R E S EA R C H A R T I C L E

Intracallosal Neuronal Nitric Oxide Synthase Neurons Colocalize With Neurokinin 1 Substance P Receptor in the Rat Paolo Barbaresi,1* Emanuela Mensa,1 Vincenzo Lariccia,2 Genni Desiato,1 Mara Fabri,1 and Santo Gratteri3 1

Department of Experimental and Clinical Medicine, Section of Neuroscience and Cell Biology, Marche Polytechnic University, I-60020 Ancona, Italy 2 Department of Biomedical Sciences and Public Health, Marche Polytechnic University, I-60020 Ancona, Italy 3 Department of Health Sciences, University “Magna Grecia”, 88100 Catanzaro, Italy

ABSTRACT The corpus callosum (cc) contains nitric oxide (NO)-producing neurons. Because NO is a potent vasodilator, these neurons could translate neuronal signals into vascular responses that can be detected by functional brain imaging. Substance P (SP), one of the most widely expressed peptides in the CNS, also produces vasomotor responses by inducing calcium release from intracellular stores through its preferred neurokinin 1 (NK1) receptor, thus inducing NO production via activation of neuronal NO synthase (nNOS). Single- and double-labeling experiments were performed to establish whether NK1-immunopositive neurons (NK1IP-n) are found in the rat cc and the extent of NK1 colocalization with nNOS. NK1IP-n were seen to constitute a large neuronal population in the cc and had a distribution similar to that of nNOSIP neurons (nNOSIP-n). NK1IP-n

were numerous in the lateral cc and gradually decreased in the more medial portions, where they were few or absent. Intracallosal NK1IP-n and their dendritic trees were intensely labeled, allowing classification into four morphological types: bipolar, round, polygonal, and pyramidal. Confocal microscopic examination demonstrated that nearly all NK1IP-n contained nNOS (96.43%) and that 84.59% of nNOSIP-n coexpressed NK1. These data suggest that the majority of intracallosal neurons can release NO as a result of the action of SP. A small proportion of nNOSIP-n does not contain NK1 and is not activated by SP; these neurons may release NO via alternative mechanisms. The possible mechanisms by which intracallosal neurons release NO are also reviewed. J. Comp. Neurol. 523:589–607, 2015. C 2014 Wiley Periodicals, Inc. V

INDEXING TERMS: corpus callosum; nitric oxide; immunocytochemistry; NK1; colocalization; immunofluorescence

The corpus callosum (cc), the largest neural pathway interconnecting the two cerebral hemispheres (Innocenti, 1986), is made up of myelinated and unmyelinated axons and glial cells (Innocenti, 1986). Callosal axons originate from neurons located in layers II/III and V of the cerebral cortex (Innocenti, 1986). The overwhelming majority of such axons use glutamate (Glu) as a neurotransmitter (Barbaresi et al., 1987), whereas a very small percentage use g-aminobutyric acid (GABA; Gonchar et al., 1995; Fabri and Manzoni, 2004; Higo et al., 2009). Glu is released in the cc by unmyelinated fibers at specific axon–glia synaptic junctions (Kukley et al., 2007; Ziskin et al., 2007). Aside from glial cells, the cc also contains neurons. Studies of cc organization or studies reporting occasional data have indentified intracallosal neurons. Some C 2014 Wiley Periodicals, Inc. V

multipolar neurons the ventral portion (Malobabic et al., Revishchin et al.

were described in the core and in of human cc with the Golgi method 1984). Riederer et al. (2004) and (2010) used immunocytochemical

Additional Supporting Information may be found in the online version of this article. Grant sponsor: Universita Politecnica delle Marche, Ricerca Scientifica d’Ateneo 2013; Grant sponsor: MIUR-Prin 2009. G. Desiato’s current address is Laboratory of Signal Transduction in Cardiac Disease, National Research Council, Humanitas Clinical and Research Center, Milano, Italy *CORRESPONDENCE TO: Paolo Barbaresi, Department of Experimental and Clinical Medicine, Section of Neuroscience and Cell Biology, Marche Polytechnic University, Via Tronto 10/A-Torrette di Ancona, I-60020 Ancona, Italy. E-mail: p.barbaresi@univpm Received July 10, 2014; Revised October 9, 2014; Accepted October 9, 2014. DOI 10.1002/cne.23695 Published online October 14, 2014 in Wiley (wileyonlinelibrary.com)

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techniques to study the localization of microtubuleassociated protein 2 (MAP2) and calretinin-positive cells in the cat and rat cc, respectively. A recent immunocytochemical study showed cholinergic cells in the monkey cc (Rockland and Nayyar, 2012). Finally, two papers have reported nitric oxide (NO)-producing neurons in the cc of monkey and rat (Rockland and Nayyar, 2012; Barbaresi et al., 2014). Many NO-producing neurons are close to intracallosal blood vessels (Rockland and Nayyar, 2012; Barbaresi et al., 2014). The physiological sphere of influence of NO has been estimated at about 200–400 lm (Estrada and DeFelipe, 1998; Wood and Garthwaite, 1994; Laranjnha et al., 2012), so NO has the potential to affect several blood vessels. Given that NO is a potent vasodilator, nNOS-containing neurons are thought to be involved in coupling metabolic changes related to neuronal function with local increases in blood flow (Iadecola, 2004). Another compound that can produce vasomotor responses is substance P (SP), one of the most widely expressed peptides in the CNS. Together with neurokinins A and B (NKA and NKB) it belongs to the tachykinin family, whose biological actions are mediated through G-protein-coupled tachykinin receptors designated NK1, NK2, and NK3 (Maggi, 1994; Harrison and Geppetti, 2001). The highest affinity of SP is for NK1 receptor (NK1R), whereas NKA and NKB have the highest affinity for NK2 and NK3 receptor, respectively (Maggi, 1994; Harrison and Geppetti, 2001). SP-containing fibers have been found around cerebral blood vessels by using immunocytochemistry (Uddman et al., 1981; Edvinsson et al., 1981, 1982; Itakura et al., 1984). Moreover dilation of brain arteries and veins is induced by application of perivascular and intravascular SP (Edvinsson et al., 1982; McCulloch et al., 1986; Jansen et al., 1991; Beattie et al., 1993; Rosenblum et al., 1993; Kobari et al., 1996). Finally pharmacological studies indicate that the vasodilator action of SP on cerebral vessels is mediated by activation of NK1R (Jansen et al., 1991; Beattie et al., 1993; Kobari et al., 1996). SP-NK1R interaction activates phospolipase C to produce inositol trisphosphate, which induces calcium

