Journal of Chemical Neuroanatomy

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1 Journal of Chemical Neuroanatomy (2013) Contents lists available at SciVerse ScienceDirect Journal of Chemical Neuroanatomy jo ur n al ho mep ag e: om /lo cate/jc h emn eu Pattern of distribution of serotonergic fibers to the orbitomedial and insular cortex in the rat Stephanie B. Linley a,b, Walter B. Hoover b, Robert P. Vertes b, * a Department of Psychology, Florida Atlantic University, Boca Raton, FL 33431, United States b Center for Complex Systems and Brain Sciences, Florida Atlantic University, Boca Raton, FL 33431, United States A R T I C L E I N F O Article history: Received 14 September 2012 Received in revised form 13 December 2012 Accepted 14 December 2012 Available online 18 January 2013 Keywords: Medial prefrontal cortex Orbital cortex Serotonin Limbic forebrain Learning/memory Affective behavior A B S T R A C T As is well recognized, serotonergic (5-HT) fibers distribute widely throughout the brain, including the cerebral cortex. Although some early reports described the 5-HT innervation of the prefrontal cortex (PFC) in rats, the focus was on sensorimotor regions and not on the limbic PFC or on the medial, orbital and insular cortices. In addition, no reports have described the distribution of 5-HT fibers to PFC in rats using antisera to the serotonin transporter (SERT). Using immunostaining for SERT, we examined the pattern of distribution of 5-HT fibers to the medial, orbital and insular cortices in the rat. We show that 5- HT fibers distribute massively throughout all divisions of the PFC, with distinct laminar variations. Specifically, 5-HT fibers were densely concentrated in superficial (layer 1) and deep (layers 5/6) of the PFC but less heavily so in intermediate layers (layers 2/3). This pattern was most pronounced in the orbital cortex, particularly in the ventral and ventrolateral orbital cortices. With the emergence of granular divisions of the insular cortex, the granular cell layer (layer 4) was readily identifiable by a dense band of labeling confined to it, separating layer 4 from less heavily labeled superficial and deep layers. The pattern of 5-HT innervation of medial, orbital and insular cortices significantly differed from that of sensorimotor regions of the PFC. Serotonergic labeling was much denser overall in limbic compared to non-limbic regions of the PFC, as was striking demonstrated by the generally weaker labeling in layers 1 3 of the primary sensory and motor cortices. The massive serotonergic innervation of the medial, orbital and insular divisions of the PFC likely contributes substantially to well established serotonergic effects on affective and cognitive functions, including a key role in many neurological and psychiatric diseases. ß 2012 Elsevier B.V. All rights reserved. 1. Introduction The prefrontal cortex (PFC) of the rat consists of the medial (mpfc), orbital (ORB) and insular (INC) prefrontal cortices and extends from the frontal pole of the brain to level of the septum. Abbreviations: 5-HT, serotonin; ac, anterior commissure; ACd,v, anterior cingulate cortex, dorsal, ventral divisions; ACC, nucleus accumbens; AGl, lateral agranular (motor) cortex; AGm, medial agranular (motor) cortex; AId, dorsal agranular insular cortex; AIp, posterior agranular insular cortex; AIv, ventral agranular insular cortex; CLA, claustrum; C P, caudate putamen; DA, dopamine; DI, dysgranular insular cortex; DLO, dorsal lateral orbital cortex; DR, dorsal raphe nucleus; EP, endopiriform nucleus; IL, infralimbic cortex; INC, insular cortex; GI, granular insular cortex; LO, lateral orbital cortex; MO, medial orbital cortex; mpfc, medial prefrontal cortex; MR, median raphe nucleus; NE, norephinephrine; OFC, orbital frontal cortex; PFC, prefrontal cortex; PIR, piriform cortex; PL, prelimbic cortex; SERT, serotonin transporter protein; SSI, primary somatosensory cortex; VLO, ventrolateral orbital cortex; VO, ventral orbital cortex. * Corresponding author. Tel.: ; fax: address: vertes@ccs.fau.edu (R.P. Vertes). Each cortical region has several subdivisions, compromised of unique