Journal of Chemical Neuroanatomy 61–62 (2014) 107–119

Neuropeptide Y immunoreactivity in the cat claustrum:A light- and electron-microscopic investigation

D.V. Hinova-Palova a, B. Landzhov a,*, E. Dzhambazova b, M. Minkov c,L. Edelstein d, L. Malinova a, A. Paloff a, W. Ovtscharoff a

a Department of Anatomy, Histology and Embryology, Medical University of Sofia, 1431 Sofia, Bulgariab Department of Chemistry, Biochemistry, Physiology and Pathophysiology, Sofia University ‘‘St. Kliment Ohridski’’, 1407 Sofia, Bulgariac Department of Anatomy, Histology and Embryology, Medical University of Varna, 9002 Varna, Bulgariad Medimark Corporation, P.O. Box 2316, Del Mar, CA 92014, USA

A R T I C L E I N F O

Article history:

Received 19 May 2014

Received in revised form 15 August 2014

Accepted 16 August 2014

Available online 23 August 2014

Keywords:

Cat

Claustrum

Neuropeptide Y

Light microscopy

Electron microscopy

Ultrastructure

A B S T R A C T

The claustrum is a telencephalic nucleus located ventrolateral to the basal ganglia in the mammalian

brain. It has an extensive reciprocal connectivity with most if not all of the cerebral cortex, in particular,

primary sensory areas. However, despite renewed and growing interest amongst investigators, there

remains a paucity of data concerning its peptidergic profile. The aim of the present study was to examine

the presence, morphology, distribution and ultrastructure of neuropeptide Y-immunoreactive (NPY-ir)

neurons and fibers in the claustrum of the cat. Ten adult healthy cats from both sexes were used. All

animals received human and ethical treatment in accordance with the Principles of Laboratory Animal

Care. Subjects were irreversibly anesthetized and transcardially perfused with fixative solution

containing glutaraldehyde and paraformaldehyde. Brains were promptly removed, postfixed and

sectioned. Slices were incubated with polyclonal anti-NPY antibodies according to the standard avidin–

biotin–peroxidase complex method adopted by our Department of Anatomy, Histology and Embryology.

NPY-ir neurons and fibers were found to be diffusely distributed throughout the claustrum, with no

obvious topographic or functional patterning other than larger numbers in its central/broadest part

(stereotaxic planes A12–A16). Neurons were generally classified by diameter into three sizes: small

(under 17 mm), medium (17–25 mm) and large (over 25 mm). Staining density is varied with some

neurons appearing darker than others. At the electron-microscopic level NPY immunoproduct was

observed within neurons, dendrites and terminal boutons, each differing relative to their ultrastructural

attributes. Two types of NPY-ir synaptic boutons were found. Lastly, it is of interest to note that gender-

specific differences were not observed.

� 2014 Elsevier B.V. All rights reserved.

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Journal of Chemical Neuroanatomy

jo ur n al ho mep ag e: www .e lsev ier . c om / lo cate / jc h emn eu

1. Introduction

The claustrum is a telencephalic nucleus in the mammalianbrain that has long been known as a site of extensive reciprocalheterosensory convergence (Olson and Graybiel, 1980; Ashwellet al., 2004; Hinova-Palova et al., 2007, 2008). Typically, it issubdivided into the dorsal claustrum (or ‘‘insular claustrum’’) andventral claustrum (or ‘‘endopiriform nucleus’’) (Guirado et al.,2003; Edelstein and Denaro, 2004a; Ashwell et al., 2004). The

* Corresponding author at: Medical University of Sofia, Faculty of Medicine,

Department of Anatomy, Histology and Embryology, 2 Zdrave Str., 1431 Sofia,

Bulgaria. Tel.: +359 29172608; fax: +359 29172601.

E-mail address: landzhov_medac@abv.bg (B. Landzhov).

http://dx.doi.org/10.1016/j.jchemneu.2014.08.007

0891-0618/� 2014 Elsevier B.V. All rights reserved.

dorsal claustrum is located deep to the insular cortex and isextensively connected with most if not all neocortex (Druga,1966a,b; Otellin and Makarov, 1972; Kunzle, 1975, 1978; Norita,1977; Riche and Lanoir, 1978; Olson and Graybiel, 1980; Careyet 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; Ashwell et al., 2004). The ventral claustrum issituated beneath the piriform cortex; its interconnections withprepiriform and entorhinal cortex are well documented (Druga,1966a,b, 1971; Sherk, 1986; Witter et al., 1988; Dinopoulos et al.,1992).

From a phylogenetic perspective, the dimensions of theclaustrum vary greatly. In lower mammals such as the mouseand rat it is a small and discrete nucleus ventrolateral to the

D.V. Hinova-Palova et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 107–119108

caudoputamen. In the cat it is a relatively large structure thatrivals, or even exceeds, the size of the putamen. In primates it is athin gray slab varying in thickness up to several millimeters,bounded laterally by the extreme capsule and medially by theexternal 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).

