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ORIGINAL PAPER
Neuronal nitric oxide synthase immunopositive neurons in catclaustrum—a light and electron microscopic study
Dimka Hinova-Palova Æ Lawrence Edelstein ÆAdrian Paloff Æ Stanislav Hristov Æ Vassil Papantchev ÆWladimir Ovtscharoff
Received: 12 April 2008 / Accepted: 15 July 2008
� Springer Science+Business Media B.V. 2008
Abstract Nitric oxide is a unique neurotransmitter,
which participates in many physiological and pathological
processes in the organism. Nevertheless there are little data
about the neuronal Nitric Oxide Synthase immunoreactive
(nNOS-ir) neurons and fibers in the dorsal claustrum (DC)
of a cat. In this respect the aims of this study were: (1) to
demonstrate nNOS-ir in the neurons and fibers of the DC;
(2) to describe their light microscopic morphology and
distribution; (3) to investigate and analyze the ultrastruc-
ture of the nNOS-ir neurons, fibers and synaptic terminals;
(4) to verify whether the nNOS-ir neurons consist a specific
subpopulation of claustral neurons; (5) to verify whether
the nNOS-ir neurons have a specific pattern of organization
throughout the DC. For demonstration of the nNOS-ir the
Avidin-Biotin-Peroxidase Complex method was applied.
Immunopositive for nNOS neurons and fibers were present
in all parts of DC. On the light microscope level nNOS-ir
neurons were different in shape and size. According to the
latter they were divided into three groups—small (with
diameter under 15 lm), medium-sized (with diameter from
16 to 20 lm) and large (with diameter over 21 lm). Some
of nNOS-ir neurons were lightly-stained while others were
darkly-stained. On the electron microscope level the
immunoproduct was observed in neurons, dendrites and
terminal boutons. Different types of nNOS-ir neurons dif-
fer according to their ultrastructural features. Three types
of nNOS-ir synaptic boutons were found. As a conclusion
we hope that the present study will contribute to a better
understanding of the functioning of the DC in cat and that
some of the data presented could be extrapolated to other
mammals, including human.
Keywords Cat � Claustrum � Nitric oxide � Nitric oxide
synthase � NO � NOS � Light microscopy � Ultrastructure
Introduction
The claustrum is a small telencephalic structure, which is
subdivided into two parts: dorsal (called also proper or
insular claustrum) and ventral (called also endopiriform
nucleus; Guirado et al. 2003; Edelstein and Denaro 2004;
Ashwell et al. 2004). The dorsal claustrum (DC) is located
deep to the insular cortex and it is extensively connected
with the neocortex (Druga 1966a, b, 1968, 1975; Otelin and
Makarov 1972; Kunzle 1975, 1978; Norita 1977; Riche and
Lanoir 1978; Olsen and Graybiel 1980; Carey et al. 1980;
Hinova-Palova et al. 1980a, b; Hinova-Palova 1981;
Hinova-Palova and Paloff 1982, 1984; Carey and Neal
1985; Edelstein 1986; Neal et al. 1986; Sloniewski et al.
1986; Tanne-Gariepy et al. 2002; Edelstein and Denaro
2004; Ashwell et al. 2004). On the other hand, the ventral
claustrum (endopiriform nucleus) is located deep to the
piriform cortex, and its interconnections with prepiriform
and entorhinal cortex are well documented (Druga 1966a,
b; Druga 1971; Sherk 1986; Witter et al. 1988; Dinopoulos
et al. 1992).
D. Hinova-Palova � A. Paloff � V. Papantchev (&) �W. Ovtscharoff
Department of Anatomy and Histology, Medical University,
1431 Sofia, Bulgaria
e-mail: [email protected]
L. Edelstein
P.O. Box 2316, Del Mar, CA 92014, USA
S. Hristov
Deparment of Forensic Medicine, Medical University,
1431 Sofia, Bulgaria
123
J Mol Hist
DOI 10.1007/s10735-008-9184-z
The size and form of the claustrum varies greatly
throughout the mammalian phylogenetic scale. In lower
mammals it is a small, ventrally located nucleus. In car-
nivores it is a representative structure that rivals, or even
exceeds, the size of the putamen. In primates it is a thin
gray slab, bounded laterally by the extreme capsule and
medially by the external capsule (De Vries 1910; Landau
1923; Berlucchi 1927; Loo 1931; Brockhaus 1940; Macchi
1984; Rae 1954; Stelmasiak 1955; Berke 1960; Pilleri
1961, 1962; Filimonoff 1966; Druga 1974, 1975; Zilles and
Zilles 1980; Paxinos and Watson 1989; Kowianski et al.
