistochemistry for tyrosine hydroxylase using
immuno-gold visualisation. AS/AGU mutant rats had
less than half of the synaptic vesicles of AS controls;
this was not only true of “readily-releasable” zones
adjacent to the synaptic cleft but also “storage pool”
zones. The findings support the hypothesis that the
initial bar to dopamine availability in the striatum is
the reduced release, with nigral cell death being a
later phenomenon.
Keywords: PKC-Gamma; Nigral DA Neurons and
Terminals; PKC-Gamma; SNC Neurons; Dopaminergic
Terminals
1. INTRODUCTION
Recently there has been increased interest in the links
between neurodegenerative conditions, protein kinases
and kinase signaling pathways [1,2] including the protein
kinase C (PKC) family [3].
The AS/AGU rat is an Albino Swiss-derived mutant
which carries a recessive mutation (agu) in the gene cod-
ing for the gamma isoform of protein kinase C [4]. The
rats are characterized by movement impairments includ-
ing rigidity of the hind limbs, a staggering gait, a ten-
dency to fall over every few step, a slight whole body
tremor and difficulty in initiating movements [5,6] and
by progressive dysfunction of the nigro-striatal dopa-
minergic [DA] and raphe-striatal serotonergic (5-HT)
systems. The chief defect in both systems is a failure to
release transmitter within the striatum under normal
physiological conditions. Thus, extracellular dopamine
levels in the mutant [measured using microdialysis with
HPLC-ECD in conscious animals] are only 10% - 20%
of control levels [7] and 5-HT is similarly reduced [8].
There is also a marked depletion in utilization of 2-de-
oxy-glucose in the substantia nigra pars compacta, sub-
thalamic nucleus and ventrolateral thalamus [9]. At later
ages, there is a loss of cell bodies within the SNc [5].
There are no cellular inclusions such as Lewy bodies,
though some molecules associated with Lewy bodies,
such as ubiquitin and parkin, are elevated [10].
It is not clear whether the AS/AGU rat represents a
spontaneous useful laboratory model of striatal disorders
or an example of accelerated ageing, in which degenera-
tion of neural systems would be anticipated. Neverthe-
less, the mutant presents an opportunity to examine
dopaminergic cell bodies and terminals in a naturally
occurring rat model which combines striatal dopamine
*Corresponding a uthor.

A. G. Al-Kushi et al. / J. Biomedical Science and Engineering 6 (2013) 1129-1136 1130
dysfunction with motor disturbance. This study was un-
dertaken to examine the nigral cell bodies and striatal
dopaminergic terminals of the mutant and to compare
them with control [unaffected] animals. Animals aged
twelve months were used, as all mutants are reliably
symptomatic at this age.
2. MATERIALS AND METHODS
This work was carried out at Glasgow University and has
approval from the Ethical Review Process Application
Panel; it conforms to the Eu ropean Community Directive
86/609/EC and the UK Animals [Scientific Procedures]
Act [1986].
2.1. Stereological Examination of Cell Bodies in
the Substantia Nigra Pars Compacta
Five AS control and 5 AS/AGU mutant male rats aged
12 months were used. All rats were killed with carbon
dioxide euthanasia and perfused via the left ventricle
with mammalian Ringer solution [200 ml] containing the
vasodilator Lignocaine followed by 500 ml 3% glutaral-
dehyde [Agar-Aldrich Inc, P6148] in 0.1 M phosphate
buffer (PB), blood and excess fluid being drained via an
incision through the right atrium. The brain was dis-
sected out and immersion-fixed in 3% glutaraldehyde in
0.1 M of phosphate buffer overnight. Pieces of brain con-
taining areas of interest were serially sectioned at 70 µm
using a Vibratome (Agar Scientific LTD).