We have long been interested in the claustrum’s cytoarchi-tecture, ultrastructure, immunocytochemistry and connectivity(Hinova-Palova et al., 1979, 1980a,b, 1988, 1997, 2001, 2007, 2008,2012, 2013a,b,c; Hinova-Palova, 1981, 1986; Hinova-Palova andUsunoff, 1981; Hinova-Palova and Christova, 1988; Hinova-Palovaand Braak, 1993; Edelstein et al., 2011a,b, 2012a,b). Theclaustrum’s syncytium of GABAergic interneurons and glutama-tergic projection neurons, as well as its heterosensory reciprocalconnectivity, leads one to ponder its functional interplay with therest of the brain.

Neuropeptide Y is a novel 36-amino acid peptide and one of thekey regulators of food intake, circadian rhythm and reproduction inmany mammalian species, including humans. Originally isolatedfrom the porcine hypothalamus (Tatemoto, 1982; Tatemoto et al.,1982), NPY is widely distributed throughout the central andautonomic nervous system of various vertebrates, such asmammals (Adrian et al., 1983; Allen et al., 1983; Whale andAlbus, 1984; Emson and De Quidt, 1984; Smith et al., 1985;Chronwall et al., 1985; Sasek and Elde, 1985; de Quidt and Emson,1986; Dumont et al., 1992; Krivukuca et al., 2010), amphians(Danger et al., 1985; Perroteau et al., 1988), teleosts (Pontet et al.,1989; Danger et al., 1991), elasmobranchs (Vallarino et al., 1988;Chiba and Honma, 1992), cyclostomes (Chiba et al., 1993) and thewhite sturgeon (Chiba and Honma, 1994). With respect tophylogenetic comparisons specific to the neurochemical architec-ture of the claustrum, a major focus of our lab, there are but ahandful of studies on NPY immunoreactivity, namely, in thesquirrel monkey (Smith et al., 1985), the cat (Edelstein et al.,2011b) and humans (Edelstein et al., 2010).

The aim of the present study is to make evident the features andcharacteristics of NPY-immunoreactive neurons and fibers in thecat claustrum at the light- and electron-microscopic level, as wellas to provide a description of their distribution.

2. Material and methods

2.1. Perfusion protocol

Ten adult healthy cats from both sexes with average weight 2.3 kg (from 1.9 to

3.0 kg) were used. Our perfusion protocol was previously described in detail

(Papantchev, 2008). In brief, all animals were deeply and irreversibly anesthetized

with an intraperitoneal injection of Urethan (40 mg/kg) and transcardially perfused

with 500 ml of heparinized saline, followed by 3000 ml of phosphate buffered

saline (PBS; pH 7.4) containing 2.5% glutaraldehyde and 4% paraformaldehyde.

After 2 h the brains were removed and postfixed in the same perfusate for

additional 2 h. The portion of each cerebral hemisphere containing the claustrum

was removed, dissected and cut into a total of fourteen blocks. Each block was then

sectioned in the coronal plane at 40–80 mm on a Vibratome (Technical Products

International, St. Louis, MO, USA). The claustrum was examined in accordance with

the stereotaxic atlas of Reinoso-Suarez (1961).

2.2. Immunohistochemistry

Our protocol for the immunohistochemical visualization of NP immunoreactivity

was previously described in detail (Paloff et al., 2004). In brief, all coronal sections as

described above were treated with sodium borohydride for 45 min followed by

three consecutive 2-min rinses in 0.01 M PBS. This was immediately followed by

30 min in a solution of 1% bovine serum albumin (BSA) and overnight incubation in

a 1:1000 solution of a polyclonal anti-NPY-antibody (Sigma, St. Louis, MO, USA).

Afterwards, sections were given three consecutive 2-min rinses in 0.01 M PBS,

incubated for 20 min in 1% BSA in 0.01 M PBS and followed by a 2-h incubation in

1:500 biotinylated anti-mouse IgG (Vector, Burlingame, CA, USA). This was then

followed by three consecutive 2-min rinses in 0.01 M PBS, with sections incubated

for 1-h in a solution of avidin–biotin–peroxidase complex (Vector, Burlingame, CA,

USA). All incubations were carried out on a shaker table at room temperature. For

controls, fifteen sections were incubated as described but with the omission of the

primary or secondary antibody. All controls were negative.