2004).
Nitric oxide (NO) is a unique gasous neurotransmitter
(Holstein et al. 2001, Martinelli et al. 2002). It was shown
that it takes part in many processes in the central nervous
system like synaptic plasticity in the hippocampus
(Krushkov et al. 1996), claustral functioning (Hinova-Pal-
ova et al. 1997), eye movements (Moreno-Lopez et al.
1996, 1998, 2001), thalamic functioning (Krushkov et al.
1996), blood pressure control (Maeda et al. 1999), inferior
colliculus functioning (Paloff and Hinova-Palova 1998),
vestibular complex functioning (Papantchev et al., 2005,
2006) and so forth. NO is synthesized by the enzyme Nitric
oxide synthase (NOS). There are at least three isoforms of
NOS: NOS I or neuronal NOS (nNOS), which is mainly
present in neurons (Dawson et al. 1991; Hope et al. 1991),
skeletal muscular cells (Dahrmann and Gossrau 1996) and
heart muscle cells (Sears et al. 2004); NOS II or inducible
(iNOS), which can be found in macrophages (Sugiyama
et al. 2003); NOS III or endothelial (eNOS), which is
present in endothelial cells (Seidel et al., 1997).
During the last years a lot of studies on NOS and NO
have been performed (Mizukawa et al. 1989, Mizukawa
1990, Dawson et al. 1991a, 1991b, 1993, 1994, Hope et al.
1991, Rodrigo et al. 1994, Kullo et al. 1997, Soares et al.
2003, Marino and Cudeiro 2003, Papantchev et al. 2003,
2005, 2006, Sears et al. 2004, Seyidova et al. 2004).
Nevertheless there are little data about the neuronal Nitric
oxide synthase immunoreactive (nNOS-ir) neurons and
fibers in the dorsal claustrum (DC) of a cat. In fact
according to the best of our knowledge there is a single
work describing the nNOS-ir in the DC of a cat (Rahman
and Baizer 2007). In one of our previous works we reported
the morphology and distribution of NADPH diaphorase
(NADPHd) positive neurons in DC of a cat (Hinova-Palova
et al. 1997). Some of present data were already reported
(Hinova-Palova et al. 2005).
The aims of this study were: (1) to demonstrate nNOS-ir
in the neurons and fibers of the DC; (2) to describe and
analyze their light microscopic morphology and distribu-
tion; (3) to investigate the ultrastructure of the nNOS-ir
neurons, fibers and synaptic boutons; (4) to verify whether
the nNOS-ir neurons consist a specific subpopulation of
claustral neurons; (5) to verify whether the nNOS-ir neu-
rons have a specific pattern of organization throughout the
DC.
Material and methods
Perfusion protocol
Eight adult healthy cats from both sexes with average
weight 2.4 kg (from 2.0 to 2.9 kg) were used. All animals
received humane care in compliance with the ‘‘Principles
of laboratory animal care’’ formulated by the National
Society for Medical Research and the ‘‘Guide for the care
and use of laboratory animals’’ prepared by the National
Institute of Health (NIH publication No. 86–23, revised
1996). Our perfusion protocol was described in details
elsewhere (Papantchev et al. 2006). In brief—all animals
were deeply anesthetized with intraperitoneal injection of
Urethan (40 mg/kg) and transcardialy perfused with
500 ml heparinized saline followed by 3,000 ml phosphate
buffered saline (PBS; pH 7.4) containing 2.5% glutaral-
dehyde and 4% parafomaldehyde. Two hours later the
brains were removed and postfixed in the same solution for
the next 2 h. The part of each cerebral hemisphere, con-
taining DC was removed, dissected and cut into tissue
blocks. Thus 16 tissue blocks, containing DC were
obtained. All tissue blocks were cut in coronal plane on a
Vibratome (Technical Products International, St. Louis,
MO, USA; http://www.vibratome.com). The thickness of
slices was 40 lm.
Immunohistochemistry
Our protocol for immunohistochemical visualization of
nNOS was already described elsewhere (Papantchev et al.
2006). In brief—all slices, prepared as described above,
were treated with sodium borohydride for 45 min followed
by three consecutive rinses in 0.01 M PBS, each for 2 min.