Electron Microscopy
Midbrains from AS (control) and AS/AGU (mutant) rats
were rinsed with PB and placed in a solution of 1% os-
mium tetroxide in PB for 20 minutes in an agitator. The
sections were rinsed with distilled water (3 × 30 mins)
and dehydrated through a series of graded concentrations
of acetone from 70% to 100%, followed by a descending
ratio of acetone to durcupan resin (3:1, 1:1, 1:3) and two
changes of durcupan resin. The sections were flat-em-
bedded in durcupan resin between two small sheets of
acetate, sandwiched between two glass slides, weighted
down with metal weights and heated at 60˚C overnight in
an oven. The top acetate sheet were peeled off, stock
embedded sections attached onto the end of a blank em-
bedding block using RS adhesive and left for at least 30
min in an oven. The block containing the area of interest
was trimmed and semi-thin sections (1 µm) cut and
stained with 1% Toluidine Blue buffered to pH 8.5 with
sodium borate and examined under the light microscope
to confirm the area of the SNC to be thin sectioned and
used in stereology. Ultrathin sections (80 - 90 nm thick-
ness) were cut from selected blocks using diamond
knives on a Reichert-Jung Ultracut E ultramicrotome.
Ultrathin sections were collected on 300 mesh-coated
copper grids and double stained with uranyl acetate and
lead citrate [11] and examined at a magnification of
5900× using a transmission electron microscope (JEOL
JEM-100S, No. IEM 10 0S-4, JEO L LTD , To k yo, Jap an) .
All those SNC cell body profiles containing a nucleolus
were photographed and analysed.
The volume fractions (Vv) of mitochondria and rough
endoplasmic reticulum were analysed through a random
point counting method on 30 SNC cells per animal. A 1
cm square grid was superimposed randomly on each mi-
crograph three times. The number of grid points that fell
on the feature of interest [mitochondria, RER, lipofuscin
granules] and the number of grid points that fell on the
reference space [cell and cytoplasm] were counted and
the Vv were calculated by the Cavalieri principle [12,
13].
2.2. Examination of Dopaminergic Terminals in
the Caudate-Putamen
It is essential to be able to distinguish dopaminergic ter-
minals within the striatum from other terminals. To do
this, we used immunohistochemical labelling of termi-
nals with an antibody to tyrosine hydroxylase followed
by secondary labelling with immuno-gold particles (1 nm)
with silver enhancement. To ensure labelling, the fixa-
tion regimen used a low percentage of glutaraldehyde
consistent with the retention of ultrastructural detail—a
mixture o f 1 % glutara ldehyd e: 4% paraformalde hyde.
Control procedures included not only confirmation
that omission of TH primary antibody would lead to no
gold particles being seen, but also the exclusion of any
sections where gold particles could be seen in the lumen
of blood vessels. To exclude very peripheral or oblique
sections, terminal profiles were only used for analysis if
they showed ten or more vesicles and a post-synaptic
density.
Measurements of Synaptic Vesicle Numbers
Synaptic vesicles were counted using a modification of
the method of Tao-Cheng [14], (see Figure 1). Two par-
allel lines were superimposed on the terminal, perpen-
dicular to the synap tic cleft [defined by the post-synaptic
density, PSD]. This area was then divided into four zones
parallel to the synaptic cleft:
Zones I and II (each 42 nm wide) contain the two
rows of synaptic vesicles immediately adjacent to the
presynaptic membrane known as the “readily releasable”
pool. Zone III is 84 nm wide, and contains almost all the
remaining vesicles of the reserve pool. Zone IV is up to
134 nm wide.
The visible length of the synapse (L) on the section
was also measured, allowing a calculation of the number
of vesicles per synaptic length. A minimum of 10 syn-
apses per animal were analysed.