After this 1-h incubation a new series of rinses were performed according to a

protocol used in our Department (Paloff et al., 2004; Hinova-Palova et al., 2007) –

first in 0.01 M PBS and then in Tris buffer, pH 7.6 – which preceded the visualization

of peroxidase activity using H2O2 and 3,30-diaminobenzidine as substrates. The

protocol continued with three 2-min rinses in Tris buffer followed by another three

2-min rinses in phosphate buffer. Afterwards, some of the sections was processed

for electron microscopy. Sections were postfixed for 1-h with 1% OsO4 in phosphate

buffer, dehydrated in graded series of ethanol and flat-embedded in Durcupan

(Fluka, Buchs, Switzerland) between plastic sheets (Paloff et al., 2004; Hinova-

Palova et al., 2007). Sections were trimmed-out under a dissecting microscope and

cemented to epoxy blanks. Thin sections were cut with an ultramicrotome (LKB,

Stockholm-Bromma, Sweden), counterstained with uranyl acetate and lead citrate

according to a standard protocol used in our Department (Paloff et al., 2004;

Hinova-Palova et al., 2007) and examined with an Hitachi H-500 electron

microscope (Hitachi, Tokyo, Japan). A light microscope (Olympus, Tokyo, Japan)

was used for the low-power examination of NPY-ir neurons.

One-hundred neuronal perikarya were identified and measured via electron

micrographs. The NPY-ir neurons (or neuronal groups) were first identified by light

microscope and then serial sections were performed. All consecutive measure-

ments were performed on sections where a prominent nucleolus was present. The

ratio of the mean nuclear diameter to the mean diameter of the perikaryon was

calculated as the nucleocytoplasmic ratio (Paloff et al., 2004; Hinova-Palova et al.,

2007) using an ultrastructural size calculator (Ted Pella, Tustin, CA, USA). In

addition, a total of 200 NPY-ir terminal boutons were analyzed in order to evaluate

the synaptic morphology and characteristics of the vesicular populations.

2.3. Morphometric analysis

Our morphometric analysis protocol was previously detailed (Hinova-Palova

et al., 2007, 2008). Briefly, stereotaxic planes from A10 to A19 (Reinoso-Suarez,

1961) were examined using an image analyzer (CUE-2, Olympus America, Center

Valley, PA, USA) and a 40� objective. A total of 42 slides (3 randomly selected slides

from each of the 14 tissue blocks examined) per stereotaxic plane were studied. The

number of NPY-ir neurons seen in each section were counted and stored in the

database. The number of NPY-ir neurons was calculated for each plane as an average

of the number of counted neurons from all analyzed sections per plane. The number

of NPY-ir neurons counted was presented as a percentage of all NPY-ir neurons seen

in the entire claustrum. Afterwards, standard planar morphometry and linear

analysis (i.e. line length and width) were performed. The maximum diameter of 500

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 those neurons with clearly visible nuclei were used.

Explanatory marks were added to all images using Adobe Photoshop CS3.

3. Results

3.1. Light microscopy

Neurons and fibers immunopositive for NPY-ir were foundthroughout the claustrum, from rostral to caudal planes (Figs. 1and 2). In general, the NPY-ir neurons were typically stained withimmunoproduct visible in the cell cytoplasm and processes, whilethe nucleus remained free of label (Fig. 3). Labeling intensity,however, varied. Some neurons were lightly stained (Fig. 4), whilein others the staining was so dense that they appeared as if theywere silver impregnated (Fig. 5). Immunopositive neurons werealso found proximal to and within the external and extremecapsules (Fig. 6).

Although NPY-ir neurons were seen throughout the claustrum,their distribution displayed a lack of uniformity. Based on ourmorphometric analysis, approximately 70% of labeled cells werelocated in stereotaxic planes A12–A16. Of these cells, the majorityappeared clustered within the claustrum’s central triangle region(planes A13–A15; Fig. 7), with but a handful found proximal to theexternal and extreme capsules. Most commonly, the neurons weresituated parallel to the fiber tracts, though some were positioned ina perpendicular fashion. Moving caudally, the number of NPY-irneurons gradually diminished (Fig. 8), and at the level ofstereotaxic planes A10–A11 only 15% of all counted NPY-irneurons were identified. Approximately 10% of all NPY-ir neurons

Fig. 1. Coronal section through claustrum in different planes from rostral to caudal:

(A) 19 mm in front of the interaural line (arrows delineate claustrum). Nissl staining

20�; (B) 15 mm in front of the interaural line (arrows delineate claustrum). Nissl

staining 20�; (C) 11 mm in front of the interaural line (arrows delineate claustrum).

Nissl staining 20�.

Fig. 2. Schematic drawings of 5 coronal sections through the cat claustrum showing

the distribution of NPY-positive neurons (black dots) from rostral to caudal planes.

Cn – caudate nucleus, Ic – internal capsule, Put – putamen, Sp – septum pellucidum,

Pal – pallidum (globus pallidus), Amg – amygdala, Th – thalamus, Ac – anterior

commissure, Hy – hypothalamus, Ent – entopeduncular nucleus, NII – second

cranial nerve (optic).