Incubation for 30 min in a solution of 1% bovine serum
albumin (BSA) was followed by incubation overnight in a
solution of a monoclonal anti-nNOS antibody (clone NOS-
3F7-B11-B5, ascites fluid; product number: N218-200UL;
MDL number MFCD01324876; Sigma, St. Louis, MO,
USA), in a dilution of 1:1000. Afterwards three consecu-
tive rinses in 0.01 M PBS (2 min each) were performed
and sections were incubated for 20 min in 1% BSA in PBS,
followed by incubation for 2 h in biotinylated anti-mouse
IgG (Vector, Burlingame, CA, USA) in a dilution of 1:500.
After three consecutive rinses in 0.01 M PBS (2 min each),
sections were incubated in a solution of avidin–biotin–
peroxidase complex (Vector, Burlingame, CA, USA) for
1 h. All incubations were carried out on a shaker at room
J Mol Hist
123
temperature. A new series of rinses were performed
according to the prescription used in the Department of
Anatomy and Histology (Paloff et al. 2004; Papantchev
et al. 2006; Hinova-Palova et al. 2007), first in PBS and
then in Tris buffer, pH 7.6, preceded visualization of per-
oxidase activity with H2O2 and 3,30-diaminobenzidine as
substrates. The procedure was continued with three rinses
in Tris buffer (2 min each) followed by another three in
phosphate buffer (2 min each). Afterwards part of the
sections was processed for electron microscopy. Sections
were postfixed with 1% OsO4 in phosphate buffer for 1 h,
dehydrated in graded series of ethanol and flat embedded in
Durcupan (Fluka, Buchs, Switzerland) between sheets
(Paloff et al. 2004; Papantchev et al. 2006; Hinova-Palova
et al. 2007). Sections were trimmed out under a dissecting
microscope and glued to epoxy blanks. Thin sections were
cut with an ultramicrotome (LKB, Stockholm-Bromma,
Sweden) and counterstained with uranyl acetate and lead
citrate according to the prescription used routinely in the
Department of Anatomy and Histology (Paloff et al. 2004;
Papantchev et al. 2006; Hinova-Palova et al. 2007) and
examined with a Hitachi 500 electron microscope (Hitachi,
Tokyo, Japan).
Thirteen sections were used as controls. All controls
were incubated in the way described, but omitting the
primary or secondary antibody. All controls were negative.
Sixty-five nNOS-ir neuronal perikarya were measured
on the electron micrographs. The nNOS-ir neurons (or
neuronal groups) were first identified by light microscope
and then serial sections were performed. All consecutive
measurements were performed on sections where a prom-
inent nucleolus was present. The ratio of the mean nuclear
diameter and the mean diameter of the perikaryon was
calculated as nucleocytoplasmic ratio (Paloff et al. 2004;
Papantchev et al. 2006; Hinova-Palova et al. 2007). Cal-
culation was made with an ultrastructural size calculator
(Ted Pella, Tustin, CA, USA). In addition a total number of
200 nNOS-ir terminal boutons were analyzed in order to
determine synaptic morphology and characteristics of the
vesicular population.
Light microscope (Olympus, Tokyo, Japan) was also
used for slice examination. The DC was examined
according to stereotaxic atlas of Reinozo-Suarez (1961).
Morphometrical study
Our morphometrical protocol was described in details
elsewhere (Hinova-Palova et al. 2007). In brief—all ste-
reotaxic planes from A10 to A19 (Reinozo-Suarez 1961)
were examined using an image analyzer (CUE-2, Olympus
America, Center Valley, PA, USA) and a 40 objective. A
total number of 48 slides (3 randomly selected slides from
each of all 16 tissue block examined) per stereotaxic plane
were studied. The number of nNOS-ir neurons were
counted out for each section separately and collected in
database. For each stereotaxic plane the amount of nNOS-ir
cells were calculated as an average from the number of
enumerated neurons from all sections per plane. Data for
the amount of neurons for each stereotaxic plane were
presented as a percentage from all nNOS-ir neurons
counted out throughout the entire DC. Afterwards, standard
planar morphometry, including linear analysis (i.e., line
length and width) was performed. The maximum diameter
of 550 neurons was measured, and the cells were divided
into groups. A mean of the maximum and minimum
diameter of all neurons in each group was then calculated.
For all measurements only neurons with well visible
nucleus were used.
