Porosome in the rat brain: effect of hypokinetic stress (electron microscopic study)
1.
Introduction
It is now well established that permanent supramolecular lipoprotein structures called “porosomes”, are present at the cell
plasma membrane in neurons, exocrine, endocrine, and neuroendocrine cells, where membrane-bound secretory vesicles
transiently dock and fuse to expel their contents to the outside during cell secretion (Schneider et al. 1997; Cho et al. 2002
a,b; Cho et al. 2004; Cho et al. 2007; Cho et al. 2008; Sicsou et al. 2008; Jena et al. 2003). Porosome therefore have been
demonstrated to be the universal secretory machinery in cells (Cho et al. 2010; Jena, 2007, 2008, 2009; Paknikar, 2007;
Paknikar et al. 2007; Alison et al. 2006; Anderson, 2004, 2006; Cracium et al. 2006). The overall morphology, composition,
and reconstitution of porosome in exocrine pancreas, neuroendocrine cells, and in neurons are well documented (Cho et al.
2002 a, b; Cho et al. 2004; Cho et al. 2007; Cho et al. 2008; Sicsou et al. 2008; Jena et al. 2003) and the 3D contour map
of the assembly of proteins within the rat brain porosome complex, has been determined (Cho, Ren, Jena, 2008).
Porosomes are composed of cholesterol (Cho, Jeremic, Jin, Ren, Jena, 2007) and several proteins (Cho et al. 2004; Jena
et al. 2003; Jeremic et al. 2003), such as SNAP-23/25, syntaxin, synaptotagmin, the ATPase NSF, cytoskeletal proteins
such as actin, alpha-fodrin, and vimentin, calcium channels
3 and 1c, and chloride ion channels CIC2, CIV3, and their
isoforms; t-SNAREs and calcium channels are present at the base of the porosome compex (Jena et al. 2003; Cho et al.
2005) and actin regulates the opening and closing of the structure during cell secretion (Schneider et al. 1997; Jena et al.
2003; Cho et al. 2010). Recent electron microscopy (EM) 3D tomography in rat brain demonstrate the presence of 12-17
nm permanent presynaptic densities to which 45-50 nm synaptic vesicles are found to dock (Siksou et al., 2007). Since the
resolution of the EM tomography is < 10 nm, it precludes a detailed analysis of the porosome structure (Sicsou et al. 2007).
Nonetheless, this 3D EM tomography of the presynaptic membrane clearly reveals the morphological outline of the
porosome complex at the presynaptic membrane. In recent studies (Cho et al. 2010), using atomic force microscopy,
ultrahigh resolution imaging of the presynaptic membrane of isolated synaptosome preparations from the rat brain has been
demonstrated the presence of neuronal porosome in open, partially open, and close conformations. Results from this study
suggests that the central plug is retracted into the porosome cup when it is in its fully open conformation, and pushed
outward to seal the porosome opening in its close status, supporting the hypothesis that the central plug operates as an exit
door for the structure. Results of our EM study further confirm the cup-shaped morphology of porosomes in the rat brain,
and demonstrate their similar shape and size in the cat nerve terminal. This research also demonstrates for the first time,
the universal presence of similar porosome structures in different species of mammals for neurotransmitter release
(Okuneva et al. 2011 a, b).
In further evaluation of the porosome structure at the presynaptic membrane in the rat brain, and to determine if the
porosome is changed as a result of pathological conditions, the comparative electron microscopic study of porosome’ s
structure in the central nucleus of amygdale in normal rat and rat subjected to chronic restraint stress was undertaken.
Earlier we described the number of alterations that take place in the synapses of abovementioned region (one of the brain
strutures, to which the stress response “sends a quick message”) as a result of this pathological condition (Zhvania 1991,
1996). The present experiments were designed to show if such alterations are associated with the changes on the
porosome structure: its depth and diameter.
2. Material and methods
2.1. Experimental design: simulation of hypokinesia
85-90 d old male Wistar rats, weighting 280-300 g were used for this study. Hypokientic rats were kept for 90 day (d)
in small individual Plexiglas cages. Cages dimensions of 195 x 80 x 95 allowed movements to be restricted in all
directions without hindering food and water consumption. The cages were housed under normal controlled
environment (temperature 20-22 0C, humidity 55-60%, light on 07.30 – 19.30). Five, age-matching control rats were
kept in ordinary vivarium conditions. Experimental procedures were, approved by Animal Studies Committee of
Georgian Life Science Research Center.