Fig. 3. NPY-immunoproduct is clearly visible in the cell cytoplasm and processes,

with the nucleus stain-free. Scale bar = 50 mm.

D.V. Hinova-Palova et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 107–119 109

were identified in the more rostral stereotaxic planes A17–A18.Finally, the rostral pole of the claustrum contained no more than5% of NPY-ir neurons.

The NPY-ir neurons differed in size and were generallysubdivided into large (over 25 mm), medium (17–25 mm), small(under 17 mm) and dwarf cells (under 8 mm). They were furthercategorized into spiny and aspiny types relative to their dendriticarchitecture. The various shapes of NPY-ir neurons seen was notunlike what has previously been noted in similar studies (perhapsowing to the likelihood that most if not all claustral neurons co-express proteins and peptides of one type or another): polygonal(multipolar), triangular, fusiform (bipolar), globular, elliptical, ovaland round.

3.1.1. Large spiny neurons

Large spiny NPY-ir neurons were classified as being greater than25 mm in diameter, with polygonal (multipolar), triangular,fusiform, or globular perikarya (Figs. 9 and 10). They were seengiving rise to as many as five thick dendritic processes, each withmultiple and multidirectional branches. These branches weretypically quite long and could sometimes be followed up to700 mm from the cell body. The secondary and tertiary dendriteswere covered with spines. Axons with highly visible hillocks andinitial segments were frequently seen emanating from these cells.The intensity of immunostaining varied widely – from highlydense (i.e. impregnation-like) to weakly stained. Typically, thenuclei were non-immunoreactive.

3.1.2. Medium spiny neurons

Medium spiny NPY-ir neurons measured from 18 to 24 mm indiameter with multipolar, fusiform, or oval perikarya. They usuallyhad long bifurcated dendrites, each covered with well-developedspines (Fig. 11).

Fig. 4. Large NPY-positive lightly stained neuron. Scale bar = 50 mm.

Fig. 5. Large spiny NPY-positive darkly stained neuron with long dendrites. Scale

bar = 50 mm.

Fig. 6. Large fusiform NPY-positive neuron close to external capsule. Scale

bar = 50 mm.

Fig. 7. Low magnification of triangular part of claustrum showing the distribution of

NPY-positive neurons. Scale bar = 1 mm.

Fig. 8. Low magnification of the caudal part of claustrum showing a lesser number

of NPY-positive neurons. Scale bar = 1 mm.

Fig. 9. Large spiny NPY-positive neuron with fusiform cell body. Scale bar = 50 mm.

D.V. Hinova-Palova et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 107–119110

Fig. 10. Large spiny pear-shaped NPY-positive neuron. Scale bar = 50 mm.

Fig. 11. Two medium spiny NPY-positive neurons with long bifurcated dendrites

rich with spines. Scale bar = 50 mm.

Fig. 12. Small spiny NPY-positive neurons with long spiny dendrites. Scale

bar = 50 mm.

Fig. 13. Large aspiny NPY-positive neuron with long prominent bulbous dendrites

(arrows show the varicosities) Scale bar = 50 mm.

Fig. 14. Medium aspiny multipolar NPY-positive neuron with varicose dendrites

(arrows). Scale bar = 50 mm.

D.V. Hinova-Palova et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 107–119 111

3.1.3. Small spiny neurons

Small spiny NPY-ir neurons measured from 15 to 17 mm indiameter and were typically oval or fusiform in shape with two tofour long and thin dendrites covered with spines (Fig. 12).

3.1.4. Large aspiny neurons

Large aspiny NPY-ir neurons were similar to the large spinyneurons. These cells also averaged over 25 mm in diameter. Theirperikarya were multipolar, triangular, fusiform, or oval in shape(Fig. 13), each with as many as five dendritic processes which –unlike the large spiny neurons – were rarely seen to branch.

3.1.5. Medium aspiny neurons

Medium aspiny NPY-ir neurons ranged from 18 to 23 mm indiameter. Typically, they had three or four dendrites (Fig. 14), withthe primary dendrites being smooth and normally non-branching,becoming progressively thinner with irregularly spaced varicosi-ties along their lengths.

3.1.6. Small aspiny neurons

Small aspiny NPY-ir neurons were typically 14–17 mm indiameter (Fig. 15) with predominantly oval or slightly ellipticalperikarya and typically two or three thin, varicose, non-branchingdendrites.

Fig. 15. Small aspiny elliptical NPY-positive neuron with short varicose dendrites.

Scale bar = 50 mm.

Fig. 17. Part of large NPY-positive neuron. Immunoproduct is seen diffusely

distributed throughout the cytoplasm (C), while the nucleus (N) is free of label.