Later, explanatory marks were added to all images using
Adobe Photoshop 7.0.
Results
Light microscopy
Neurons and fibers immunopositive for nNOS were found
throughout the entire DC (Fig. 1). In general the nNOS-ir
neurons were typically stained—the immunoproduct was vis-
ible in the cell cytoplasm and processes, while the cell nucleus
remained free (Fig. 1). Intensity of immunolabeling was dif-
ferent amongst the nNOS-ir neuronal population. Some nNOS-
ir neurons were lightly stained, while others were so darkly
labeled that they looked more like silver impregnated (Fig. 1b–
f). Immunopositive neurons were found also near to and inside
the external and extreme capsule (Fig. 1a, e, f).
The distribution of the neurons was not uniform
throughout the DC. Approximately 60% of nNOS-ir neu-
rons were present at the stereotaxic planes A12–A16. The
majority of nNOS-ir neurons were present as clusters in the
central triangle of DC (planes A13–A15; Fig. 1d) while
only few were found near to the external and extreme
capsule (Fig. 1e, f). There was some nNOS-ir within the
external and/or extreme capsule (Fig. 1e). The neuronal
orientation was parallel to the capsular fibers (Fig. 1e).
However, some neurons were oriented perpendicular to
them (Fig. 1f). Moving caudally the number of nNOS-ir
neurons gradually diminishes. Thus at the level of stereo-
taxic planes A10-A11 only 25% of all nNOS-ir neurons
were identified. In the stereotaxic planes A17-A18 (rostral
portion of the DC) approximately 10% of all nNOS-ir
neurons were identified. Finally, the rostral pole of the DC
contains no more than 5% of all nNOS-ir neurons.
The nNOS-ir neurons were different in shape and size.
Oval, fusiform, triangular, elongated, multipolar and irreg-
ularly shaped nNOS-ir neurons were observed (Fig. 1).
J Mol Hist
123
According to their size the nNOS-ir neurons were subdivided
into small (under 15 lm), medium-sized (16–20 lm) and
large (over 21 lm). The small nNOS-ir neurons were always
oval or fusiform in shape, while medium-sized and large
nNOS-ir neurons show all described shapes (Fig. 1). The
large neurons have up to six long dendrites with varicose
appearance (Fig. 1b–d). Usually the secondary and tertiary
dendrites were present (Fig. 1b–d). On the contrary the
dendrites of the small neurons were always short. The nNOS-
ir fibers were found to form a background staining (Fig. 1).
Many puncta were also present (Fig. 1b–d).
Electron microscopy
The ultrastructural analysis demonstrated immuneproduct
in neuronal perikarya, dendrites, dendritic spines, axons
(both myelinated and unmyelinated), and terminal synaptic
boutons (SB; Figs. 2 and 3). The ultrastructural features of
different types of nNOS-ir neurons were also different.
Small nNOS-ir neurons
Approximately 30 % of nNOS-ir neurons were classified in
this group (Fig. 2a). Most of these neurons were darkly-
stained. They showed the following ultrastructural char-
acteristics: size under 15 lm; relatively large cell nucleus;
a great amount of heterochromatin; thin rim of cytoplasm
around the nucleus (less than 1 lm); a limited number of
organelles (few small mitochondria, a low number of free
ribosomes, scattered cisterns of granular endoplasmatic
reticulum); relatively well developed Golgi apparatus; a
low number of SB (labeled and unlabeled) on the neuronal
surface. Usually, a lot of sections were necessary for their
visualization. The heavily stained neurons usually had a
deep invagination on the nuclear envelope (Fig. 2a).
Medium-sized nNOS-ir neurons
Approximately 40 % of all nNOS-ir neurons were classi-
fied as medium-sized (Fig. 2b, c). These neurons were
usually darkly-stained and have diameter from 16 to
20 lm; a relatively large cell nucleus, with a high amount
of euchromatin; many mitochondria; well developed Golgi
apparatus; a small amount of Nissl bodies; a greater
number of SB (both labeled and unlabeled) on the neuronal
surfaces compared with small neurons (Fig. 2b, c).