2.2. Electron microscopic examination
Following pentobarbital injection (100mg/kg), animals to have EM examination of their brains underwent transcardiac
perfusion with heparinized 0.9% NaCl, followed by 500 ml of 4% paraphormaldehyde and 2,5% glutaraldehyde in 0, 1
M phosphate buffer (PB), pH-7,4, at a perfusion pressure 120 mm Hg. The brains were removed from skull and
placed in the same fixative overnight. The right hemispheric tissue blocks containing hippocampi, were cut into 400
micron-thick coronal slices. Slices were washed in cold 0,1 M PB and kept in 2,5% glutaraldehyde in 0,1 M PB until
processing; When processing, the slices were washed in cold PB, post-fixed in 1% osmium tetroxide in cold PB for 2
h and again washed in 0,1 M PB. The hippocampus was identified with an optical microscope Leica, cut out from the
coronal slices, dehydrated in graded series of ethanol and acetone and embedded in araldite. Blocks were trimmed
and 70-75-nm-thick sections were cut with an ultra-microtome (Reichert), picked up on 200-mesh copper grids,
double-stained with uranyl-acetate and lead-citrate and examined with a JEM 100 C (JEOL, Japan), Hitachi (Japan)
and Tesla (Czechoslovakia) transmission electron microscopes. For each case 115 sections were observed.
2.3. Morphometric analysis of porosome diameter & depth
A morphometric analysis of the porosomes in presynaptic membrane of central nucleus of amygdale in normal rats
and rats subjected to hypokinetic stress in order to identify any differences in size with regard to diameter and depth
was performed. The following abbreviations of parameters will be used bellow: DiP – Diameter of Porosome, DeP – Depth of
Porosome. Totally 184 synaptic terminals were observed: n = 91 - in experimental rats and n = 90 - in normal rats. 70
neuronal porosomes for each case were identified and measured with ImageJ (version 1.41).
2.3. Statistical analyses
One – way ANOVA was performed to determine whether parameters of neuronal porosomes - diameter of opening and
depth – differ from each other in the brain of normal rat and rat subjected to hypokinetic stress. The statistical significance
of differences between two groups of measurements was calculated by two sample t-test with a p-value threshold of <
0.05.
2.
Results:
In view of the 10-18 nm size of neuronal porosome structure, it has been difficult to observe these structures in electron
micrographs. The difficulty in imaging neuronal porosomes is further compounded by the presence of high concentration of
proteins at the presynaptic membrane. Consequently, in normal brain totally 196 axo-dendritic synapses were observed
and only 28 porosomes were found; in the rats subjected to hypo kinetic stress 200 axo-dendritic synapses were studied
and 29 porosomes were found. As in our previous studies (Okuneva et al. 2011 a, b), the cup-shaped neuronal porosomes
in normal and experimental brain were clearly observable with or without docked synaptic vesicles (Figure 1a, b, g);
a.
b.
c.
Figure 1. Electron micrographs of neuronal porosome complex in the central nucleus of amygdale in normal rat brain. The
10-17 nm cup-shaped porosome (P) at the pre synaptic membrane (pre-SM), with (a, b) and without (c) docked synaptic
vesicles. The post synaptic membrane is shown in green (a).
One-way ANOVA doesn’t reveal significant difference between diameter and depth of porosome in normal and
experimental rats. Therefore, these parameters of porosome in the central nucleus of amygdale are not affected by
hypokinetic stress (Table 1).
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1
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7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Dimensions of neuronal porosome complex
cont diam
exp diam
cont depth
exp depth
17
16
12
14
18
13
9
10
13
14
19
19
7
9
15
15
17
12
14
17
15
15
18
17
17
17
16
17
18
16
12
11
10
11
13
12
13
14
14
14
14
10
16
17
18
16
16
17
18
16
16
17
15
15
12
11
12
9
7
8
14
8
19
15
14
9
11
14
11
7
21
19
16
15
21
14
10
9
9
9
6
6
6
6
6
9
9
8
9
9
15
10
10
17
11
14
14
13
13
18
11
10
14
14
27
28
29
19
15
18
16
11
14
13
14
20
One–way ANOVA results.
Dimensions of
neuronal porosome
complex
F (3,252)
Depth of porosome
P
0.093
2.92
Diameter of
0.02
0.890
porosome
Table. 1. Depth and diameter (nm) of the neuronal porosome complex from the normal and pathological brain
(90 days of hypokinetic stress) and summary of one –way ANOVA results. cont diam – diameter of porosome in
the control group of animals; exp diam - diameter of porosome in the experimental group of animals; cont depth –
depth of porosome in the control group of animals; exp depth - depth of porosome in the experimental group of
animals; F – variance from one-way ANOVA; P – probability.
Because of the heterogeneity of porosomal dimensions, the size distribution percentage histograms were
constructed and porosomal dimensions from normal and experimental animals were grouped in following
bins: (1) 5-10 nm, (2) 10-15 nm, (3) 15-20 nm and (4) 20-25 nm. In control animals, porosomes, based on
their diameter (a) and depth (c) were grouped into three (a) and four (c) bins: (a) In the first bin (5-10 nm)
- 10%, in the second bin (10-15 nm) – 30%, in the third bin (15-20 nm); (b) the distribution of porosome
depth revealed 4 groups: (I) 5-10 nm - 25%; (2) 10-1`5 nm - 45%; (3) 15-20 nm -25% and (4) 20-25 nm 5%. nm (IV). After the 90 days of stress porosomes of I group in (a) and III ,IV group in (d) expressed a
rightward and a leftward shift correspondingly of percentage towards the II group in (b) and to the I group
in (d), but the changes of porosome’ s diameter are not statistically significant in experimental rats vs.
control rats (14.75 + 0.61 vs. 14.86 + 0.48, p>0.05), as well as in the case of porosomes’ depth (12.893+
3.98 vs.11.14 + 3.78, p>0.05) (Fig. 2). The diameter of porosome in normal and experimental rats is
almost the same; these data are consistent with evidences obtained with atomic force microscopic and
photon correlation spectroscopic studies, according which the mean value of porosome diameter is 12–16
nm (Cho et al., 2007).