A. G. Al-Kushi et al. / J. Biomedical Science and Engineering 6 (2013) 1129-1136 1131
3. RESULTS
3.1. SNC Cell Bodies in AS and AS/AGU Rats
Aged 12 Months (See Table 1)
Table 1 shows significant differences between the two
groups in relation to the volume of SNC cell bodies and
their nuclei. In each case, AS/AGU mutants have smaller
cell bodies and nuclei than AS rats of the same age. Re-
garding cell organelles, the volume density of mitochon-
dria and rough endoplasmic reticulum (either as a frac-
tion of the whole cell body or of the cytoplasm) was
raised in mutants. Nuclei often exhibited indented enve-
lopes, but there was no strain difference in the number of
indentations. There were no obvious Lewy body inclu-
sions. Many microglial cells were seen near SNC cells in
AS/AGU rats (see Figure 2).
3.2. Synaptic Terminals and Vesicles (See Table
2)
Low power surveys of the dorsal caudate-putamen
showed that AS/AGU mutants possessed significantly
fewer TH positive terminals per unit area than AS con-
trols (23 ± 2.1 compared to 43.7 ± 2 p < 0.01). When
terminals were analysed at high magnification, AS rats
had some 16% of terminal vesicles in zone I and a simi-
lar percentage in zone II, with 42% in zone III and 26%
in zone IV. This pattern of distribution was similar for
AS/AGU rats, although a higher proportion of vesicles
were found in zone III (48%) and a smaller percentage in
zone IV (10%). In all zones, and overall, the number of
vesicles was significantly reduced in AS/AGU mutants
compared to AS controls.
4. DISCUSSION
Two effects of the agu mutation require discussions: 1)
the morphology of cell bodies in the SNC and 2) the ef-
fects on synaptic terminals within the caudate-putamen.
In each case, the results obtained here can be compared
with studies on brain ageing, studies on diseases of the
nigro-striatal system, and studies involving treatment
with toxins to dopaminergic neurons e.g. MPTP or 6-
OH-DA (f or review see [ 15,16] ).
The SNC cell bodies in the parent AS strain resemble
existing reports for the rat in consisting of medium-sized
neurons with a round, eccentric nucleus, slight nuclear
indentations and abundant endoplasmic reticulum [17,
18]. SNC cell bodies were reduced in size in the AS/
AGU mutant strain, as were their nuclei. Cell shrinkage
has been reported in animals treated with MPTP [19] or
6-OH-DA [20], as well as in human Parkinson’s dis-
ease patients [21].
Nuclear indentations are often held to be a characteris-
tic of pathological change [20,22]. However, in the pre-
Zone II
Zone III
Zone I
Zone IV
PSD
Figure 1. Schematic diagram of synaptic measurement zones.
Two parallel lines (A + B) perpendicular to the segment of the
presynaptic membrane define the two sides of the area of meas-
urement; the distance between them is the index length (L).
Three parallel bands with increasing distance from the pre-
synaptic membrane were marked in dotted lines: Zone I, 0 - 42
nm; Zone II, 42 - 84 nm; Zone III, 84 - 168 nm and Zone IV,
168 - 300 nm. The postsynaptic density (PSD) is shown as a
dark gray rectangle (modified from Tao-Cheng, 2006).
N
NE
M
Figure 2. Electron micrograph showing SNC cell in an AS/
AGU [mutant] rat aged 12 months. Black arrows show in-
dented nuclear envelope; white arrow shows lipofuscin deposits;
M = microglial cell; N = nucleus; NE = nucleolus [Bar = 5
m].
sent study, both strains showed low, comparable levels
of indentation. There was no evidence of chromatin

A. G. Al-Kushi et al. / J. Biomedical Science and Engineering 6 (2013) 1129-1136 1132
Table 1. Volume fraction [Vv] and volume [V] of the SNC cell and its organelles in the midbrain of AS control and AS/AGU mutant
rats aged 12 months [n = 5 per group]. All values are mean ± SEM. All comparisons are two-sample t-tests. RER = rough endoplas-
mic retic ulum.