Scale bar = 3 mm.

D.V. Hinova-Palova et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 107–119112

3.1.7. ‘‘Neurogliaform’’ or ‘‘Dwarf’’ cells

While rarely seen in our sections, we made note of particularlysmall cells, averaging under 8 mm, which we termed ‘‘neuroglia-form’’ or ‘‘dwarf’’. They had round or oval perikarya with numerousshort and intensely branching processes, most with varicosities ofdiffering shape and size (Fig. 16).

3.2. Electron microscopy

The ultrastructural analysis of NPY-ir neurons in our sectionsclearly demonstrated immunoproduct within perikarya, dendrites,dendritic spines, axons (both myelinated and unmyelinated), andterminal synaptic boutons. The ultrastructural features of theseneurons differed relative to their assigned type (i.e. large spiny,medium spiny, etc.).

3.2.1. Large NPY-ir neurons

Approximately 50% of all NPY-ir neurons were included in thisgroup (Fig. 17). These neurons were usually heavy stained and haddiameters over 27 mm. Ultrastructurally, they were seen to containan abundance of cytoplasm and organelles: mitochondria, Nisslbodies, a well-developed Golgi apparatus, many free ribosomes, alarge centrally located nucleus (filled with diffusely distributed

Fig. 16. Two ‘‘dwarf’’ cells with irregular cell bodies and intensely branching short

varicose dendrites (arrows). Scale bar = 50 mm.

euchromatin), and a prominent nucleolus. Many labeled andunlabeled synaptic boutons terminated on the cell surface.

3.2.2. Medium NPY-ir neurons

Approximately 40% of all NPY-ir neurons were classified asmedium in size with a diameter from 18 to 22 mm (Fig. 18).Typically, these neurons were darkly stained with a relatively largecell nucleus, a large amount of euchromatin, many mitochondria, awell-developed Golgi apparatus, and a small amount of Nisslbodies. A greater number of synaptic boutons (both labeled andunlabeled) were noted to have terminated on neuronal surfacescompared with small NPY-ir neurons.

3.2.3. Small NPY-ir neurons

Approximately 10% of NPY-ir neurons were classified as smallin size, usually under 16 mm (Fig. 19). Most of these neurons were

Fig. 18. Medium fusiform NPY-positive neuron. Immunoproduct is distributed

throughout the cytoplasm and nucleus. Scale bar = 5 mm.

Fig. 19. Two small NPY-positive neurons. One of them contains heavy a deposition

of immunoproduct (D), the other, less so (L). Scale bar = 5 mm.

Fig. 20. Large NPY-positive dendrite (D) on which terminate two unlabeled boutons

(T). Scale bar = 1 mm.

Fig. 21. Large NPY-positive dendrite (D) on which terminates a large NPY-positive

synaptic terminal (T). Scale bar = 0.5 mm.

D.V. Hinova-Palova et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 107–119 113

darkly stained and evinced several defining characteristicsincluding a relatively large cell nucleus, a large volume ofheterochromatin, a thin rim of cytoplasm surrounding the nucleus,a paucity of organelles (with few small mitochondria and freeribosomes), scattered cisterns of granular endoplasmatic reticu-lum, and a well-developed Golgi apparatus. Also, a comparativelysmaller number of synaptic boutons (labeled and unlabeled) wereseen to have terminated on the neuronal surface. More often thannot, we had to view quite a few sections in order to spot one withthese cells. These heavily stained neurons usually had a deepnuclear invagination.

3.2.4. Lightly and darkly stained NPY-ir neurons

Lightly and darkly stained NPY-ir neurons were analyzedindividually. In general, their ultrastructural appearance wasdissimilar. Lightly stained neurons were usually medium or largein size, while darkly stained neurons were small and rarelymedium in size. The darkly stained small and medium neuronsoften had a deep invagination of the nucleolar envelope. There wasalso a difference in electron density, which was significantlygreater in the subgroup of darkly stained NPY-ir neurons.

3.2.5. NPY-ir dendrites

NPY-immunoreactive dendrites differed in size from 0.5 mm(small) up to 3 mm in diameter (large). Immunoproduct wasassociated with the axial neurofilaments in the dendrites (Fig. 20),and was seen in both spiny and aspiny types. The majority of bothlabeled and unlabeled synaptic boutons were found to terminateon NPY-ir dendrites (Figs. 21–23).

3.2.6. NPY-ir synaptic boutons

With regard to synaptic terminals, three main categories werenoted: axo-somatic, axo-dendritic and axo-spinous. They varied inshape, size and vesicular morphology (Figs. 21, 22 and 24). In thecontext of synaptic terminals, two distinct types of boutons wereidentified relative to vesicular diameter: large round or ‘‘LR’’(Figs. 21, 22 and 24) and small round or ‘‘SR’’ (Fig. 26).