(Ca21) release from intracellular stores by binding to its receptors on the endoplasmic reticulum.The increase in cytoplasmic Ca21 activates NOS, a Ca21/calmodulindependent enzyme, to produce NO (Bredt and Snyder, 1990; Vincent, 1994; Khawaja and Rogers, 1996). To test whether SP might play a role in cc blood flow regulation through NK1R, immunocytochemical experiments were performed in the rat cc to localize NK1Rexpressing neurons. In addition, to investigate the possible influence of SP on intracallosal NO-producing neurons, colocalization of NOS and NK1R immunoreactivity was investigated using a double-immunofluorescence method.

MATERIALS AND METHODS The study involved eight adult male Sprague-Dawley albino rats (weight 250–300 g) whose care and handling were approved by the Animal Research Committee of Marche Polytechnic University in accordance with National Institutes of Health guidelines. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Light microscopy nNOS-NK1 experiments Five rats (CC-nNOS-1–5;CC-NK1-1–5) were perfused transcardially with saline followed by a solution of 4% paraformaldehyde, 0.5% glutaraldehyde, and 40% saturated picric acid in PB (0.1 M, pH 7.4). Brains were removed and postfixed for 12 hours in the same fixative used for perfusion. After postfixation brains were cryoprotected in increasing concentrations of a sucrose solution (10%, 20%, 30% in 0.1 M PB at 4 C) until they sank and then freeze-sectioned in the sagittal plane (four consecutive sections, two 60 lm and two 40 lm in thickness) on a sliding microtome. The two 60-lm sections from both hemispheres were used for nNOS and NK1 immunocytochemistry (nNOSIcc and NK1Icc). They were first placed in a solution of 3% H2O2 in phosphate-buffered saline (PBS) for 30 minutes to inhibit endogenous peroxidase activity, then incubated for 1 hour in a blocking solution consisting of 20%

Abbreviations CA1 CA2 CA3 CA4 cc Cl CPu df dhc fi

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field CA1 of Ammon’s horn field CA2 of Ammon’s horn field CA3 of Ammon’s horn field CA4 of Ammon’s horn corpus callosum claustrum caudate-putamen nucleus dorsal fornix dorsal hippocampal commissure fimbria of the hippocampus

fmi fmj gcc LSD LSI LV scc SFi vhc 3V

The Journal of Comparative Neurology | Research in Systems Neuroscience

forceps minor of the corpus callosum forceps major of the corpus callosum genu lateral septal nucleus, dorsal part lateral septal nucleus, intermediate lateral ventricle splenium of the corpus callosum septofimbrial nucleus ventral hippocampal commissure third ventricle

nNOS and NK1 Coexpression in Rat cc

normal goat serum (NGS) in PBS, rinsed in phosphate buffer (PB), and then incubated for 18–24 hours in the primary antibodies: nNOS antibody (Swant, Belinzona, Switzerland; code No. CB-38a) diluted 1:1,000 and NK1 (Shigemoto et al., 1993) diluted 1:1,000. Sections were then reacted with goat anti-rabbit serum (diluted 1:100; 1 hour at room temperature; Vector, Burlingame, CA). After washing, sections were immersed in a solution of avidin-biotin complex (ABC; 1:100; Vector; Hsu et al., 1981), rinsed again in PBS, and reacted with 3,3’-diaminobenzidine (DAB; 0.025%; Sigma, St. Louis, MO) and H2O2 (0.0008%; Merck KGaA, Damstadt, Germany). To define cc borders, the first 40-lm-thick section was reacted for cytochrome oxidase (CO) histochemistry (COHi); the second was counterstained with neutral red (1% in aqueous solution; Fluka Chemie GmbH, Buchs, Switzerland; Barbaresi et al., 2014).

CO staining For COHi, 40-lm-thick sections were incubated at 37 C in the dark for 10–12 hours in a solution containing 50 mg DAB, 30 mg cytochrome C (type III; Sigma) and 4 g sucrose dissolved in 90 ml PB (0.1 M, pH 7.4; Wong-Riley, 1979). Incubation was arrested when a clear differentiation between cerebral cortex and cc was visible. Sections were rinsed many times in PB, mounted on subbed slides, air dried, dehydrated in xylene, and then coverslipped.

Immunofluorescence experiments Three further animals (CC-Fl-1–3) were used for this series of experiments. Rats were deeply anesthetized with chloral hydrate and then transcardially perfused with saline, followed by 4% paraformaldehyde in PB. After the brains had been removed, they were postfixed overnight in the same fixative and then cut as described above into three consecutive sections (one 60-lm and two 40-lm thick). The former sections were first transferred to a solution of 3% H2O2 in PBS for 30 minutes to inhibit endogenous peroxidase activity and then incubated for 1 hour in blocking solution. After these steps, sections were rinsed several times in PBS and then incubated overnight in a cocktail of primary antibodies containing nNOS made in mouse (Invitrogen-Zymed, Carlsbad, CA; clone 3G6B10, catalog No. 37-2800; 1:1,000) and NK1 made in rabbit (Shigemoto et al., 1993; 1:800). After being washed in PB, sections were incubated in a mixture of species-specific secondary antibodies (1:150) conjugated to fluorescein (FITC) and rhodamine (TRITC; both from Invitrogen, Chicago, IL) for 1 hour at room temperature. Sections were washed in PB, mounted on slides, dried and coverslipped with Vecta-

shield (Vector). Then, 40-lm-thick sections were reacted for COHi, followed by neutral red counterstaining. The overlying cerebral cortex was used as a positive control.

