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Journal of Chemical Neuroanatomy 61–62 (2014) 107–119 Contents lists available at ScienceDirect Journal of

Contents lists available at ScienceDirect

Journal of Chemical Neuroanatomy

j o u r n al ho mep a g e: www .elsevier . c om /lo cate/jc h emn e u

al ho mep a g e: www .elsevier . c om /lo cate/jc h emn e

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

, L. Malinova a , A. Paloff a , W. Ovtscharoff a a Department of Anatomy,

a Department of Anatomy, Histology and Embryology, Medical University of Sofia, 1431 Sofia, Bulgaria

b Department of Chemistry, Biochemistry, Physiology and Pathophysiology, Sofia University ‘‘St. Kliment Ohridski’’, 1407 Sofia, Bulgaria

c Department of Anatomy, Histology and Embryology, Medical University of Varna, 9002 Varna, Bulgaria

d 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 m m), medium (17–25 m m) and large (over 25 m m). 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.

1. Introduction

The claustrum is a telencephalic nucleus in the mammalian brain that has long been known as a site of extensive reciprocal heterosensory convergence ( Olson and Graybiel, 1980; Ashwell et al., 2004; Hinova-Palova et al., 2007, 2008 ). Typically, it is subdivided into the dorsal claustrum (or ‘‘insular claustrum’’) and ventral 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).

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

dorsal claustrum is located deep to the insular cortex and is extensively 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; 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; Ashwell et al., 2004 ). The ventral claustrum is situated beneath the piriform cortex; its interconnections with prepiriform 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 the claustrum vary greatly. In lower mammals such as the mouse and rat it is a small and discrete nucleus ventrolateral to the

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caudoputamen. In the cat it is a relatively large structure that rivals, or even exceeds, the size of the putamen. In primates it is a thin gray slab varying in thickness up to several millimeters, 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 ). 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 and Usunoff, 1981; Hinova-Palova and Christova, 1988; Hinova-Palova and Braak, 1993; Edelstein et al., 2011a,b, 2012a,b ). The claustrum’s syncytium of GABAergic interneurons and glutama- tergic projection neurons, as well as its heterosensory reciprocal connectivity, leads one to ponder its functional interplay with the rest of the brain. Neuropeptide Y is a novel 36-amino acid peptide and one of the key regulators of food intake, circadian rhythm and reproduction in many mammalian species, including humans. Originally isolated from the porcine hypothalamus ( Tatemoto, 1982; Tatemoto et al., 1982 ), NPY is widely distributed throughout the central and autonomic nervous system of various vertebrates, such as mammals ( Adrian et al., 1983; Allen et al., 1983; Whale and Albus, 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 the white sturgeon ( Chiba and Honma, 1994 ). With respect to phylogenetic comparisons specific to the neurochemical architec- ture of the claustrum, a major focus of our lab, there are but a handful of studies on NPY immunoreactivity, namely, in the squirrel 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 and characteristics of NPY-immunoreactive neurons and fibers in the cat claustrum at the light- and electron-microscopic level, as well as 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 m m 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 H 2 O 2 and 3,3 0 -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% OsO 4 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 found throughout the claustrum, from rostral to caudal planes ( Figs. 1 and 2 ). In general, the NPY-ir neurons were typically stained with immunoproduct visible in the cell cytoplasm and processes, while the nucleus remained free of label ( Fig. 3 ). Labeling intensity, however, varied. Some neurons were lightly stained ( Fig. 4 ), while in others the staining was so dense that they appeared as if they were silver impregnated ( Fig. 5 ). Immunopositive neurons were also found proximal to and within the external and extreme capsules ( Fig. 6 ). Although NPY-ir neurons were seen throughout the claustrum, their distribution displayed a lack of uniformity. Based on our morphometric analysis, approximately 70% of labeled cells were located in stereotaxic planes A12–A16. Of these cells, the majority appeared clustered within the claustrum’s central triangle region (planes A13–A15; Fig. 7 ), with but a handful found proximal to the external and extreme capsules. Most commonly, the neurons were situated parallel to the fiber tracts, though some were positioned in a perpendicular fashion. Moving caudally, the number of NPY-ir neurons gradually diminished ( Fig. 8 ), and at the level of stereotaxic planes A10–A11 only 15% of all counted NPY-ir neurons were identified. Approximately 10% of all NPY-ir neurons