chemical and morphological features. The subdivisions of the rat are largely functionally homologous to those of the primate PFC, and each subserves distinct affective and cognitive functions (Chudasama and Robbins, 2006; Dalley et al., 2004; Ongür and Price, 2000; Seamans et al., 2008; Uylings et al., 2003). The mpfc consists of four main divisions which dorsally to ventrally include the medial (frontal) agranular cortex (AGm), the anterior cingulate cortex (AC), the prelimbic cortex (PL) and the infralimbic cortex (IL) (Figs. 1 and 2). The dorsal (AGm and AC) and ventral (PL and IL) mpfc reportedly serve separate and distinct functions, with the dorsal mpfc primarily involved in motor behavior and the ventral mpfc in diverse emotional, cognitive and mnemonic processes (Heidbreder and Groenewegen, 2003; Hoover and Vertes, 2007; Vertes, 2006). More specifically, the AGm, or secondary motor area, mainly participates in motor planning and execution, and the AC in motor initiation/impulsivity and attention (Chudasama et al., 2003; Dalley et al., 2004; Kesner and Churchwell, 2011; Muir et al., 1996). By contrast, the PL is /$ see front matter ß 2012 Elsevier B.V. All rights reserved.

2 30 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) Fig. 1. Color coded maps (A E) of the rostral prefrontal cortex (PFC) depicting divisions of the medial, orbital and insular cortices taken from adjacent Nissl-stained sections to the SERT illustrated cases (Figs. 5 15). Shown are the infralimbic, prelimbic and anterior cingulate cortices of the medial PFC; the medial, ventral, ventrolateral, lateral and dorsolateral orbital cortices of the orbital PFC; and the dorsal agranular insular and dysgranular insular cortices for the insular PFC. involved in executive functions including decision making and working memory, while the IL modulates autonomic and visceral activity and fear extinction (Dalley et al., 2004; Sotres-Bayon and Quirk, 2010; Vertes, 2006). The ORB of the rat extends medial-laterally across the ventral surface of the rostral pole of the PFC and consists (medial to lateral) of the medial orbital (MO), ventral orbital (VO), ventrolateral orbital (VLO), lateral orbital (LO) and dorsolateral orbital (DLO) cortices (Figs. 1 and 2) (Krettek and Price, 1977; Price, 2007; Ray and Price, 1992; Van De Werd and Uylings, 2008). The ORB has been shown to serve a prominent role in behavioral flexibility, as commonly demonstrated by orbitalassociated deficits in reversal learning, impulsivity and compulsive behaviors (Fineberg et al., 2010; Kesner and Churchwell, 2011; Schoenbaum et al., 2009; Winstanley et al., 2010). The INC surrounds the rhinal fissure and includes the dorsal (AId), ventral (AIv) and posterior (AIp) agranular insular cortices, and the dysgranular (DI) and granular (GI) insular cortices (Figs. 1 and 2). The INC is reciprocally connected with the gustatory and visceral cortices and among its various functions is thought to serve as a visceral or multisensory receptive zone (Kobayashi, 2011; Saper, 1982). Serotonin (5-HT) innervates the entire neuroaxis including the PFC. The dorsal (DR) and median (MR) raphe nuclei are the primary origins of 5-HT projections to the forebrain, with the DR the main source of 5-HT projections to the PFC (Morin and Meyer-Bernstein, 1999; Vertes, 1991; Vertes and Linley, 2008). 5-HT is a key modulator of various homeostatic, affective and cognitive functions (Cools et al., 2008; Homberg, 2012; Monti, 2011; Robbins and Roberts, 2007; Sanford et al., 2008). This control, in part, is mediated by 5-HT DR projections to the PFC and return PFC modulatory influences on the DR and MR (Gonçalves et al., 2009;

