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: vassil_papanchev@yahoo.com

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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