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of Chemical Neuroanatomy 61–62 (2014) 107–119 109 Fig. 1. Coronal section through claustrum in different

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 .

line (arrows delineate claustrum). Nissl staining 20 . Fig. 2. Schematic drawings of 5 coronal sections

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).

were identified in the more rostral stereotaxic planes A17–A18. Finally, the rostral pole of the claustrum contained no more than 5% of NPY-ir neurons. The NPY-ir neurons differed in size and were generally subdivided into large (over 25 m m), medium (17–25 m m), small (under 17 m m) and dwarf cells (under 8 m m). They were further categorized into spiny and aspiny types relative to their dendritic architecture. The various shapes of NPY-ir neurons seen was not unlike what has previously been noted in similar studies (perhaps owing 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, oval and round.

3.1.1. Large spiny neurons Large spiny NPY-ir neurons were classified as being greater than 25 m m in diameter, with polygonal (multipolar), triangular, fusiform, or globular perikarya ( Figs. 9 and 10 ). They were seen giving rise to as many as five thick dendritic processes, each with multiple and multidirectional branches. These branches were typically quite long and could sometimes be followed up to 700 m m from the cell body. The secondary and tertiary dendrites were covered with spines. Axons with highly visible hillocks and initial segments were frequently seen emanating from these cells. The intensity of immunostaining varied widely – from highly dense (i.e. impregnation-like) to weakly stained. Typically, the nuclei were non-immunoreactive.

stained. Typically, the nuclei were non-immunoreactive. Fig. 3. NPY-immunoproduct is clearly visible in the cell

Fig. 3. NPY-immunoproduct is clearly visible in the cell cytoplasm and processes, with the nucleus stain-free. Scale bar = 50 m m.

3.1.2. Medium spiny neurons Medium spiny NPY-ir neurons measured from 18 to 24 m m in diameter with multipolar, fusiform, or oval perikarya. They usually had long bifurcated dendrites, each covered with well-developed spines ( Fig. 11 ).

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/ Journal of Chemical Neuroanatomy 61–62 (2014) 107–119 Fig. 4. Large NPY-positive lightly stained neuron. Scale

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

NPY-positive lightly stained neuron. Scale bar = 50 m m. Fig. 7. Low magnification of triangular

Fig. 7. Low magnification of triangular part of claustrum showing the distribution of NPY-positive neurons. Scale bar = 1 mm.

the distribution of NPY-positive neurons. Scale bar = 1 mm. Fig. 5. Large spiny NPY-positive darkly

Fig. 5. Large spiny NPY-positive darkly stained neuron with long dendrites. Scale bar = 50 m m.

stained neuron with long dendrites. Scale bar = 50 m m. Fig. 8. Low magnification of

Fig. 8. Low magnification of the caudal part of claustrum showing a lesser number of NPY-positive neurons. Scale bar = 1 mm.

a lesser number of NPY-positive neurons. Scale bar = 1 mm. Fig. 6. Large fusiform NPY-positive
a lesser number of NPY-positive neurons. Scale bar = 1 mm. Fig. 6. Large fusiform NPY-positive

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of Chemical Neuroanatomy 61–62 (2014) 107–119 111 Fig. 10. Large spiny pear-shaped NPY-positive neuron. Scale

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

spiny pear-shaped NPY-positive neuron. Scale bar = 50 m m. Fig. 12. Small spiny NPY-positive neurons

Fig. 12. Small spiny NPY-positive neurons with long spiny dendrites. Scale bar = 50 m m.

neurons with long spiny dendrites. Scale bar = 50 m m. Fig. 11. Two medium spiny

Fig. 11. Two medium spiny NPY-positive neurons with long bifurcated dendrites rich with spines. Scale bar = 50 m m.

3.1.3. Small spiny neurons

Small spiny NPY-ir neurons measured from 15 to 17 m m in diameter and were typically oval or fusiform in shape with two to four 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 spiny neurons. These cells also averaged over 25 m m in diameter. Their perikarya 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 m m in diameter. Typically, they had three or four dendrites ( Fig. 14 ), with the 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 m m in diameter ( Fig. 15 ) with predominantly oval or slightly elliptical

perikarya and typically two or three thin, varicose, non-branching dendrites.

two or three thin, varicose, non-branching dendrites. Fig. 13. Large aspiny NPY-positive neuron with long

Fig. 13. Large aspiny NPY-positive neuron with long prominent bulbous dendrites (arrows show the varicosities) Scale bar = 50 m m.