3 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) Fig. 2. Color coded maps (A D) of the caudal prefrontal cortex (PFC) depicting divisions of the medial, orbital and insular cortices taken from adjacent Nissl-stained sections to the SERT illustrated cases (Figs. 5 15). Shown are the infralimbic, prelimbic and anterior cingulate cortices of the medial PFC; the ventrolateral and lateral orbital cortices of the orbital PFC; and the dorsal and ventral agranular insular, dysgranular and granular insular cortices of the insular PFC. Hoover and Vertes, 2007; Lowry et al., 2008; Peyron et al., 1998; Vertes, 1991; Vertes and Linley, 2007, 2008). With respect to cognitive processes, 5-HT has been shown to be critical for decision-making, behavioral flexibility, behavioral inhibition and impulsivity (Chudasama and Robbins, 2006; Clark et al., 2004; Cools et al., 2011; Robbins and Roberts, 2007). Each of these behaviors is dependent upon 5-HT input to specific subregions of the PFC. For instance, 5-HT afferents to the mpfc modulate decision-making and impulsivity, while those to the ORB are involved in behavioral flexibility (Chudasama and Robbins, 2006; Clark et al., 2004; Robbins and Roberts, 2007). Intracerebroventricular or local injections of the 5-HT neurotoxin, 5,7- dihydroxytryptamine (5,7-DHT) into the mpfc markedly enhanced impulsive motor choices in the 5-choice serial reaction time test, as well as impulsive behavior in delay discounting paradigms (Harrison et al., 1997; Mobini et al., 2000; Winstanley et al., 2004). In addition, several reports using in vivo microdialysis demonstrated increases in 5-HT in the mpfc during performance of these tasks (Chudasama and Robbins, 2006; Clark et al., 2004; Dalley et al., 2004, 2008; Robbins and Roberts, 2007; Winstanley et al., 2006). Van der Plasse et al. (2007) reported that serotonergic denervation of the mpfc produced impairments in decisionmaking which were dependent on the salience of an appetitive reward. Serotonergic afferents to the ORB are crucial for cognitive flexibility. Excitotoxic lesions of the ORB in the rat significantly altered reversal learning in the intradimensional extradimensional (IED) olfactory set shifting task but left other attentional measures unimpaired (McAlonan and Brown, 2003). Reversal learning was similarly disrupted following 5-HT loss in the ORB in a primate analog of the task (Clarke et al., 2004, 2005, 2007). The application of 5-HT 2A, 5-HT 2C, 5-HT 6, 5-HT 7 receptor antagonists or 5-HT 1A receptor agonists to the ORB improved reversal learning using various paradigms (Boulougouris et al., 2008; Boulougouris and Robbins, 2010; Hatcher et al., 2005; McLean et al., 2009). By contrast, decreases in levels of dopamine (DA) or norepinephrine (NE) in the ORB had no effect on reversal learning in the IED task or other tasks. This is unlike the

4 32 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) recognized effects of catecholamines (DA and NE) on attentional tasks in other regions of the PFC (Robbins and Roberts, 2007; Roberts et al., 1994). Finally, acute tryptophan depletion in healthy volunteers produced impairments in reversal learning (Clark et al., 2004). No reports have comprehensively investigated the distribution of serotonergic afferents to the mpfc, ORB and INC in the rat. Some early reports described overall patterns of distribution of 5-HT fibers to regions of the cortex but without specific attention to the PFC (Audet et al., 1989; Berger et al., 1988; Lidov et al., 1980; Steinbusch, 1981) while others have concentrated on a particular subregion of the PFC (Miner et al., 2000). On the other hand, several studies, using anatomical tracers, have described DR and MR projections to the PFC but for the most part made no distinction between 5-HT and non-5-ht fibers (Azmitia and Segal, 1978; Morin and Meyer-Bernstein, 1999; Vertes, 1991; Vertes and Martin, 1988; Vertes et al., 1999). A recent report, however, described the concentration/distribution of 5-HT fibers over regions of the PFC in primates (Way et al., 2007). The present study examined, compared, and contrasted the distribution and laminar organization of 5-HT fibers to the medial, orbital and insular divisions of the PFC using immuno-procedures for the detection of the serotonin transporter protein (SERT). Nielsen et al. (2006) showed that SERT as a biomarker for 5-HT produces a stronger signal than antisera to serotonin. We found that both 5-HT and SERT immunohistochemistry produce similar patterns of serotonergic labeling, but that SERT immunostaining, by heightening signal to background, provided a bolder representation of labeled 5-HT fibers (Vertes et al., 2010). 