Confocal microscopy Images from sections stained for nNOS/NK1 were acquired with a Zeiss LSM 510 module mounted on an Axiovert 200M microscope equipped with argon and helium/neon lasers (Carl Zeiss Microimaging, Jena, Germany). The cc was divided into seven or eight mediolateral sectors; different z-stack projections were taken at various sites across the slice surface. Orthogonal zstack profiles were acquired with a Plan Neofluar 320/ 0.5 air objective at 4-lm intervals from the slice bottom (which contained no in-focus cells) to the top (the last focal plane containing cells). Unless otherwise specified, each image was 636.4 3 636.4 lm in size. Acquisition settings were adjusted to optimize fluorescence intensities. Staining was analyzed at room temperature, and image processing was performed in LSM software and Adobe Photoshop Elements 6. Immunoreactivity was investigated independently in each color channel, and a visual-based approach was used for quantification. To ensure that every stained cell was counted and none was counted twice, once identified, each cell was numbered. A cell was considered positive (i.e., to exhibit colocalization) if it stained with both the red and the green fluorescence dyes and negative if only one or neither fluorescent dye was detected. The images acquired were examined by two independent blinded raters, who obtained identical results.

Antibody characterization The nNOS polyclonal antibody (Cayman, Ann Arbor, MI; catalog No. 160870) was made in rabbit against a peptide corresponding to amino acids 1422–1433 of human nNOS; on Western blots of protein extracts from rat mesencephalon, it reacted with a band of 160 kDa (Barbaresi et al., 2013) as stated by the manufacturer (Table 1). This antibody does not cross-react with related endothelial NOS (eNOS) or inducible NOS (iNOS) proteins. The nNOS antibody used in fluorescence experiments was a monoclonal antibody made in mouse against a recombinant protein derived from the N-terminal region of rat nNOS protein (Table 1; Invitrogen-Zymed; catalog No. 37–2800, clone 3G6B10). The antibody recognizes only nNOS based on amino acid sequence homology. This antibody has been used in previous studies for both immunocytochemistry and Western blotting, producing a band of 160 kDa as stated by the

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TABLE 1. List of Primary Antibodies Used in This Study Antigen

Source/catalog No./RRID/ references

Immunogen

Neuronal nitric oxide synthase (nNOS) Neuronal nitric oxide synthase (nNOS) Substance P receptor (SPR-NK1)

Human nNOS amino acids 1422–1433 Recombinant protein from N-terminal region of rat nNOS protein Bacterial fusion protein of trpE and SPR-C terminus peptide (amino acid residues 349–407)

Cayman/160870/AB_10080041/ Barbaresi et al., 2013, 2014 Zymed Laboratories Invitrogen/37– 2800/AB_431433/Barbaresi et al., 2012, 2014 Prof. Ryuichi Shigemoto/Shigemoto et al., 1993; Kaneko et al., 1994; Barbaresi, 1998

manufacturer (Barbaresi et al., 2012, 2013); moreover, both nNOS antibodies were successfully used in a previous study on the organization of nitrergic neurons of the rat cc (Barbaresi et al., 2014). In the present experiments, they produced a pattern of immunoreactivity identical to that described previously (Barbaresi et al., 2014). The NK1R antibody (generously provided by Prof. Ryuchi Shigemoto, Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki, Japan) was made in rabbit against a peptide corresponding to amino acid residues 349–407 of rat SP receptor. The specificity of this antibody has been verified by preabsorbion with trp E-SPR fusion protein, which abolished all staining (see Fig. 2c of Shigemoto et al., 1993) and has successfully been used in previous studies (Table 1; Shigemoto et al., 1993; Kaneko et al., 1994; Barbaresi, 1998). The overlying cerebral cortex was used as a positive control. The pattern of both antibodies and NK1-positive neurons was consistent with previous studies (Valtschanoff et al., 1993; Shigemoto et al., 1993; Kaneko et al., 1994; De Vente et al., 1998; Vruwink et al., 2001).

Data analysis The distribution of NOSIP-n and NK1IP-n in the cc was drawn with a camera lucida attached to a Leitz Orthoplan microscope equipped with a 325 objective (Leica, Wetzlar, Germany). Callosal boundaries were obtained by comparing the sections reacted for COHi with those counterstained with neutral red. The reconstructions thus obtained were then compared with those of the atlases of Paxinos and Watson (1982) and Zilles (1985). Counts and distributions were examined in 96 sagittal sections (48 for nNOS and 48 for NK1) from three animals (CC-NOS/NK1-1–3); counts were obtained by pooling data from two adjacent sections for each stereotaxic level (Fig. 2). Microscopic studies of the morphology and percentage of intracallosal neurons (three

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Host and type

Dilution

Rabbit polyclonal

1:1,000

Mouse monoclonal

Rabbit polyclonal

1:800

1:1,000

cases, CC-NOS/NK1–3–5; see Tables 3, 4) were performed using staining criteria similar to those of previous Golgi and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) studies (Jacobs and Scheibel, 1993; Jacobs et al., 1993; Phillips et al., 2000; Barrera et al., 2001). Selected neurons, drawn with a camera lucida and a 3100 oil immersion objective, exhibited the following characteristics: labeled neurons had a clearly distinguishable morphology; cell bodies were located in the central part of the 60-lm section depth, to minimize cutting of dendritic branches near the section surface; dendrites were not overly obscured by other heavily stained processes from nearby cells; and dendritic trees were intensely labeled and did not show discontinuity with their cell bodies. With these criteria we calculated the percentage of each morphological type of intracallosal NOSIP-n.