(arrows show the varicosities) Scale bar = 50 m m. Fig. 14. Medium aspiny multipolar NPY-positive

Fig. 14. Medium aspiny multipolar NPY-positive neuron with varicose dendrites (arrows). Scale bar = 50 m m.

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/ Journal of Chemical Neuroanatomy 61–62 (2014) 107–119 Fig. 15. Small aspiny elliptical NPY-positive neuron with

Fig. 15. Small aspiny elliptical NPY-positive neuron with short varicose dendrites. Scale bar = 50 m m.

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

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

3.2. Electron microscopy

The ultrastructural analysis of NPY-ir neurons in our sections clearly demonstrated immunoproduct within perikarya, dendrites, dendritic spines, axons (both myelinated and unmyelinated), and terminal synaptic boutons. The ultrastructural features of these neurons 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 this group ( Fig. 17 ). These neurons were usually heavy stained and had diameters over 27 m m. Ultrastructurally, they were seen to contain an abundance of cytoplasm and organelles: mitochondria, Nissl bodies, a well-developed Golgi apparatus, many free ribosomes, a

large centrally located nucleus (filled with diffusely distributed

located nucleus (filled with diffusely distributed Fig. 16. Two ‘‘dwarf’’ cells with irregular cell

Fig. 16. Two ‘‘dwarf’’ cells with irregular cell bodies and intensely branching short varicose dendrites (arrows). Scale bar = 50 m m.

short varicose dendrites (arrows). Scale bar = 50 m m. Fig. 17. Part of large NPY-positive

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 m m.

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

3.2.2. Medium NPY-ir neurons

Approximately 40% of all NPY-ir neurons were classified as medium in size with a diameter from 18 to 22 m m ( Fig. 18 ). Typically, these neurons were darkly stained with a relatively large cell nucleus, a large amount of euchromatin, many mitochondria, a well-developed Golgi apparatus, and a small amount of Nissl bodies. A greater number of synaptic boutons (both labeled and unlabeled) were noted to have terminated on neuronal surfaces compared with small NPY-ir neurons.

3.2.3. Small NPY-ir neurons

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

under 16 m m ( Fig. 19 ). Most of these neurons were Fig. 18. Medium

Fig. 18. Medium fusiform NPY-positive neuron. Immunoproduct is distributed throughout the cytoplasm and nucleus. Scale bar = 5 m m.

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of Chemical Neuroanatomy 61–62 (2014) 107–119 113 Fig. 19. Two small NPY-positive neurons. One of them

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 m m.

(D), the other, less so (L). Scale bar = 5 m m. Fig. 20. Large NPY-positive

Fig. 20. Large NPY-positive dendrite (D) on which terminate two unlabeled boutons (T). Scale bar = 1 m m.

darkly stained and evinced several defining characteristics including a relatively large cell nucleus, a large volume of heterochromatin, a thin rim of cytoplasm surrounding the nucleus, a paucity of organelles (with few small mitochondria and free ribosomes), scattered cisterns of granular endoplasmatic reticu- lum, and a well-developed Golgi apparatus. Also, a comparatively smaller number of synaptic boutons (labeled and unlabeled) were seen to have terminated on the neuronal surface. More often than not, we had to view quite a few sections in order to spot one with these cells. These heavily stained neurons usually had a deep nuclear invagination.

3.2.4. Lightly and darkly stained NPY-ir neurons

Lightly and darkly stained NPY-ir neurons were analyzed individually. In general, their ultrastructural appearance was dissimilar. Lightly stained neurons were usually medium or large in size, while darkly stained neurons were small and rarely medium in size. The darkly stained small and medium neurons often had a deep invagination of the nucleolar envelope. There was also a difference in electron density, which was significantly greater in the subgroup of darkly stained NPY-ir neurons.

3.2.5. NPY-ir dendrites

NPY-immunoreactive dendrites differed in size from 0.5 m m (small) up to 3 m m in diameter (large). Immunoproduct was associated with the axial neurofilaments in the dendrites ( Fig. 20 ),

and was seen in both spiny and aspiny types. The majority of both labeled and unlabeled synaptic boutons were found to terminate on NPY-ir dendrites ( Figs. 21–23 ).