2. Materials and methods Ten (5 male, 5 female) naïve Sprague Dawley rats (Harlan, Indianapolis, IN) weighing g on arrival were housed in pairs on a 12:12 light cycle for 7 days, during which food and water were given ad libitium. These experiments were approved by the Florida Atlantic University Institutional Animal Care and Use Committee and conform to all federal regulations and National Institutes of Health guidelines for the care and use of laboratory animals. Rats were deeply anaesthetized with an intraperitoneal injection of sodium pentobarbital (Nembutal, 75 mg/kg). Rats were first perfused transcardially with ml of ice cold heparinized 0.1 M phosphate buffered saline (PBS) followed by ml of chilled 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at a ph = 7.4. The brains were removed and postfixed for h in 4% paraformaldehyde in 0.1 M PB. Brains were then placed in a 30% sucrose solution for another 48 h. Following sucrose cryoprotection, 50 um sections were cut on a freezing sliding microtome. Sections were collected in a six well plate using 0.1 M PB as a storage solution, so that every sixth section was represented throughout the brain for each series of sections. Sections were stored in 0.1 M PB at 4 8C until the tissue was prepared for immunohistochemistry. Fig. 3. (A and B) Low power Nissl-stained transverse sections from the rostral prefrontal cortex showing the laminar organization and cytoarchitectural divisions of the orbital (A) and insular (B) cortices. (A) Taken from Fig. 1B; (B) taken from Fig. 2B. Scale bar for (A) and (B) = 250 mm. See list for abbreviations. Fig. 4. Low power Nissl-stained transverse section from the prefrontal cortex showing the laminar organization and cytoarchitectural divisions of the medial prefrontal cortex. Taken from Fig. 2B. Scale bar = 250 mm. See list for abbreviations.

5 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) SERT immunohistochemistry For each rat, immunohistochemical analysis to detect serotonergic fibers was conducted with an antiserum to SERT using an avidin biotin protein complex protocol. First, sections were treated with 1% sodium borohydride (NaBH 4 ) in 0.1 M PB to remove excess aldehydes. Following copious 0.1 M PB washes, sections were treated for 1 h in 0.5% bovine serum albumin (BSA) in 0.1 M Tris buffered saline (TBS; ph = 7.6) containing 0.25% Triton X-100. Sections were then incubated in the primary polyclonal antibody, rabbit anti- SERT (Immunostar, Hudson, WI), in a diluent of 0.1% BSA TBS containing 0.25% Triton X-100 at a concentration of 1:5000 at room temperature for h. Following further washes, sections were placed in a secondary antibody, biotinylated goat anti-rabbit immunoglobulin (Vector Labs, Burlingame, CA) in diluent at a 1:500 concentration for 2 h. This was followed by another series of PB washes. Sections were then incubated for 2 h in biotinylated horse anti-goat immunoglobulin (Vector Labs, Burlingame, CA) in diluent at a 1:500 concentration. After washing the tissue in 0.1 M PB, sections were incubated for 1 h in an avidin biotin complex (ABC) using the ABC Elite kit (Vector Labs, Burlingame, CA) in a diluent of 0.1% BSA in TBS containing 0.25% Triton X-100 at a 1:200 concentration. Following final 0.1 M PB washes, brown serotonin fibers expressing the serotonin transporter protein were visualized with the chromagen: 0.022% 3,3 0 -diaminobenzidine (DAB) (Aldrich, Milwaukee, WI) and 0.003% hydrogen peroxide in TBS for approximately 4 6 min. Sections were stored in 0.1 M PB at 4 8C until mounted onto chrome-alum gelatin-coated slides, dehydrated using graded methanols and coverslipped with Permount. Sections that were reacted without either the primary or secondary antibodies did not show immunoreactivity (data not shown). Fig. 5. (A and B) Pattern of distribution of serotonergic fibers to the rostral pole of the PFC showing a massive 5-HT innervation of divisions of the medial (PL) and orbital (MO, VO, VLO, LO, DLO) prefrontal cortices at these levels. Scale bar for (A) and (B) = 600 mm. See list for abbreviations.