The diameter of porosomes in the experimental group
60
50
50
40
40
P e rc e n t
P e rc e n t
The diameter of porosomes in the control group
60
30
30
20
20
10
10
0
0
5
10
15
20
25
5
nm
10
15
20
25
nm
a.
b.
The depth of porosomes in the control group
The depth of porosomes in the experimental group
50
40
40
30
P e rc e n t
P e rc e n t
30
20
20
10
10
0
0
5
10
15
20
25
nm
5
10
15
20
25
nm
c.
d.
Figure 2. Histograms showing the distribution of neuronal porosome based on the diameter (a, b) and depth (c, d)
value in the synaptic boutons of brain in the control (a, c) and experimental (b, d) group of animals. All
measured porosomes are separated by the diameter and depth’s size and binned by area in 5 nm natural
log increment.
Our results demonstrate that despite the high dynamism of neuromal porosome complex the range of
dimensions’ fluctuation (diameter – 12-16 nm, depth – 5-20 nm) is the same in normal rat brain and the
brain of rats subjected to the chronic hypokinetic stress. Therefore, they aren’t affected by experimental
conditions.
Discussion:
In the present EM research for the first time the effect of pathological condition on the ultrastructure of neuroporosome
ultrastructure was analyzed. Specifically, the morphometric measurement of the diameter and depth of this cup-shaped
supromolecular structure at the presynaptic membrane of central nucleus of amygdale in normal rat and rat subjected to 90
d hypo kinetic stress was made.
It is well known that the amygdale, and specifically, the central nucleus, plays a crucial role in the orchestration and
modulation of the organism response to aversive, stressful events (Beretta 2005; Carrasco, Van de Kar 2003; Carter
et al. 2004). Particularly, besides numerous other processes stimulated by chronic stress, this condition is known to
cause cortisol/corticosterone-induced released of epinephrine and nor epinephrine that are specifically modulate the
function of this nucleus (Mo et al.2008). Furthermore, recent results indicate that the restraint stress rapidly and
selectively increases serotonin release in the central nucleus of amygdale by the activation of central corticotropinreleasing factor receptors and this activity is considered as an important component of a stress response (Beretta
2005; Carrasco, van de Kar, 2003). Earlier significant alterations in neurons, glial elements and synapses of the
central nucleus of amygdala provoked by 90 d hypo kinetic stress was described ((Zhvaniia, 1996; Zhvaniya, Bliadze,
1991; Zhvaniya, Kakabdze, 1996). Thus, among synapses, several presynaptic terminals were characterized with
clearly defined pathological changes, such as agglutination of synaptic vesicles, the presence of large osmyophilic
bodies, destruction of vacuolization of mitochondria; in some others, polymorphism of synaptic vesicles, including the
occurrence of large irregular forms or relatively large dense-core synaptic vesicles was observed. Moreover,
significant alterations in the number of several types of synapses, such as increase of the number of symmetric forms
on dendritic shafts that are extremely rare in normal brain, or the decrease of the total number of asymmetrical forms
were revealed (Zhvania, 1996). Such alterations should reflect modifications in synaptic transmission provoked by
chronic hypokinetic stress in abovementioned limbic region. Taking into consideration that porosome complex at the
presynaptic membrane of neuron represents the place, where membrane-bound provoke synaptic vesicles transiently
dock and fuse to expel their contents to the outside during process of neurotransmission, we were especially
interested to clarify if alterations in the structure of synapses “catch up” the porosome complex, or, in other words, if
they are reflected on porosome structure.
Comparative analysis of depth and diameter of the porosome in normal brain and brain of rats subjected to chronic hypo
kinetic stress doesn’t reveal any difference in quantitative characteristics of these parameters. Therefore, our data indicate
that despite significant alterations in several types of synapses, the main structural parameters of porosome: depth and
diameter remain unchangeable. It s very likely, that more fine alterations take place before synaptic vesicles dock with
synaptic membrane.
Our results point out to the heterogeneity of porosome dimension but the range of fluctuation (diameter: 12016 nm, depth –
5-20 nm) and averages are not affected by experimental condition. The heterogeneity of porosomal dimensions could
provide by vesicles size, curvature and highly dynamic structure of neural porosomes, connected to the different
conformational state (open, partially open or closed) of central plug and a whole porosome too.
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