3.2.6. NPY-ir synaptic boutons

With regard to synaptic terminals, three main categories were noted: axo-somatic, axo-dendritic and axo-spinous. They varied in shape, size and vesicular morphology ( Figs. 21, 22 and 24 ). In the context of synaptic terminals, two distinct types of boutons were identified 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 diameter from 1.5 to 3.5 m m ( Figs. 21, 22 and 24 ). Their vesicles were round with an average diameter of 40 nm ( Figs. 21 and 24 ) and mitochondria 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

unlabeled boutons were also found ( Fig. 23 ). On occasion Fig. 21. Large NPY-positive dendrite

Fig. 21. Large NPY-positive dendrite (D) on which terminates a large NPY-positive synaptic terminal (T). Scale bar = 0.5 m m.

an LR bouton was seen to terminate on more than one dendrite whiles some formed axo-somatic synapses ( Fig. 25 ). Synaptic contacts by LR boutons were of the asymmetric type. Approximately 25% of all NPY-ir boutons were of the SR type with a diameter from 0.5 to 1.2 m m ( Fig. 26 ). The synaptic vesicles were somewhat smaller in size than the LR type, with an average diameter of 30 nm ( Fig. 26 ). On rare occasion we observed heavily labeled ‘‘en passant’’ boutons ( Fig. 27 ).

3.2.7. NPY-ir neuropil Immunoproduct was prevalent in dendrites, myelinated axons, and terminal boutons throughout the claustrum.

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/ Journal of Chemical Neuroanatomy 61–62 (2014) 107–119 Fig. 22. NPY-positive terminal bouton (T) establishes

Fig. 22. NPY-positive terminal bouton (T) establishes synaptic contact with a large labeled NPY-positive dendrite (D). Scale bar = 1 m m.

labeled NPY-positive dendrite (D). Scale bar = 1 m m. Fig. 23. NPY-positive dendrite (D) on

Fig. 23. NPY-positive dendrite (D) on which terminate two unlabeled boutons (T). Scale bar = 2 m m.

terminate two unlabeled boutons (T). Scale bar = 2 m m. Fig. 24. Labeled NPY-positive large

Fig. 24. Labeled NPY-positive large round synaptic bouton (T) terminates on two unlabeled medium dendrites (D). Scale bar = 1 m m.

two unlabeled medium dendrites (D). Scale bar = 1 m m. Fig. 25. Labeled NPY-positive bouton

Fig. 25. Labeled NPY-positive bouton (T) contacting an NPY-positive neuron. Scale bar = 1 m m.

4. Discussion

To the best of our knowledge, the present study is the first to provide detailed insight into the topographical distribution and morphological characteristics of NPY-ir neurons in the cat claustrum at the light- and electron-microscopic level. Overall, our findings are consistent with those reported for the squirrel monkey, Saimir sciureus ( Smith et al., 1985 ), cat ( Edelstein et al., 2011b ) and human ( Edelstein et al., 2010 ) in which NPY-ir neurons were recognized in the claustrum of those species. In this investigation, we report the existence of at least seven readily 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 subtypes based on diameter: large, medium, small and ‘‘dwarf,’’ and then into spiny and aspiny relative to their dendritic architecture. The observed population of NPY-ir neurons was heterogeneous in composition which, though seen in higher numbers within the triangular dorsal region (A13–A15) in comparison with more rostral and caudal planes, was reflected throughout the claustrum. When ruminating on the function of the claustrum, we must surely take into consideration its anatomy and connectivity, as

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of Chemical Neuroanatomy 61–62 (2014) 107–119 115 Fig. 26. NPY-positive dendrites (D) and terminal boutons of

Fig. 26. NPY-positive dendrites (D) and terminal boutons of differing size and shape (arrows). Scale bar = 1 m m.

of differing size and shape (arrows). Scale bar = 1 m m. Fig. 27. Heavy labeled

Fig. 27. Heavy labeled ‘‘en passant’’ NPY-positive bouton (arrows) terminates on the periphery of an NPY-positive neuron. Scale bar = 2 m m.