Large nNOS-ir neurons
Approximately 30% of all nNOS-ir neurons were included
in this group (Fig. 2d). These neurons were usually lightly-
stained and have size greater than 21 lm. However as an
exception darkly-labeled large neurons were also found
(Fig. 2d). Ultrastructurally these neurons contained an
abundance of cytoplasm and organelles—mitochondria
(sometimes over 150 in number), many Nissl bodies, well
developed Golgi apparatus, a lot of free ribosomes, a large
centrally located cell nucleus filled with euchromatin, with
prominent nucleolus; many labeled and unlabeled SB on
the cell surface (Fig. 2d).
Lightly and darkly stained nNOS-ir neurons
Both lightly and darkly stained nNOS-ir neurons were
analyzed separately. In general the ultrastructural appear-
ance of both type nNOS-ir neurons was not similar (Fig. 2).
The lightly-stained neurons were usually medium or large
in size, while the darkly-stained neurons were usually small
Fig. 1 Light microscopic appearance of nNOS-ir neurons and fibers of
dorsal claustrum (DC). (a) Low magnification of DC. Bar 500 lm; (b)
An oval, darkly-stained medium-sized nNOS-ir neuron form DC. Note
the tertiary dendrites and their varicose appearance. Bar 30 lm; (c) Two
nNOS-ir neurons form DC – one large nNOS-ir neuron multipolar in
shape (black arrowhead) and one medium-sized nNOS-ir neuron
triangular in shape (white arrowhead). Note the typical dendritic
arborization of the large nNOS-ir neuron and the small dendrite of the
medium-sized one. Bar 30 lm; (d) A cluster of nNOS-ir neurons for
DC. There are three lightly-stained nNOS-ir neurons (one medium-
sized and two large) and one large darkly-stained nNOS-ir neuron. Note
the long dendrite of the darkly-stained neuron (small arrowheads). Bar
30 lm; (e) Low magnification of the DC and external capsule. Note the
nNOS-ir neurons located in the capsule (black arrowhead). Bar 200 lm;
(f) Small darkly-stained fusiform nNOS-ir neuron from DC. Note the
short dendrites of this neuron. Bar 10 lm
J Mol Hist
123
and rarely medium-sized. The darkly-stained small and
medium-sized neurons usually have a deep invagination on
the nucleolar envelope (Fig. 2a, b). There was also a dif-
ference in electron density, which was significantly greater
in the subgroup of darkly-stained nNOS-ir neurons
(Fig. 2).
Neuronal NOS immunopositive structures
of the neuropil
The immunoproduct was seen in dendrites, myelinated
axons, and terminal boutons throughout the entire DC
(Figs. 3 and 4).
Neuronal NOS immunopositive dendrites
Dendrites immunopositive for nNOS were different in
size—from small (0.5–1 lm) to large (up to 3 lm). The
immunoproduct was associated with the axial neurofila-
ments in the dendrites (Figs. 3a–g and 4d). Immunolabeled
dendrites were both spiny (Fig. 3f, g) and aspiny (Fig. 3a–
e). A great number of both labeled (Figs. 3d–g and 4d and
4f) and unlabeled (Fig. 3a–c) SBs were found to terminate
on nNOS-ir dendrites.
Neuronal NOS immunopositive synaptic boutons
A variety of synaptic terminals were found (Figs. 3 and 4).
There were axo-somatic; axo-dendritic and axo-spinous
synaptic terminals. SBs varied in shape, size and vesicular
morphology (Figs. 3 and 4). Following synaptic terminals
were found—large round (LR), small round (SR) and
pleomorphic (P) (Figs. 3 and 4).
Approximately 70% of all nNOS-labeled terminal bou-
tons contained LR synaptic vesicles (Figs. 3d, e, and 4c–f).
These terminals have irregular appearance and diameter
between 1.5 and 3.5 lm (Figs. 3d, e, 4c–f). Their vesicles
were round, with average diameter of approximately 40 nm
(Figs. 3d, e, 4c–f). As a rule mitochondria were present in
the terminal (Figs. 3d, e, 4c–f). Unlabeled terminals with
LR appearance were also found (Fig. 3a–d). Most often LR
synaptic boutons terminate on medium and large nNOS-ir
dendrites (Figs. 3a–g and 4d). Sometimes a LR synaptic
bouton terminates on more than one dendrite (Fig. 4f).
Some LR terminal boutons form axo-somatic synapses
(Fig. 4c, e). Synaptic contacts were of the asymmetric type
(Figs. 3d, e and 4c, d–f).
Approximately 25% of all terminal boutons contain SR
vesicles (Figs. 3g and 4a). These terminals were small with
diameter between 0.5 and 1.2 lm (Figs. 3g and 4a). The
synaptic vesicles were with average diameter of approxi-
mately 30 nm (Figs. 3g and4a).