RESULTS Distribution and morphology of nNOSIP neurons As in a previous study by our group, the nNOS antibody stained a population of intracallosal neurons scattered throughout the anteroposterior rat cc, whose distribution showed regional variation along the lateromedial cc (Fig. 1A; Barbaresi et al., 2014). Overall, 718 intracallosal nNOSIP-n were counted in three experiments. Small and roughly equal numbers of nNOSIP-n were detected in the stereotaxic planes between 3.9 and 2.9; nNOSIP-n increased at about 2.4, peaked at about 1.9–2.0, declined at more medial levels (Fig. 2), and virtually disappeared near 0. nNOSIP-n were intensely labeled (Fig. 3), and their dendrites could be followed for tens of micrometers, branching in all directions and in some cases reaching the white matter, the caudate-putamen nucleus, or the alveus of the hippocampus, depending on their location in the cc (Fig. 3). A dense network of labeled dendrites was found on the

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nNOS and NK1 Coexpression in Rat cc

Figure 1. A: Distribution of nNOSIP neurons in the rat corpus callosum (cc) from lateral to medial. B: Distribution of NK1IP neurons in the rat cc from lateral to medial. Bottom left: Stereotaxic coordinates and abbreviations according to the atlas of Paxinos and Watson (1982). Scale bar 51 mm.

ependymal border of the cc (Fig. 3E). Dendrites often bore spines or fine dendritic processes. As documented in our previous article (Barbaresi et al., 2014), nNOSIP-n showed different morphologies (see Fig. 3): bipolar (Fig. 3A,B,E), round (Fig. 3A,D,E), polygonal (Fig. 3A,D), and pyramidal (Fig. 3B,D,F).

Distribution and morphology of NK1IP neurons The distribution of NK1IP-n was similar to that of nNOSIP-n; the neurons were found along the rostrocaudal extension of the rat cc (Figs. 1B, 4A,B) and showed a different distribution along its lateromedial dimension

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Figure 2. Numbers of nNOSIP and NK1IP neurons found in different stereotaxic planes. Stereotaxic planes from lateral (3.9) to medial (0.4) according to the atlas of Paxinos and Watson (1982). The number of neurons per level was obtained by pooling data from two adjacent sections.

(Fig. 1B). They increased from lateral to medial (from stereotaxic planes 3.9 to 2.4, according to the atlas of Paxinos and Watson, 1982), peaking at about 2; they decreased again, especially at the more medial levels (from 1.4 to 0.4), and then virtually disappeared at about 0 (Figs. 1, 2; see also Fig. 7A,B). The overwhelming majority of NK1IP-n were strongly labeled; although a count was not performed, weakly labeled NK1IP-n were very rare. NK1IP-n often formed clusters of three to five elements whose dendrites intersected to form a dense network (see Fig. 6C). The morphology of NK1IP-n was identical to that of nNOSIP-n; this allowed the morphological classification used for nNOS neurons to be applied to NK1IP-n (Barbaresi et al., 2014): bipolar (fusiform and rectangular), round, polygonal (quadrangular), and pyramidal (triangular-pyriform).

Bipolar neurons Fusiform neurons (average length 3 width 25.775 3 11.68 lm; 90 neurons) These neurons showed an elliptical cell body, with the minor and major axis measuring on average 11.6 lm and 25.7 lm, respectively. From each pole of the perikaryon one to three principal dendrites emerged

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that gave rise to secondary and tertiary dendrites bearing spines (Figs. 4F1, 5B1) and fine dendritic processes. Some dendrites bore swellings (Figs. 4C,F,G, 5B1). Often a primary dendrite emerged from the middle of the cell body (Fig. 5B1). In some cases, dendrites could be followed into the overlying white matter or the caudate-putamen nucleus, depending on their location in the cc. These neurons were often observed in the ependymal region of the rat cc (Fig. 4D,F).

Rectangular neurons (average length 3 width 28.633 3 6.09 lm; 18 neurons; 1.18% of the NK1IP-n population) This rare type of neuron (18/1522; see Table 2) had a long and very narrow cell body with one or two principal dendrites emerging from the two poles of the perikaryon.

Polygonal neurons (average length 3 width 21.738 3 14.404 lm; 75 neurons) These neurons had a polygonal or quadrangular perikaryon (Fig. 5C1). Dendrites emerged from the vertices of the soma and radiated in all directions and in some cases could be followed into the overlying white matter or the caudate-putamen nucleus, depending on their location in the cc.

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nNOS and NK1 Coexpression in Rat cc

Figure 3. Photomicrographs of nNOSIP neurons in the rat corpus callosum (cc). A: Low-power photomicrograph showing different morphological types of nNOSIP neurons found in the rostral portion of the rat cc. B: Triangular and bipolar nNOSIP neuron. C: Inverted triangular nNOSIP neuron. D: Low-power photomicrograph showing nNOSIP neurons in the middle cc; boxed area is enlarged in F. E: Three nNOSIP neurons near the ependymal region of the rat cc, where their dendrites form an extensive network. F: Inverted drop-like nNOSIP neuron. An apical dendrite splits into two branches parallel to the ependymal surface; dendrites running toward the dorsal portion of the cc issue from each branch. Stereotaxic coordinates and abbreviations according to the atlas of Paxinos and Watson (1982). Cx, cerebral cortex; wm, white matter. Scale bars 5250 lm in A; 50 lm in B,C,E; 500 lm in D; 25 lm in F.

Round neurons (average length 3 width 17.352 3 15.252 lm; 72 neurons) These neurons had a round cell body with two to four principal dendrites radiating in opposite directions (Fig. 5D1,E1). Those found in the splenium and genu formed a wide, roughly circular dendritic field, whereas those detected in the central one-third of the cc formed a narrow dendritic field oriented in rostrocaudal direction (Fig. 5D1). Dendrites bore some spines and showed several swellings in their distal portions (Fig. 5D1,E1).