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

dendrites of these large neurons – especially those of the spiny variety – offer support the contention that these neurons receive a significant volume of synaptic input. Both the light- and electron- microscopic characteristics of these large cells are indicative of projection neurons rather than interneurons. This was confirmed by our previous research ( Hinova-Palova, 1986 ). Medium NPY-ir neurons typically had a lesser volume of cytoplasm with a relative paucity of granular endoplasmic reticulum. In point of fact, several subtypes of medium neurons have been found in the cat claustrum ( Hinova-Palova, 1986 ). However, for the purposes of this study, we were unable to confirm that the medium NPY-ir neurons we found corresponded to a specific subtype. Nonetheless, it is not unreasonable to assume that some of these cells are projection neurons owing to their light- microscopic and ultrastructural morphology. Small NPY-ir neurons corresponded to the subtypes previously described by Hinova-Palova (1986) . These neurons displayed a relatively large nucleus, a thin rim of cytoplasm, a paucity of organelles and very few axo-somatic synapses – morphological criteria that are commonly attributed to local circuit neurons, also referred to as interneurons ( Paloff, 1985; Paloff et al., 1989 ). Furthermore, most of the small NPY-ir neurons that we found were seemingly filled or impregnated with immunoproduct. On the basis of their light-microscopic and ultrastructural characteristics, we propose that these densely labeled small NPY-ir neurons are part 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 the striatum by using the Golgi impregnation method. In previous investigations ( Hinova-Palova, 1986; Hinova-Palova et al., 1987 ) we demonstrated the existence of dwarf cells in the cat claustrum via light- and electron-microscopy. Mounting evidence leads us to believe that these cells are also interneurons. To elaborate, the small and medium aspiny NPY-ir neurons seen in the present investigation were predominantly oval-to-fusiform in shape. In contrast, the large and medium spiny NPY-ir neurons were often multipolar-to-pyramidal in shape. Based on the extant literarture on this topic, it is not unreasonable to assume that the aspiny neurons represent a subpopulation of local circuit inhibitory interneurons (along with the dwarf cells), while the spiny neurons represent projection neurons. It was previously reported that the large spiny neurons in the cat claustrum receive direct input from cortex and were also shown to project back to the same 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 monkey claustrum, Brand (1981) reported that the axons of large neurons were efferent in nature, projected out of the claustrum. Moreover, in some species, it was reported that the overwhelming majority of claustral neurons are glutamatergic projection neurons, with only 6–12% of cells engaged in local network activity ( Wojcik et al., 2004 ). The existence of projection neurons in the cat claustrum was also supported by our present study concerning the features of large, medium and small spiny NPY-ir neurons. In some sections, the entire initial segment of an NPY-ir myelinated axon was present. However, it must be noted that some of these axons were found not to originate in the claustrum, and thus were afferent in nature. Neverthless, the existence of afferent and efferent projection fibers in claustrum has been suggested by Wojcik et al. (2004) . Golgi impregnation studies of the mammalian claustrum have lead to the description of two major functional classes of neurons – projection neurons with polymorphic perikarya and spiny dendrites, 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; Druga et al., 1993; Wojcik et al., 2004 ). Our current findings also reveal two major classes of NPY-ir neurons in the cat claustrum: spiny and aspiny. In a previous study on the ultrastructure of the NPY- labeled cat claustrum ( Edelstein et al., 2011b ), we reported on the existence of immunoproduct within dendritic spines of differing shape and size. Thus, our light- and electron-microscopic investigations strongly suggest that NPY-immunoreactivity is present in all neuronal types within the claustrum, and that NPY-ir neurons are not reflective of a specific subpopulation. The fact that NPY-immunoreactivity was observed in neurons of varying shapes and sizes suggests that these cells play differing roles in the context of claustral function ( Hinova-Palova et al., 2007, 2008 ). Present results were comparable to our previous study of the synaptic organization of the cat claustrum ( Hinova- Palova, 1986 ). It has been established that neurons of differing shapes and sizes can have different functions ( Papantchev et al., 2006 ). One of the aims of the present study was to identify whether NPY immunoproduct was present in possibly functionally distinct subpopulations of claustral neurons. It is generally accepted that the number and types of synapses are principal factors with respect to influencing neuronal function ( Paloff, 1985; Paloff et al., 1989 ). This explains