Fig. 2 Electron microscope appearance of nNOS-ir neuron. (a) Small
nNOS-ir neuron. Note the small amount of cytoplasm and the
relatively large nucleus. Note the deep invagination of the nuclear
envelope (white arrowhead). Bar 3 lm; (b) Medium-sized nNOS-ir
neuron. This neuron was darkly-labeled on the light microscope level.
Note the deep invagination of the nuclear envelope (white arrow-
head). Bar 5 lm; (c) Medium-sized nNOS-ir neuron. This neuron was
lightly-labeled on the light microscope level. Bar 5 lm; (d) Large
nNOS-ir neuron. This neuron was darkly-labeled on the light
microscope level. Note the large amount of cytoplasm and the large
number of terminal boutons on the neuron’s surface. Bar 5 lm
J Mol Hist
123
No more than 5% of all terminals contain P type vesicles
(Fig. 3f). These terminals have longer diameter of
approximately 4 lm (Fig. 3f) and they contain different
types of vesicles—oval (with diameter 35–40 nm), ellip-
tical (with diameter 30–40 nm) and typically elongated
(with longer diameter 35–40 nm).
Discussion
Our present data are compared with all major published
data on cat (Rahman and Baizer 2007), rats (Rodrigo
et al. 1994; Guirado et al. 2003), rabbits (Pro-Sistiaga
et al. 2002) and mice (Kowianski et al. 2002). Rahman
and Baizer (2007) are first who mentioned the existence
of nNOS-ir neurons in cat’s claustrum. These authors
reported that nNOS-ir neurons have size of ‘‘about
20–25 lm’’. These sizes correspond with large and med-
ium-sized neurons described here. The authors do not
mentioned anything about the presence of small nNOS-ir
neurons (size under 15 lm). Since Rahman and Baizer
(2007) do not give a detailed description of the light
microscopic characteristics of the nNOS-ir neurons we
could suggest that the presence of small neurons was
overlooked. This suggestion is also based on the fact that
the study of Rahman and Baizer (2007) look much like
mapping study. Another important difference between our
results and those reported by Rahman and Baizer (2007)
is the fact that these authors do not mentioned anything
about lightly and darkly stained nNOS-ir neurons. Finally,
the mentioned study does not deal with the ultrastructural
characteristics of the nNOS-ir neurons (Rahman and Ba-
izer 2007).
Our present data are in a good agreement with our
previous works (Hinova-Palova et al. 1997) where the
throughout distribution of NADPHd positive neurons in the
DC of cat was reported. In spite of the good correlation
between both NADPHd staining and nNOS-ir (Dawson
et al. 1991b; Hope et al. 1991; Lysakowski and Singer
2000), some remarks must be made:
Fig. 3 Ultrastructural
characteristics of nNOS-ir
structures of the neuropile. (a)
Longitudinal section through a
nNOS-ir dendrite (D). Bar
3 lm; (b) Transverse section
through a small nNOS-ir
dendrite (D). Two unlabeled LR
synaptic boutons (SB)
terminates on its surface. Bar
0.5 lm; (c) Transverse section
through a large nNOS-ir
dendrite (D). Note the four
unlabeled LR synaptic terminals
on its surface (SB). Bar 0.5 lm;
(d) Transverse section through a
large nNOS-ir dendrite (D).
Note the nNOS-ir terminal
bouton, containing large round
vesicles (LR). Bar 0.5 lm; (e)
Small nNOS-ir dendrite (D) on
which terminates a single
terminal bouton, containing
large round vesicles (LR). Bar
0.5 lm; (f) Transverse section
through a large nNOS-ir
dendrite (D) in contact with a
nNOS-ir synaptic terminal,
containing pleomorphic vesicles
(P). Note the dendritic spine
(Sp). Bar 1 lm; (g) Transverse
section through a large nNOS-ir
dendrite (D) on which surface
terminates a single synaptic
terminals, containing small
round (SR) vesicles. Note the
dendritic spine (Sp). Bar 1 lm
J Mol Hist
123
First, the NADPHd staining results in significant back-
ground staining and it reveals more neuronal processes
with axonal appearance. Similar observations were repor-
ted also by others (Guirado et al. 2003).
Secondary, as we reported earlier (Papantchev et al.