Approximately 75% of NPY-ir boutons were of the LR type(Figs. 21, 22 and 24) with an irregular appearance and a diameterfrom 1.5 to 3.5 mm (Figs. 21, 22 and 24). Their vesicles were roundwith an average diameter of 40 nm (Figs. 21 and 24) andmitochondria were always present (Figs. 21 and 24). Most often,LR boutons terminated on medium and large dendrites (Fig. 23),although unlabeled boutons were also found (Fig. 23). On occasion

an LR bouton was seen to terminate on more than one dendritewhiles some formed axo-somatic synapses (Fig. 25). Synapticcontacts by LR boutons were of the asymmetric type.

Approximately 25% of all NPY-ir boutons were of the SR typewith a diameter from 0.5 to 1.2 mm (Fig. 26). The synaptic vesicleswere somewhat smaller in size than the LR type, with an averagediameter of 30 nm (Fig. 26). On rare occasion we observed heavilylabeled ‘‘en passant’’ boutons (Fig. 27).

3.2.7. NPY-ir neuropil

Immunoproduct was prevalent in dendrites, myelinated axons,and terminal boutons throughout the claustrum.

Fig. 22. NPY-positive terminal bouton (T) establishes synaptic contact with a large

labeled NPY-positive dendrite (D). Scale bar = 1 mm.

Fig. 23. NPY-positive dendrite (D) on which terminate two unlabeled boutons (T).

Scale bar = 2 mm.

Fig. 24. Labeled NPY-positive large round synaptic bouton (T) terminates on two

unlabeled medium dendrites (D). Scale bar = 1 mm.

Fig. 25. Labeled NPY-positive bouton (T) contacting an NPY-positive neuron. Scale

bar = 1 mm.

D.V. Hinova-Palova et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 107–119114

4. Discussion

To the best of our knowledge, the present study is the first toprovide detailed insight into the topographical distribution andmorphological characteristics of NPY-ir neurons in the catclaustrum at the light- and electron-microscopic level. Overall,our findings are consistent with those reported for the squirrelmonkey, Saimir sciureus (Smith et al., 1985), cat (Edelstein et al.,2011b) and human (Edelstein et al., 2010) in which NPY-ir neuronswere recognized in the claustrum of those species.

In this investigation, we report the existence of at least sevenreadily distinguishable types of NPY-ir neurons in the cat

claustrum based on shape, namely: polygonal (multipolar),triangular, fusiform (bipolar), globular, elliptical, oval and round.We further categorized each of these cells into four main subtypesbased on diameter: large, medium, small and ‘‘dwarf,’’ and theninto spiny and aspiny relative to their dendritic architecture. Theobserved population of NPY-ir neurons was heterogeneous incomposition which, though seen in higher numbers within thetriangular dorsal region (A13–A15) in comparison with morerostral and caudal planes, was reflected throughout the claustrum.

When ruminating on the function of the claustrum, we mustsurely take into consideration its anatomy and connectivity, as

Fig. 26. NPY-positive dendrites (D) and terminal boutons of differing size and shape

(arrows). Scale bar = 1 mm.

Fig. 27. Heavy labeled ‘‘en passant’’ NPY-positive bouton (arrows) terminates on the

periphery of an NPY-positive neuron. Scale bar = 2 mm.

D.V. Hinova-Palova et al. / Journal of Chemical Neuroanatomy 61–62 (2014) 107–119 115

well as the ultrastructure of its neurons. Our electron-microscopicanalysis of the cat claustrum revealed that large NPY-ir neuronscontained an abundance of cytoplasm rich in a variety oforganelles. In addition, their nuclei displayed a preponderanceof euchromatin, with mitochondria-laden terminal boutons.Characteristics such as these are reflective of cells with significantmetabolic activity. Furthermore, the long and multibranched

dendrites of these large neurons – especially those of the spinyvariety – offer support the contention that these neurons receive asignificant volume of synaptic input. Both the light- and electron-microscopic characteristics of these large cells are indicative ofprojection neurons rather than interneurons. This was confirmedby our previous research (Hinova-Palova, 1986).

Medium NPY-ir neurons typically had a lesser volume ofcytoplasm with a relative paucity of granular endoplasmicreticulum. In point of fact, several subtypes of medium neuronshave been found in the cat claustrum (Hinova-Palova, 1986).However, for the purposes of this study, we were unable to confirmthat the medium NPY-ir neurons we found corresponded to aspecific subtype. Nonetheless, it is not unreasonable to assumethat some of these cells are projection neurons owing to their light-microscopic and ultrastructural morphology.