Pyramidal neurons (triangular-pyriform; average length 3 width 25.606 3 14.886 lm; 58 neurons) This class of neurons had a triangular or drop-like (pyriform) perikaryon with two basal and one apical dendrite (Fig. 5A1). Apical dendrites often bifurcated into two thinner branches that crossed the cc toward its dorsal portion and in some cases reached the overlying white matter. In other cases apical dendrites headed toward the ventral portion of the cc (inverted pyriform). After a few tens of

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Figure 4. Photomicrographs of NK1IP neurons in the rat corpus callosum (cc). A: Low-power photomicrograph showing the distribution of NK1IP neurons in the central portion of the rat cc. Boxed area is enlarged in A1. A1: Two NK1IP neurons whose dendrites reach the ependymal region of the rat cc. B: Photomicrograph of the splenium showing several NK1IP neurons. C: Round intracallosal NK1IP neuron whose dendrites are studded with swellings. D: Bipolar NK1IP neuron in the ependymal layer of the rat cc. In this layer longitudinally oriented dendrites form a dense network with dendrites originating from other portions of the rat cc. E: Intracallosal bipolar NK1IP neuron. Dense network ofNK1IP dendrites in the dorsal region of the cc. F: Ovoid intracallosal NK1IP neurons. F1: Enlarged portion of the soma and proximal dendrite of the neuron shown in F. Proximal dendrites bear several spines. G: Bipolar intracallosal NK1IP neuron found in the rostral portion of the medial stereotaxic plane. Ax, axon. Stereotaxic coordinates according to the atlas of Paxinos and Watson (1982). Cx, cerebral cortex; wm, white matter. Scale bars 5 500 lm in A,B; 25 lm in A1,C–G.

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nNOS and NK1 Coexpression in Rat cc

Figure 5. Camera lucida reconstruction of five morphologically different NK1IP neurons from different portions of the rat cc. A1: Triangular neuron in the caudal portion of the rat cc. One basal dendrite is directed toward the rostral portion of the cc; the other folds back toward the caudal cc portion. B1: Bipolar neuron. A dendrite emerging from the middle of the soma runs toward the ependymal region. C1: Polygonal neuron with many dendrites emerging from its vertices. D1: Round neuron in the middle portion of the rat cc. Two principal dendrites run in opposite directions and another toward the ependymal region. E1: Round neuron in the middle cc showing four principal dendrites going in opposite directions. Black dots in A–E indicate neuron location. Stereotaxic coordinates and abbreviations according to the atlas of Paxinos and Watson (1982). Scale bars 51 mm in A–E; 50 lm in A1–E1.

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Figure 6. Camera lucida reconstructions showing the tangle of NK1IP fibers found in dorsal (A), ependymal (B), and splenium (C) portions of the rat cc. Stereotaxic coordinates and abbreviations according to the atlas of Paxinos and Watson (1982). Black dots in A1–C1 indicate neuron and dendrite locations. wm, White matter. Scale bars 550 lm in A–C; 1 mm in A1–C1.

micrometers, the apical dendrite branched into thinner dendrites that coursed along the cc in a rostrocaudal direction. Rare spines were seen on dendrites.

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We studied the morphology of 1,522 NK1IP-n from three cases (see Table 2) using the criteria described in Materials and Methods. Similar proportions of round,

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nNOS and NK1 Coexpression in Rat cc

Figure 7. Photomicrographs of NK1IP neurons and dendrites found at the most medial stereotaxic levels. A,B: Two different stereotaxic levels; in both cases neurons are located between the dorsal edge of the cc and the overlying cerebral cortex (indusium griseum according to Fig. 48 of Zilles, 1985). Boxed areas in A,B enlarged in A1,B1, respectively. Asterisk in B indicates an NK1IP neuron that is shown enlarged in Figure 4G. Note the absence and/or small number of NK1IP neurons at these levels. Scale bars 5250 lm in A,B; 50 lm in A1,B1.

polygonal, and bipolar intracallosal neurons were detected (round 27%, 411/1,522; polygonal 26.28%, 400/1,522; bipolar 27%, 411/1522; see Table 2); pyramidal neurons accounted for about 19.71% of the NK1IP–n population. Regardless of morphological class, most NK1IP–n found at medial levels lay between the dorsal boundary of the cc and the overlying cerebral cortex (or induseum griseum; Zilles, 1985; Fig. 7). Their dendrites formed a long, narrow network parallel to the longitudinal cc axis and sometimes entered the cc (Fig. 7B1). A dense dendrite network was often noted also in the dorsal cc (up to the boundary with white matter) or in its ventral portion, close to the lateral ventricle (Figs.

4A1,E, 6A,B). In the former case, the network was formed by dendrites from neighboring cells and likely also from dendrites of distant neurons that could not be followed to the perikaryon of origin (Figs. 4E, 6A). In the latter case, the network was formed both by dendrites belonging to neurons whose perikaryon was located in the ependymal layer and by neurons whose perikaryon lay in middle or dorsal cc portions (Figs. 4A1,D–F, 5D1, 6B). NK1IP-n cell bodies and dendrites were often detected near the wall of intracallosal blood vessels; also in these cases most of the dendrites found in the vicinity of blood vessels could not be tracked to their cell bodies (Fig. 8).

TABLE 2. Number and Percentage of Subtypes of Intracallosal NK1IP Neurons1 Bipolar Case

Round N (%)

Polygonal N (%)

CC-NK1–2

139 (26.17)

145 (27.30)

CC-NK1–3

153 (27.56)

142 (25.58)

CC-NK1–4

119 (27.29)

113 (25.91)

411 (27)

400 (26.28)

Total

Fus N (%)

Pyramidal Rec N (%)

144 (27.11) 138 (25.98) 6 (1.12) 145 (26.12) 137 (24.68) 8 (1.44) 122 (27.98) 118 (27.06) 4 (0.91) 411 (27) 393 (25.82) 18 (1.18)

Trian N (%)

Pyr N (%)

103 (19.39) 35 (6.59) 68 (12.80) 115(20.72) 33(5.94) 82(14.77) 82(18.80) 26(5.96) 56(12.84) 300(19.71) 94(6.17) 206(13.53)

Total 531 555 436 1,522

1

Fus, fusiform; Rec, rectangular; Trian, triangular; Pyr, pyriform (drop-like).