2003; Papantchev et al. 2006) NADPHd staining could
result in some significant artifacts.
Thirdly, the NADPHd staining results in more uniform
and heavy labeling of the neurons and the difference in
staining (lightly versus darkly) is masked. Similar obser-
vations were reported also by others (Guirado et al. 2003).
Guirado et al. (2003) reported in mouse claustrum the
presence of darkly and lightly stained nNOS-ir neurons. In
addition, these authors proved that most of the darkly-
stained nNOS-ir neurons were also GABAergic, while
most of the lightly-stained nNOS-ir neurons were non-
GABAergic (Guirado et al. 2003). In our present work both
darkly and lightly-stained nNOS-ir neurons were found.
We also observed that the darkly-stained neurons were
mainly small or medium-sized neurons, while lightly-
stained nNOS-ir neurons were medium-sized or large.
It is well known that differences in the shape and size of
neurons have different functions in the nervous system
(Blumcke et al. 1991; Vater and Braun 1994; Paloff et al.
2004; Papantchev et al. 2005, 2006). One of the aims of the
present study was to verify whether nNOS-ir is present in
distinct subpopulations of claustral neurons. As we repor-
ted above, the observed population of nNOS-ir neurons
was not homogeneous. It consisted of small, medium-sized
and large neurons.
Ultrastructural analysis demonstrated that large nNOS-ir
neurons contained an abundance of cytoplasm, and were
rich in organelles (e.g. large mitochondria, granular endo-
plasmic reticulum with distinct Nissl bodies, and a well-
defined Golgi complex). Their nucleus displayed a pre-
ponderance of euchromatin. These entire characteristics
respond to neurons with a significant metabolic activity.
Furthermore, a long branching dendrites of large nNOS-ir
neurons show that these neurons received a significant
synaptic input. All these light and electron microscope
characteristics of the large nNOS-ir neurons show that
these neurons are projective. This suggestion was proved
by our previous works (Hinova-Palova et al. 1988). Med-
ium-sized nNOS-ir neurons had a lesser volume of
cytoplasm, with relatively low amounts of granular endo-
plasmic reticulum. In point of fact, there are several
subtypes of medium-sized neurons in the claustrum (Hi-
nova-Palova 1986). However, for the purposes of this
study, we were not able to confirm that medium-sized
nNOS-ir claustral neurons correspond to a specific subtype.
We could suggest that some of these neurons are projec-
tive, because of their light and ultrastructural morphology.
The small nNOS-ir neurons corresponded to the subtypes
described earlier by Hinova-Palova (1986). These neurons
displayed a relatively large nucleus, small volumes of
cytoplasm, a paucity of organelles, and very few axoso-
matic synapses—morphological criteria that are commonly
attributed to local circuit neurons, also known as inter-
neurons (Morest, 1971; Lieberman 1973; Paloff 1985;
Romansky and Usunoff 1985; Paloff et al. 1989, 1992a, b,
1998; Paloff and Hinova-Palova 1998; Usunoff 1990).
Furthermore, most of small nNOS-ir neurons were darkly-
stained. In the light of observations of Guirado et al. (2003)
that most of the small darkly-stained neurons show in
Fig. 4 Ultrastructural characteristics of nNOS-ir structures of the
neuropile. (a) Showing a nNOS-ir terminal bouton, containing small
raound vesicles and forming an axo-somatic contact with lightly-
labeled large nNOS-ir neuron. Bar 1 lm; (b) Immunopositive
myelinated axon. Bar 0.5 lm; (c) Showing a nNOS-ir terminal
bouton, containing large round (LR) vesicles and forming two
asymmetrical axo-somatic contacts with lightly-labeled large nNOS-ir
neuron (Soma). Bar 1 lm; (d) Showing a pair of nNOS-ir dendrites
(D1 and D2). On one of them (D1) terminates a single nNOS-ir
bouton, containing large round vesicles (LR1). On the other nNOS-ir
dendrite (D2) terminate one nNOS-ir terminal, containing large round
vesicles (LR2) and one unlabeled terminal (LR3). Note that the nNOS-
ir terminal (LR2) makes an additional synaptic contact with unlabeled
dendrite (D3). Bar 2 lm; (e) Showing darkly-stained medium-sized
nNOS-ir neuron (Soma) on which surface terminate two large round
boutons—one labeled (LR1) and one unlabeled (LR2). There is also a
single unlabeled dendrite (D). Bar 2 lm; (f) Showing a nNOS-ir
synaptic bouton, containing large round vesicles (SB), which makes
synapses with two labeled dendrites (D). In the bottom left a single
labeled myelinated axon could be seen. Bar 1 lm
J Mol Hist
123
claustrum of mice are also neurons GABAergic we could
conclude that darkly-stained neurons in DC of a cat rep-
resent a subpopulation of local circuit inhibitory
interneurons.