AS AS/AGU p
SNC cell volume [V] 664.1 ± 25 240.3 ± 22 <0.001
SNC nuclear volume [V] 133 ± 7.3 53.13 ± 0.60 <0.001
Mitochondrial Vv: [whole cell] 0. 0 42 ± 0 .002 0.051 ± 0.002 <0.05
Mitochondrial Vv: [cytoplasm] 0.082 ± 0.001 0.089 ± 0.006 <0.05
RER Vv: [whole cell] 0.025 ± 0.0003 0.031 ± 0.0009 <0.05
RER Vv: [cytoplasm] 0.046 ± 0.0023 0.056 ± 0.011 <0.05
Table 2. Numbers of synaptic vesicles in TH +ve nigrostriatal dopaminergic terminals [identified by immunogold staining for TH]
within the DCPU in AS control and AS/AGU mutant rats aged 12 months [n = 5 per group]. All values are mean number of synaptic
vesicles [V] or numbers per synaptic length [V/L] ± SEM. All comparisons are two-sample t-tests [NS: not significant].
Terminals AS AS/AGU p
9.98 ± 0.94 5.08 ± 0.17 <0.05
Zone I
[0 - 42 nm] V V/L 36.15 ± 2.7 14.2 ± 0. 9 6 <0.05
10.11 ± 0.78 5.57 ± 0.6 <0.05
Zone II
[42 - 84 nm] V V/L 36.61 ± 1.7 15.64 ± 2.2 <0.01
26.48 ± 3.3 12.64 ± 2.6 n.s.
Zone III
[84 - 168 nm] V V/L 95.7 ± 9.3 35.7 ± 8.2 <0.05
16.63 ± 2.9 2.72 ± 0.87 <0.05
Zone IV
[168 - 300 nm] V V/L 60.9 ± 1 2 7.76 ± 2.6 <0.05
63.20 ± 4.4 26.01 ± 4.1 <0.01
Total V
V/L 229.4 ± 13 73.3 ± 14 <0.01
clumping, such as occurrences in apoptosis following
MPTP treatment [23], but this is not surprising since the
present study examined a spontaneous neurodegenerative
mutant in which the number of apoptotic events at a
given time would be few, rather than a model in which
widespread cell death had been suddenly provoked by a
toxic agent.
The presence of microglial cells near SNC cell bodies
is of interest. There is microglial activation in human PD
patients [24] as well as MPTP-treated mice [25] and
6-OH-DA-treated rats [26].
It has been shown in a previous study that levels of
ubiquitin and parkin are elevated in nigral cell bodies of
the AS/AGU mutant compared to the AS parent strain
[10]. In idiopathic Parkinson’s disease, levels of ubiq-
uitin and parkin (which contains a ubiquitin-like homol-
ogy domain at its N-terminus and may be involved in the
recognition of the substrates and the subsequent degrad-
ing of mis-folded proteins) are elevated and the proteins
incorporated into cell in clusions [27 -30]. Such inclusions
have not been found in laboratory models of Parkinson’s
disease produced through 6-OH-dop amine or MPTP tox-
icity [31,32] and they do not occur in the AS/AGU mu-
tant rat.
Nevertheless, it is of interest th at ubiquitin, parkin and
the volume fraction of endoplasmic reticulum are all
elevated in the AS/AGU rat compared to the parent AS
strain. Parkin expression is induced by ER stress [sug-
gesting an adaptive role in the ER-associated protein
degradation pathway to clear misfolded proteins], while
overexpression of Parkin suppresses cell death induced
by several ER stress—inducing agents. Moreover, He-
reditary mutations [33] and the administration of 6-OH-
DA, MPP and rotenone [34] will all lead to elevated en-
doplasmic reticulum stress. Coupled with an increase in
the volume fraction of endoplasmic reticulum, this sug-
gests that the AS/AGU rat may be dealing with increased
levels of mis-folded proteins.
Whole tissue dopamine levels in micropunch samples
from the dorsal and lateral caudate-putamen analysed by
HPLC-ECD are known to be reduced by some 30% -
40% in the AS/AGU mutant rat compared to the AS con-
trol between 6 and 12 months of age [39]. Similar reduc-
tions of dopamine levels in the dorsal striatum have been
seen in post-mortem Parkinson’s disease patients [35]
and in living patients with the disorder [36]. MPTP ex-
posure can also greatly reduce striatal dopamine [37,38].