why otherwise morphologically identical neurons function and respond differently, as they are dependent upon on the characteristics and spike codes of their synaptic inputs. Therefore, one could speculate that the morphologically similar NPY-ir neurons we have found dispersed throughout the cat claustrum may not be as functionally similar as one might think. As we have shown in this study, the observed population of NPY-ir neurons was heterogeneous in nature, consisting of small, medium and large neurons of various shapes. In support of the above-mentioned conclusions, we must emphasize that Golgi impregnation studies of the claustrum in various species of mammal have lead to the description of two major functional classes of neurons: projection neurons with polymorphic perikar- ya, spiny dendrites, prominent mitochondria and numerous axodendritic synapses, and inhibitory interneurons characterized by round or oval perikarya and aspiny dendrites ( Braak and Braak, 1982; Hinova-Palova, 1986; Wojcik et al., 2004 ). Another aim of this study was to establish whether NPY-ir neurons in the claustrum have a well-defined topographical distribution. In point of fact, such a distribution was not found. On the contrary, despite the fact that the central part of the claustrum (stereotaxic planes A12–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 was apparent within the central stereotaxic planes (which generally has the highest concentration of neurons), this was not seen within the ascribed functional zones of claustrum. Immunohistochemical and in situ hybridization studies have shown that NPY is one of most abundant and widely distributed peptides in the central and autonomic nervous system of mammals, particularly in rats and humans. In addition to the claustrum, NPY is highly expressed in the cerebral cortex, hypothalamus, amygdala, hippocampus, nucleus of the solitary tract, locus coeruleus, nucleus accumbens, periaqueductual gray, nucleus raphe pallidus, striatum and the bed nucleus of the stria terminalis ( Allen et al., 1983; Chronwall et al., 1985; Sasek and Elde, 1985; Smith et al., 1985; de Quidt and Emson, 1986; Dumont et al., 1992; Todd and Spike, 1993; Krivukuca et al., 2010; Edelstein et al., 2010, 2011a,b ). Neuropeptide Y in involved in the control of a broadly diverse array of physiological functions including feeding behavior, 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 and Bleakman, 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 disorders including depression, eating disorders, anxiety and epilepsy ( Baraban et al., 1997; Colmers and Bleakman, 1994; Grundemar et al., 1992; Hokfelt et al., 1998; Munglani et al., 1996; Wahlested and Reis, 1993 ). What more, hypothalamic injections of NPY increase food intake ( Stanley and Leibowitz, 1985 ); in the hippocampus, NPY has been suggested as an endogenous anti- epileptic ( Colmers et al., 1991 ); in the cortex NPY is co-expressed in many inhibitory GABAergic neurons that modulate pyramidal cell function in the other regions of the brain ( Kubota et al., 2011 ); NPY cells play a role in cardiovascular function and emotion ( Colmers and Wahlested, 1993 ). From a clinical perspective, given that the claustrum and NPY have been implicated in the development, symptoms and/or sequelae 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; Venneri and Shanks, 2014; Kalaitzakis, 2014; Corcoran, 2004; Halliday et al., 1990; Koide et al., 1995; Croom and Taylor, 2001; Flint and Martin, 1986; Ramos et al., 2006 ), it behooves both basic and clinical investigators to learn as much as possible about the interplay between the two.

5. Conclusion

In conclusion, this is the first detailed investigation of the light and electron-microscopic features of NPY-ir neurons and fibers in the cat claustrum. It is hoped that our results and wide-ranging discussion will provide deeper insight into the functional neuroanatomy of the claustrum, further elucidating its involve- ment with a host of critical physiological parameters and biologic processes, especially those impacted by the aforementioned neurological disorders. Lastly, much work remains to be done in terms of studying the claustrum from an ontogenetic and phylogenetic perspective, with respect to homologues in non- mammalian species as well as its contribution in the context of higher cortical functions and prevalent hypotheses ( Smythies et al., 2012, 2014a,b ). In this regard, it must be mentioned that one of the greatest minds in modern biology, the late Nobel laureate and co-discoverer of the structure of DNA, Francis Crick, spent the last hours of his remarkable life dictating corrections to a draft of what was to become his final paper, entitled ‘‘What is the function of the claustrum?’’ ( Crick and Koch, 2005; Edelstein and Denaro, 2004b ). Francis Crick and his co-author Christof Koch have laid the foundation for future investigative efforts into the claustrum’s putative role in that most complex and elusive of neurophysiologic attributes, consciousness.

Ethical statement

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

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