In support of our above mentioned conclusion we must
emphasize that the Golgi impregnation studies of the DC in
various species of mammal have lead to the description of
two major functional classes of neurons: projection neu-
rons—with polymorphic perikarya and spiny dendrites, and
inhibitory interneurons—characterized by round or oval
perikarya and aspiny dendrites (Brand 1981; LeVay and
Sherk 1981a, b; Braak and Braak 1982; Mamos 1984;
Spahn and Braak 1985; Druga et al. 1993; Rowniak et al.
1994; Wojcik et al. 2004). In the present study, we
observed both classes of neurons in the cat DC. Thus, our
light- and electron-microscopic investigation strongly
suggests that nNOS-immunostaining was present in all
neuronal types within the DC. The fact that nNOS-immu-
noreactivity was observed in neurons of varying shapes and
sizes suggest that these cells play differing roles in the
context of claustral function (Kowianski et al. 2002).
Kowianski et al. (2003) proved a co-localization
between nNOS and calbindin (CB) in DC neurons. Same
authors reported also a co-localization between nNOS and
parvalbumin (PV). This findings suggest the importance of
NO and PV for the process of maturation of DC. Later
Kowianski et al (2004) reported the presence and
co-localization between nNOS and different neurotrans-
mitters (neuropeptide Y) and calcium-binding proteins
(PV, calretinin) in ventral claustrum. Since calcium-bind-
ing proteins (CaBP) as a rule are co-localized with GABA
the presence of CaBP can be used as a marker of inhibitory
neurons (Kowianski et al., 2004).
An immunocytochemical co-localization of nNOS and
glutamate NMDA receptor 1 (NMDAR1) was already
shown in different region of the central nervous system
(Dohrn and Beitz 1994, Aoki et al. 1997, Gracy and Pickel
1997, Lin and Talman 2000). It is well known that NO
could be toxic in certain circumstances (Dawson et al.
1991a; Dawson et al. 1993, Bishop and Anderson 2005).
The importance of both neuronal and inducible isoformes
of NOS for the neuronal damage, via NO production was
also reported (Samdani et al. 1997, Wada et al. 1998,
Dalkara and Moskowitz 1994, Dawson 1994, Iadecola
1997). The presence of great amount of nNOS-ir neurons
could explain the cytotoxic effect of NO in claustral region
after excessive NMDA stimulation.
The last aim of this study was to verify whether nNOS-ir
neurons have a well-defined topographical distribution
throughout the DC. In fact, such an organization was not
found. Despite of the fact that the central part of DC
(stereotaxic planes A12 – A16) contains the majority of
nNOS-ir neurons these were distributed throughout the
entire DC. No clustering or grouping was found within
different functional regions of DC. Similar results were
reported by others in cats (Rahman and Baizer 2007) rat
(Guirado et al. 2003) and mice (Kowianski et al. 2002).
The absence of a distinct heterotopic arrangement of
nNOS-ir DC neurons does not fit with its previously
described projection zones (Carey et al. 1980; Pearson
et al. 1982; Macchi et al. 1983; Sherk 1988; Morys et al.
1996; Sadowski et al. 1997; Kowianski et al. 1998; Real
et al. 2003; Wojcik et al. 2004). On the other hand, it is
generally accepted that the number and types of synapses is
a principal factor influencing neuronal function (Czeiger
and White 1997). Stated another way, morphologically
identical neurons should function and respond differently,
provided the characteristics of their synaptic inputs are
different. Therefore, it could be speculated that the similar
nNOS-ir neurons we have found throughout the cat DC are
not as similar as one might think.
In conclusion, this is the first complex study of light and
electron microscope features of nNOS-ir neurons and fibers
in DC of the cat. We hope that this study could add
something unique to the literature, expand the data analysis
and that it could contribute to a better understanding of the
functioning of the DC. We also believe that some overall
general conclusions could be drawn from this work and
hopefully be extrapolated to other mammals, particularly
human.
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