Small NPY-ir neurons corresponded to the subtypes previouslydescribed by Hinova-Palova (1986). These neurons displayed arelatively large nucleus, a thin rim of cytoplasm, a paucity oforganelles and very few axo-somatic synapses – morphologicalcriteria that are commonly attributed to local circuit neurons, alsoreferred to as interneurons (Paloff, 1985; Paloff et al., 1989).Furthermore, most of the small NPY-ir neurons that we found wereseemingly filled or impregnated with immunoproduct. On thebasis of their light-microscopic and ultrastructural characteristics,we propose that these densely labeled small NPY-ir neurons arepart of a subpopulation of local circuit inhibitory interneurons.

We also observed NPY-ir dwarf cells in the present study.Ramon and Cajal (1911) was the first to recognize these cells in thestriatum by using the Golgi impregnation method. In previousinvestigations (Hinova-Palova, 1986; Hinova-Palova et al., 1987)we demonstrated the existence of dwarf cells in the cat claustrumvia light- and electron-microscopy. Mounting evidence leads us tobelieve that these cells are also interneurons.

To elaborate, the small and medium aspiny NPY-ir neurons seenin the present investigation were predominantly oval-to-fusiformin shape. In contrast, the large and medium spiny NPY-ir neuronswere often multipolar-to-pyramidal in shape. Based on the extantliterarture on this topic, it is not unreasonable to assume that theaspiny neurons represent a subpopulation of local circuitinhibitory interneurons (along with the dwarf cells), while thespiny neurons represent projection neurons. It was previouslyreported that the large spiny neurons in the cat claustrum receivedirect input from cortex and were also shown to project back to thesame areas of cortex, establishing excitatory synapses (Hinova-Palova, 1986; Hinova-Palova et al., 1988; Reynhout and Baizer,1999). Using the Golgi impregnation method in the monkeyclaustrum, Brand (1981) reported that the axons of large neuronswere efferent in nature, projected out of the claustrum. Moreover,in some species, it was reported that the overwhelming majority ofclaustral neurons are glutamatergic projection neurons, with only6–12% of cells engaged in local network activity (Wojcik et al.,2004).

The existence of projection neurons in the cat claustrum wasalso supported by our present study concerning the features oflarge, medium and small spiny NPY-ir neurons. In some sections,the entire initial segment of an NPY-ir myelinated axon waspresent. However, it must be noted that some of these axons werefound not to originate in the claustrum, and thus were afferent innature. Neverthless, the existence of afferent and efferentprojection fibers in claustrum has been suggested by Wojciket al. (2004).

Golgi impregnation studies of the mammalian claustrum havelead to the description of two major functional classes of neurons –projection neurons with polymorphic perikarya and spinydendrites, and inhibitory interneurons characterized by round-to-oval perikarya and aspiny dendrites (Brand, 1981; Braak and

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Braak, 1982; Spahn and Braak, 1985; Hinova-Palova, 1986; Drugaet al., 1993; Wojcik et al., 2004). Our current findings also revealtwo major classes of NPY-ir neurons in the cat claustrum: spinyand aspiny. In a previous study on the ultrastructure of the NPY-labeled cat claustrum (Edelstein et al., 2011b), we reported on theexistence of immunoproduct within dendritic spines of differingshape and size. Thus, our light- and electron-microscopicinvestigations strongly suggest that NPY-immunoreactivity ispresent in all neuronal types within the claustrum, and thatNPY-ir neurons are not reflective of a specific subpopulation. Thefact that NPY-immunoreactivity was observed in neurons ofvarying shapes and sizes suggests that these cells play differingroles in the context of claustral function (Hinova-Palova et al.,2007, 2008). Present results were comparable to our previousstudy of the synaptic organization of the cat claustrum (Hinova-Palova, 1986).

It has been established that neurons of differing shapes andsizes can have different functions (Papantchev et al., 2006). One ofthe aims of the present study was to identify whether NPYimmunoproduct was present in possibly functionally distinctsubpopulations of claustral neurons. It is generally accepted thatthe number and types of synapses are principal factors withrespect to influencing neuronal function (Paloff, 1985; Paloff et al.,1989). This explains why otherwise morphologically identicalneurons function and respond differently, as they are dependentupon on the characteristics and spike codes of their synapticinputs. Therefore, one could speculate that the morphologicallysimilar NPY-ir neurons we have found dispersed throughout thecat claustrum may not be as functionally similar as one mightthink.