By contrast, extracellular levels of dopamine in the stria-

A. G. Al-Kushi et al. / J. Biomedical Science and Engineering 6 (2013) 1129-1136 1133
tum as measured in microdialysis samples from con-
scious, freely-moving AS/AGU rats are reduced by 80%
- 90% [7]. This leaves the possibility that dopamine is
present in striatal terminals in reasonable amounts, but is
not releasable under normal physiological conditions.
The results obtained in the present study show a consid-
erable reduction in both the number of dopaminergic
synaptic terminals per unit area, and in the number of
vesicles contained within the remaining terminals. The
zonal analysis for synaptic vesicle numbers suggests that
the loss is general, is not linked particularly to either the
readily-releasable or the storage pool, and is therefore
probably not linked to a molecularly characteriseable
population of vesicles. In particular, there is no evid ence
that the region closest to the synaptic cleft is devoid of
vesicles. Reduced vesicle counts have been seen in
dopaminergic terminals of mice treated with MPTP; low
doses caused axon terminals to swell and reduced vesi-
cles; with high doses, terminals disappeared [39]. Para-
doxically, in rats treated with 6-OHDA, terminal size and
vesicle numbers may increase [40,41] a finding very
much at variance with the present study.
The identification of tyrosine hydroxylase positive
terminals is a vital element of this study, and there are
many potential difficulties associated with labeling for
electron microscopy. These include alteration of TH pro-
tein by fixation, dehydration and heating leading to de-
creased recognition by antibodies [42] and high concen-
trations of glutaraldehyde leading to increased non-spe-
cific staining and decreased antigenicity of some proteins
[43]. Avidin-biotin-peroxidase labeling may show dif-
fuse artefactual labeling [44,45] and, while immunogold-
silver labeling is more localized, it penetrates less deeply
into the tissue [42]. The low level of glutaraldehyde used
in our study of ter minals was designed to maximize sp e-
cific immunogold-silver labeling, and, in order to omit
obique sections through the periphery of a terminal 1)
terminals were considered to be labeled when they con-
tained one or more silver particles which appear as small
black aggregates [46] and 2) only labeled terminals
showing a post-synaptic density were included. Control
procedures included not only confirmation that omission
of TH primary antibody would lead to no gold particles
being seen, but also the exclusion of any sections where
gold particles could be seen in the lumen of blood ves-
sels. The proportion of TH-labeled striatal terminals in
AS [control] rats was 17%, a figure consistent with pre-
vious studies in rats of 9% - 21% [47,48] and man of
approximately 16% [49], suggesting that labeling is de-
pendable.
Given 1) that TH levels are not depleted in the AS/
AGU mutant [10] and 2) that total dopamine levels
within the striatum are less depleted than extracellular
levels, one possibility is that dopamine is produced but is
not sequestered within vesicles i.e. is free within the cy-
toplasm. This may explain both the increased striatal
levels of extracellular metabolites such as homovanillic
acid and DOPAC seen in the mutant [7] and the eventual
loss of cell bodies within the SNC [5,50] as dopamine
may cause the formation of damaging free radicals or
dopaquinones [51,52].
Taken collectively, the investigation of the SNC cell
bodies of AS/AGU rats shows a reduction in size of the
whole cell and its nucleus, but with an in creased volume
of cell organelles, suggesting a cell body which is still
capable of coping with adverse circumstances and re-
sponding to synthetic demands. By contrast, analysis of
the dopaminergic terminals within the caudate-putamen
reveals a substantial reduction in number plus a substan-
tial reduction in the number of vesicles within those ter-
minals that remain. This co uld form the basis of both the
reduced release of dopamine reported previously and the
eventual loss of dopaminergic cell bodies in older ani-
mals.
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