6 34 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) Photomicroscopy Lightfield photomicrographs at 100 magnification were taken for visualization of SERT and 5-HT fibers throughout the frontal cortex. Photomicrographs were captured using a QImaging (QICAM) camera mounted onto a Nikon Eclipse E600 microscope. Digital images were captured and reconstructed by using Nikon Elements and then imported into Adobe Photoshop (CS 4.0; Mountain View, CA), where brightness and contrast were adjusted. Representative adjacent Nissl stained sections throughout the extent of the PFC were captured and uploaded into Adobe Illustrator (CS 4.0) to map the cortical subdivisions of the PFC (Figs. 1 and 2). 3. Results 3.1. Cytoarchitectural divisions of the medial, orbital and insular cortices The prefrontal cortex (PFC) of the rat consists of three major parts, the medial (mpfc), orbital (ORB) and insular (INC) cortices, each containing various subdivisions. Figs. 1 and 2 consist of a rostral to caudally aligned series of transverse sections through the forebrain depicting divisions/subdivisions of the PFC for a representative case (case 14B). At the rostral pole of cortex, the prelimbic cortex (PL) is located dorsally and medial orbital cortex (MO) ventrally on the medial wall of the PFC. (Fig. 1A and B). The anterior cingulate cortex (AC), dorsal to PL, joins these two regions slightly caudally (Fig. 1C). At the beginning of the anterior forceps (Fig. 1E), the infralimbic cortex (IL) replaces MO. From there (Fig. 1E) to caudally through the PFC (Fig. 2D), the medial wall of the PFC consists of the dorsoventrally aligned AC, PL and IL cortices (Fig. 2A D). As depicted, PL is the largest of the three regions at these levels. The dorsomedially located AC extends caudally to the level of the rostral pole of the thalamus, where it subdivides into dorsal (ACd) and ventral (ACv) divisions (not shown in Figs. 1 and 2). The ORB extends medio-laterally along the ventral surface of the PFC and consists rostrally of MO, the ventral (VO), ventrolateral (VLO), lateral (LO) and dorsolateral (DLO) orbital cortices (Fig. 1A C). Anterior to the forceps (Fig. 1D), DLO is replaced by the dorsal agranular insular cortex (AId), and slightly caudal to this (Fig. 1E) MO gives way to IL. At the level of nucleus accumbens (Fig. 2A), VO is no longer present and just caudally (Fig. 2B) LO is replaced by the ventral agranular insular cortex (AIv). VLO continues to approximately the end of the PFC (Fig. 2C), thus occupying a considerable longitudinal extent of the PFC. The INC is located along the lateral convexity of cortex bordering the rhinal fissure and consists of AId, AIv, the dysgranular (DI) and granular (GI) insular divisions (Figs. 1D, E and 2A D). As depicted, AId begins just anterior to the forceps (Fig. 1D) and is joined caudally by DI (Fig. 1E) and then by AIv and GI at the level of nucleus accumbens (Fig. 2B). All four divisions extend to the caudal pole of the PFC. The posterior agranular insular cortex (AIp) emerges at the level of the septum and extends to mid-levels of the thalamus (not shown in Figs. 1 and 2). AId is the largest of the insular divisions extending virtually rostrocaudally throughout the PFC. The Nissl-stained sections of Figs. 3 and 4 show the laminar organization of the three divisions of the PFC: orbital (Fig. 3A), insular (Fig. 3B) and medial (Fig. 4) prefrontal cortex Pattern of distribution of SERT immunoreactive fibers in the PFC The patterns of distribution of SERT immunoreactive fibers throughout the PFC are depicted in the low and higher magnification plates of Figs As demonstrated, all divisions/subdivisions of Fig. 6. Pattern of distribution of serotonergic fibers to the rostral PFC showing a massive 5-HT innervation of divisions of the medial (PL, AC) and orbital (MO, VO, VLO, LO, DLO) prefrontal cortices at this level. Scale bar = 600 mm. See list for abbreviations.