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TABLE 3. Number (No.) and Percentage of Both Single-Labeled (SL) and Double-Labeled (DL) Neurons in the Rat Corpus Callosum Case

No. NK1IP neurons

No. nNOSIP neurons

No. DL neurons

Total SL neurons

DL/NK1IP (%)

DL/nNOSIP (%)

DL/total (%)

95 115 126 336

110 129 144 383

94 111 119 324

111 133 151 395

98.95 96.52 94.44 96.43

85.45 86.05 82.64 84.59

84.68 83.46 78.81 82.02

CC-Fl-1 CC-Fl-2 CC-Fl-3 Total

nNOS and NK1 colocalization

ferent species; 2) immunocytochemical experiments using NK1 antibody found several neurons immunopositive for this receptor in the rat cc; 3) the morphology and distribution of NK1IP-n and nNOSIP-n overlapped; and 4) immunofluorescence experiments documented that 84.59% of nNOSIP-n (324/383) were immunopositive for NK1 and that 96.43% of NK1IP-n (324/336) were nNOS positive.

The colocalization of nNOS and NK1 was investigated in three rats (CC-Fl1–3; see Fig. 10, Table 3) via confocal microscopy. The nNOSIP-n and NK1IP-n populations were clearly identifiable by their red and green fluorescence, respectively (Fig. 9). Double-labeled neurons showed a yellow-orange fluorescence (Fig. 9C1,F,I,L,O). Neurons containing both nNOS and NK1 made up 82.02% (324/395) of all immunofluorescent neurons; in addition, 84.59% (324/383) of nNOSIP-n were also immunopositive for NK1, and 96.43% (324/336) of NK1IP–n were nNOS positive (see Table 3). Doublelabeled neurons showed the same mediolateral distribution as single-labeled neurons. Moreover, 49.09% of intracallosal double-labeled neurons were bipolar, 26.66% were round, 16.96% were pyramidal, and 7.27% were polygonal (Table 4); 9.09% were found in the ependymal layer, and 80% of ependymal double-labeled neurons were fusiform. Many double-labeled neurons and dendrites were found in close proximity to blood vessels. Several double-labeled dendrites close to blood vessels were not attributable to the soma of neighboring neurons.

Intracallosal nNOSIP neurons The first series of experiments confirmed the presence of NO-producing neurons in the rat cc, described from a previous histochemical and immunocytochemical study by our group (Barbaresi et al., 2014). All nNOSIPn found in the rat cc could be classified as type I (Yan et al., 1996) by their intense labeling, morphology, and width of the dendritic tree, which made them identical to the NADPH-d-positive neurons described in that paper (Barbaresi et al., 2014). Such neurons are also found in the monkey cc (Rockland and Nayyar, 2012); in both species, NO-containing intracallosal neurons show considerable morphological heterogeneity and are associated with blood vessels, suggesting that they can translate neuronal signals into vascular responses (Iadecola, 2004). Type I nNOS neurons probably play the role they serve in the cc also in the overlying cerebral cortex. They are closely associated with blood vessels and seem to be involved in coupling metabolic changes related to neuronal function with local increases in blood flow (Suarez-Sosa et al., 2009). Moreover, it has

DISCUSSION The results of the present study can be summarized as follows. 1) Immunocytochemical experiments using nNOS antibody confirmed previous studies in monkey (Rockland and Nayyar, 2012) and rat (Barbaresi et al., 2014) showing NO-producing neurons in the cc of dif-

TABLE 4. Number and Percentage of Subtypes of Double-Labeled Intracallosal Neurons1 Case

Round N (%)

Polygonal N (%)

CC-Fl-1

13 (20.63)

5 (7.94)

CC-Fl-2

17 (32.69)

2 (3.85)

CC-Fl-3

14 (28)

5 (10)

44 (26.66)

12 (7.27)

Total

Bipolar Fus N (%) Rec N (%)

Pyramidal Trian N (%) Pyr N (%)

35 (55.55) 32 (50.79) 3 (4.76) 22 (42.30) 22 (42.30) — 24 (48) 23 (46) 1 (2) 81 (49.09) 77 (46.66) 4 (2.42)

10 (15.87) 7 (11.11) 11 (21.15) 4 (7.69) 7 (13.46) 7 (14) 4 (8) 3 (6) 28 (16.96) 11 (6.66) 17 (10.30)

1

Fus, fusiform; Rec, rectangular; Trian, triangular; Pyr, pyriform (drop-like).

600

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3(4.76)

Total 63 52 50 165

nNOS and NK1 Coexpression in Rat cc

Figure 8. Photomicrographs of NK1IP neurons and dendrites close to blood vessels. A: Large blood vessel (bv) located in the rostral portion of the rat cc surrounded by several NK1IP neurons. B: Dense network of NK1IP dendrites around a blood vessel. C: An NK1IP neuron and many labeled dendrites around two intracallosal small blood vessels. D: Two NK1IP neurons whose dendrites reach a small blood vessel. Scale bars 5250 lm in A; 50 lm in B; 25 lm in C,D.

recently been shown that cortical type I nitrergic neurons are activated during long slow-wave sleep periods following sleep deprivation (Gerashchenko et al., 2008; Kilduff et al., 2011; Perrenoud et al., 2012). According to recent immunocytochemical experiments in the mouse cerebral cortex, 91.5% of type I NO neurons express somatostatin (SOM), suggesting that type I nitrergic cerebral cells form a specific class of SOM-expressing neurons (Perrenoud et al., 2012). Further double- and/or triple-labeling immunocytochemical experiments are therefore needed to establish whether rat cc nNOSIP-n or double-labeled neurons (NOSIPNK1IP) also express SOM. nNOSIP-n were also found in the ependymal layer of the rat cc, where their dendrites formed a dense network. These neurons may be part of the cerebrospinal fluid (CSF)-contacting system found in various periventricular brain regions of vertebrates and are responsible for the transformation and emission of nonsynaptic signals between internal and external CSF and the intercellular fluid of the brain. They could also have a role in the regulation of CSF pH and osmolarity or else in the synthesis and release into the CSF of several peptides (Westergaard, 1972; Sancesario et al., 1996; Vigh et al., 2004; Xiao et al., 2005).