As we have shown in this study, the observed population ofNPY-ir neurons was heterogeneous in nature, consisting of small,medium and large neurons of various shapes. In support of theabove-mentioned conclusions, we must emphasize that Golgiimpregnation studies of the claustrum in various species ofmammal have lead to the description of two major functionalclasses of neurons: projection neurons with polymorphic perikar-ya, spiny dendrites, prominent mitochondria and numerousaxodendritic synapses, and inhibitory interneurons characterizedby round or oval perikarya and aspiny dendrites (Braak and Braak,1982; Hinova-Palova, 1986; Wojcik et al., 2004). Another aim ofthis study was to establish whether NPY-ir neurons in theclaustrum have a well-defined topographical distribution. In pointof fact, such a distribution was not found. On the contrary, despitethe fact that the central part of the claustrum (stereotaxic planesA12–A16) was seen to contain the majority of NPY-ir neurons,these cells were diffusely distributed throughout the claustrum.Indeed, though NPY-ir neuronal clustering or grouping wasapparent within the central stereotaxic planes (which generallyhas the highest concentration of neurons), this was not seen withinthe ascribed functional zones of claustrum.

Immunohistochemical and in situ hybridization studies haveshown that NPY is one of most abundant and widely distributedpeptides in the central and autonomic nervous system ofmammals, particularly in rats and humans. In addition to theclaustrum, NPY is highly expressed in the cerebral cortex,hypothalamus, amygdala, hippocampus, nucleus of the solitarytract, locus coeruleus, nucleus accumbens, periaqueductual gray,nucleus raphe pallidus, striatum and the bed nucleus of the striaterminalis (Allen et al., 1983; Chronwall et al., 1985; Sasek andElde, 1985; Smith et al., 1985; de Quidt and Emson, 1986; Dumontet al., 1992; Todd and Spike, 1993; Krivukuca et al., 2010; Edelsteinet al., 2010, 2011a,b). Neuropeptide Y in involved in the control of abroadly diverse array of physiological functions including feedingbehavior, water consumption, learning and memory, locomotion,body temperature regulation, sexual behavior, emotional behavior,

neuronal excitability, cardiovascular homeostasis, hormone secre-tion and circadian rhythms (Baraban et al., 1997; Colmers andBleakman, 1994; Hokfelt et al., 1998; Inui, 1999; Munglani et al.,1996; Vezzani et al., 1999; Wahlested and Reis, 1993). In addition,it has been suggested that NPY plays a role in psychiatric disordersincluding depression, eating disorders, anxiety and epilepsy(Baraban et al., 1997; Colmers and Bleakman, 1994; Grundemaret al., 1992; Hokfelt et al., 1998; Munglani et al., 1996; Wahlestedand Reis, 1993). What more, hypothalamic injections of NPYincrease food intake (Stanley and Leibowitz, 1985); in thehippocampus, NPY has been suggested as an endogenous anti-epileptic (Colmers et al., 1991); in the cortex NPY is co-expressed inmany inhibitory GABAergic neurons that modulate pyramidal cellfunction in the other regions of the brain (Kubota et al., 2011); NPYcells play a role in cardiovascular function and emotion (Colmersand Wahlested, 1993).

From a clinical perspective, given that the claustrum and NPYhave been implicated in the development, symptoms and/orsequelae of serious maladies such as autism, schizophrenia,epilepsy, Alzheimer’s disease, Parkinson’s disease, and Hunting-ton’s disease (Wegiel et al., 2014; Cascella and Sawa, 2014; Venneriand Shanks, 2014; Kalaitzakis, 2014; Corcoran, 2004; Hallidayet al., 1990; Koide et al., 1995; Croom and Taylor, 2001; Flint andMartin, 1986; Ramos et al., 2006), it behooves both basic andclinical investigators to learn as much as possible about theinterplay between the two.

5. Conclusion

In conclusion, this is the first detailed investigation of the lightand electron-microscopic features of NPY-ir neurons and fibers inthe cat claustrum. It is hoped that our results and wide-rangingdiscussion will provide deeper insight into the functionalneuroanatomy of the claustrum, further elucidating its involve-ment with a host of critical physiological parameters and biologicprocesses, especially those impacted by the aforementionedneurological disorders. Lastly, much work remains to be done interms of studying the claustrum from an ontogenetic andphylogenetic perspective, with respect to homologues in non-mammalian species as well as its contribution in the context ofhigher cortical functions and prevalent hypotheses (Smythieset al., 2012, 2014a,b). In this regard, it must be mentioned that oneof the greatest minds in modern biology, the late Nobel laureateand co-discoverer of the structure of DNA, Francis Crick, spent thelast hours of his remarkable life dictating corrections to a draft ofwhat was to become his final paper, entitled ‘‘What is the functionof the claustrum?’’ (Crick and Koch, 2005; Edelstein and Denaro,2004b). Francis Crick and his co-author Christof Koch have laid thefoundation for future investigative efforts into the claustrum’sputative role in that most complex and elusive of neurophysiologicattributes, consciousness.

Ethical statement

All animals in this study were afforded human care incompliance 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 bythe National Institutes of Health (NIH publication No. 86-23,revised 1996).

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