Intracallosal NK1IP neurons The present findings also document several intracallosal neurons expressing NK1R, the preferred SP receptor (Commons, 2010). Their distribution and morphology are identical to those of intracallosal nNOSIP-n. NK1IP-n were numerous in central cc portions, gradually declined at the more medial levels, and disappeared at stereotaxic levels around 0. At this level many NK1IP-n were detected in the dorsal cc, probably in the induseum griseum, where they formed a dense network with their dendrites. These extracallosal neurons were connected to the cc through several dendrites that coursed through the cc genu or trunk. These findings, together with those obtained from previous studies, suggest a preferential distribution for each class of intracallosal neurons in the cc. Indeed, neurons positive for MAP2 are found only in the anterior portion of the cc (the ventral portion of the rostrum; Riederer et al., 2004); in the monkey, NADPH-dpositive neurons were found throughout the cc body but showed a preferential distribution in the rostrum and genu (Rockland and Nayyar, 2012), whereas cc neurons positive for the calcium-binding protein calretinin lay below the cingulum, in the posterior region of the forebrain (Revishchin et al., 2010)

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Figure 9. Photomicrographs showing single- and double-labeled (DL) neurons in the rat cc; NK1IP neurons are green; nNOSIP neurons are red; DL neurons are yellow-orange. A–C: Two fluorescent intracallosal neurons (one round and one bipolar) found in of the rat cc (lateral stereotaxic plane 1.9 mm). Boxed areas in A–C are enlarged in A1–C1. D–F: Bipolar DL neuron. G–I: Two representative NK1IP neurons; one is DL (asterisk). J–L: Bipolar DL neuron in the ependymal region. M–O: Intracallosal DL neuron close to the CPu nucleus. A magentagreen version of this figure is provided in the Supporting Information online. Scale bars 525 lm in C1 (applies to A1–C1); 50 lm in F (applies to D–F); 25 lm in I (applies to G–I); 25 lm in L (applies to J–L); 25 lm in O (applies to M–O).

These neurons and GABAergic and intracallosal neurofilament M-positive neurons (DeDiego et al., 1994; Hornung and Riederer, 1999) are particularly numerous

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during early postnatal development. In this period they may be involved in a variety of processes such as axon guidance or elongation, pruning of exuberant axons,

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and functional tuning. In the adult cc they are much fewer, the soma is small, and dendrites stain poorly and tend to occupy specific regions (Riederer et al., 2004; Jovanov-Milosevic et al., 2010). Although there are no data regarding NK1IP-n in early postnatal development, in the adult cc they are a sizeable population with large somata and wide, intensely labeled dendritic trees that often form dense networks. Their persistence in the adult cc suggests that NK1IP-n may be involved in functions other than those related to callosal plasticity, and colocalization with nNOS suggests that they may be able to translate neural signals into vascular responses (see below). Although a 3D reconstruction of NK1IP-n could not be performed, the criteria applied to study their morphology, which have been used in several Golgi and NADPH-d studies (see Materials and Methods), allowed their classification into bipolar (fusiform and rectangular), round, polygonal (quadrangular), and pyramidal (triangular-pyriform). These findings suggest that intracallosal NK1IP-n, like intracallosal nNOSIP-n (see Rockland and Nayyar, 2012; Barbaresi et al. 2014), possess a high degree of morphological heterogeneity. NK1IP-n had a wide dendritic field with many dendrites that could be followed into the overlying white matter. However, it is likely that dendrites, especially those of neurons colocalizing with nNOS, can reach the inner cortical layers (Barbaresi et al., 2014), where they could be activated by SP from different sources. Scattered SP-immunoreactive axons have been described throughout the rat neocortex (Ljungdahl et al., 1978). One possible source of SP-immunoreactive axons is intrinsic neurons. Indeed, a relatively large population of neurons displaying SP-like immunoreactivity has been identified in the rat cerebral cortex (Penny et al., 1986; Vruwink et al., 2001); SPergic afferents to the cortex have been found to originate from the laterodorsal tegmental nucleus (Vincent et al., 1983); finally, SP is a potent excitatory agent whose application to the cerebral cortex causes strong excitatory responses (Dittrich et al., 2012), mostly from infragranular neurons (Lamour et al., 1983; Jones and Olpe, 1984). Regardless of the source, SP released from terminals and/or somata (Huang and Neher, 1996) of neurons found in the cortex could reach and excite the dendrites of intracallosal neurons synaptically or in a paracrine-like manner or in both ways (Vruwink et al., 2001). NK1IP-n were also detected in the ependymal layer of the lateral ventricle, where together with the dendrites of NK1IP-n located in the cc parenchyma, they formed a dense dendritic network in contact with the CSF. NK1IP-n whose dendrites extend into the CSF have been described for the dorsal columns of several mam-

mals (Abbadie et al., 1999; Ramer, 2008). These neurons receive symmetric and asymmetric synapses, have small receptive fields, and respond to innocuous and/ or mechanical stimulation of the distal extremities (Abbadie et al., 1999; Ramer, 2008). They can be activated by SP circulating in CSF and integrate these responses with those elicited by somatic stimuli (Abbadie et al., 1999; Ramer, 2008). The presence of dendritic spines on intracallosal NK1IP-n and their proximity to CSF suggest that these neurons could receive a synaptic input that can then be integrated with the responses to SP circulating in CSF. This hypothesis is further supported by an early electron microscopic study describing synapses on the perikaryon of intracallosal neurons (Ling and Ahmed, 1974).

Double-labeling experiments: nNOS and NK1 colocalization Double-labeling experiments demonstrated that on average 84.59% (324/383) of nNOSIP-n were immunopositive for NK1 and that 96.43% (324/336) of NK1IP-n were nNOS positive. These data agree with previous findings from the rat cerebral cortex (Vruwink et al., 2001; Dittrich et al., 2012). The high degree of NK1 and nNOS colocalization suggests that SP may play an important role in both the production and the release of NO in the rat cc. NO is produced from L-arginine by NOS, a Ca21/calmodulin-dependent enzyme. When the local Ca21 concentration is elevated NOS converts arginine to citrulline, generating NO. NK1R stimulation by SP leads to intracellular inositol 1,4,5-trisphosphate (IP3) turnover and elevation of intracellular Ca21 ([Ca21]i) that may stimulate NOS, inducing NO production (Bredt and Snyder, 1990; Vincent, 1994; Khawaja and Rogers, 1996). Therefore, any event causing intracortical SP release could excite those intracallosal neurons whose dendrites reach the cerebral cortex, resulting in NO release in the cc. Moreover, the present findings indicate that intracallosal nNOSIP-n are more numerous than double-labeled neurons, suggesting the existence of two populations of intracallosal nNOSIP-n, one of which is not activated by SP. The increased [Ca21]i levels required for NO production can be obtained by activation of Glu receptors such as Nmethyl-D-aspartate (NMDA), thus inducing a Ca21 inflow (Garthwaite, 1991; Christopherson and Bredt, 1997; Kiss and Vizi, 2001). Large numbers of callosal fibers originate from cortical layer II/III and V glutamatergic neurons (Barbaresi et al., 1987). Consistent with their origin, callosal fibers are rich in Glu (Barbaresi et al., 1987). Excitation of

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callosal fibers results in vesicular release of Glu (Kukley et al., 2007), which in turn may induce NO production and release in the cc via NMDA receptors. Moreover, the possibility cannot be ruled out that SP interacts with Glu in the same intracallosal neurons. A recent immunofluorescence study (Lin et al., 2008), in which both NK1R and NMDA receptor immunoreactivity colocalized in the same neurons in the rat nucleus tractus solitarii, supports this hypothesis. Therefore, an increase in cortical activity could lead to significant NO release in the cc by a number of different mechanisms. SP release in the cerebral cortex could induce depolarization of those intracallosal nNOS-NK1IP neurons whose dendrites reach the overlying cerebral cortex, which could release NO in the cc; moreover, the excitation of cortical cells that give rise to callosal projections could release Glu in the cc, exciting intracallosal NOproducing neurons through NMDA receptors: excitation of the cerebral cortex could then result in NO release in the cc through the synergistic action of Glu and SP on the same intracallosal neurons, activating both NK1R and NMDA receptors. Pharmacological studies indicate that NO produced by multiple sources in a finite volume, such as occurs in the cc, can diffuse at a significant concentration over a considerable distance from NO-releasing neurons (Lancaster, 1994; Wood and Garthwaite, 1994; Laranjinha et al., 2012), influencing a large number of neuronal elements as well as intracallosal blood vessels (Kiss and Vizi, 2001). NO is a potent vasodilator, so nNOS-containing neurons are thought to be involved in coupling metabolic changes related to neuronal function with local increases in blood flow that can be detected by functional magnetic resonance imaging (fMRI; Iadecola, 2004). The blood oxygen level-dependent (BOLD) signal is a very complex phenomenon that reflects changes in the hemodynamic responses in active brain tissue and is used in most fMRI applications. The exact mechanism underlying it has not been completely elucidated, but it has been proved to be a sensitive tool for mapping brain activation (Buxton et al., 2014). Hemodynamic changes induced by motor (Mosier and Bereznaya, 2001) and visuomotor tasks (Tettamanti et al., 2002; Omura et al., 2004) and by peripheral stimulation (D’Arcy et al., 2006; Weber et al., 2006; Mazerolle et al., 2010; Fabri et al., 2011) recorded by fMRI in specific cc portions could thus be related to callosal activation of a small group of fibers connecting the site being stimulated with the contralateral homotopic area and to the depolarization of NO-producing neurons, whose dendrites reach the overlying activated cortical areas. The activation of callosal fibers and intracallosal NO-producing neurons could therefore induce an increase in blood flow by the mechanisms hypothesized above.

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The identification of several double-labeled neurons and dendrites in close proximity to blood vessels suggests that circulating SP (Nicoll et al., 1980) may participate through NO in regulating vasomotor responses in the cc. There is indirect evidence for this hypothesis; immunocytochemical findings demonstrate NK1IP nerve fibers in the wall of cerebral blood vessels (Shimizu et al., 1999), through which SP induces vasodilation (Kobari et al., 1996), and pharmacological studies indicate that SP-induced vasodilation is inhibited by NGmonomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis (Whittle et al., 1989). Therefore, intracallosal blood vessels could be regulated primarily by neighboring neuronal elements (intracallosal neurons) and then by an integrated vascular mechanism, as proposed for other vascular beds in the CNS (Tomita et al., 2000). How and when intravascular SP gains access to intracallosal perivascular nNOS/ NK1 neurons to induce NO release remains to be determined.

CONCLUSIONS The present study confirms the findings of previous histochemical and immunocytochemical work describing NO-producing neurons in the cc of different species (see Rockland and Nayyar, 2012; Barbaresi et al. 2014). Moreover, it provides new, important information about cc organization: the cc contains a considerable neuronal population expressing NK1R that, like NOproducing neurons, shows a lateromedial gradient and marked morphological heterogeneity. Moreover, double-labeling immunofluorescence experiments indicate that nearly all NK1IP-n contained nNOS, the enzyme responsible for NO production, whereas 84.59% of nNOS-containing neurons were immunopositive for NK1. These data suggest that all NK1IP-n can release NO through the action of SP and that the cc contains two populations of intracallosal nNOSIP-n, one of which is not activated by SP but could release NO through the action of Glu from callosal fibers.

ACKNOWLEDGMENTS The authors are grateful to Prof. Ryuichi Shigemoto (Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki, Japan) for generously supplying the NK1 antibody and to Word Designs for the language review (www.silviamodena.com).

CONFLICT OF INTEREST STATEMENT All authors have disclosed any known or potential conflict of interest including any financial, personal, or other relationships with other people or organizations

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within 3 years of the beginning this work that could inappropriately influence, or be perceived to influence, this work.

ROLE OF AUTHORS All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: PB. Acquisition of data: PB, EM, GD, VL. Analysis and interpretation of data: PB, MF. Drafting of the manuscript: PB. Critical revision of the manuscript for important intellectual content: PB, MF. Statistical analysis: EM, GD. Obtained funding: PB, MF. Administrative, technical, and material support: GD, SG, VL. Study supervision: PB.

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