7 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) the medial, orbital and insular cortices contain a very dense concentration of SERT immunoreactive (SERT+) fibers, with variations across divisions and layers of the PFC. As shown rostrally in the PFC (Figs. 5A and B, and 6), a characteristic pattern of labeling within each division consisted of a dense concentration of SERT+ fibers in layer 1, a sparser distribution in layers 2/3 and intense labeling in layers 5/6. This pattern of labeling is most evident in ORB, particularly in VO, VLO and LO, but also present in the other divisions. Specifically, as depicted in the higher magnification micrographs of ORB (Fig. 7A D), the moderately dense labeling of layers 2/3 was bordered by considerably stronger labeling in layers 1 and 5. This laminar pattern is evident in MO (Fig. 7A) and DLO (Fig. 7B) but is particularly strikingly rostrally and caudally in VLO (Fig. 7C and D). As depicted, there is a clear demarcation between the very intense labeling of layers 1 and 5 and the less robust labeling of layers 2/3. A similar pattern of labeling was observed further caudally as IL emerges and replaces MO (Fig. 9), and INC begins to occupy the lateral convexity of the PFC (Figs. 8 10). As shown, labeling was dense in layer 1, especially dorsoventrally along the medial wall of the PFC (Figs. 8 10), about as equally pronounced in layers 5/6 and less marked in intermediate layers (layers 2/3). As demonstrated rostrally, laminar differences were most prominent in ORB. With the appearance of GI (and to some extent DI), labeling became intensified in layer 4 or the granular cell layer. As depicted, 5-HT fibers were densely packed in the granule cell layer in GI as it emerged on the lateral convexity of cortex dorsal to the rhinal fissure (Fig. 11A) and this pattern persisted caudally throughout GI (Fig. 11B). With the transition from DI/GI to AIp, this band of dense labeling shifts from layer 4 to deeper layers, primarily to layer 6 (Fig. 11C). In general, 5-HT labeling is much more intense in limbicassociated (mpfc, ORB, INC) cortices than in sensorimotor regions of the cortex. This is perhaps best exemplified by the striking reduction in SERT+ fibers proceeding dorsally from the INC to the primary motor cortex (or AGl) (Fig. 9), and further caudally transitioning from INC to the primary somatosensory cortex (SSI) (Figs. 10, 12 14). In effect, labeling was very light in layers 1 3 of AGl/SSI which not only contrasted with PFC patterns, but with the considerably denser labeling of deeper layers of AGl (layers 5/6) and SSI (layer 4). Interestingly, unlike AGl (or SSI), the pattern and density of labeling of the secondary motor cortex (AGm) was generally comparable to that of limbic regions of the PFC. Although, as discussed above, labeling was less intense in layers 2/3 than in bordering deep or superficial layers, this difference was not as prominent in the mpfc (IL, PL and AC) as for other PFC Fig. 7. High magnification photomicrographs showing patterns of distribution of serotonergic fibers to the medial orbital (A), the dorsolateral orbital (B) and rostral (C) and caudal (D) regions of the ventrolateral orbital cortex. Demonstrates a massive 5-HT innervation of these divisions of the ORB and further shows a considerably stronger 5-HT innervation of superficial (layer 1) and deep layers (layer 5) than intermediate layers (layers 2/3) of MO, VLO and DLO, most prominent for VLO (C and D). (A) and (B) taken from Fig. 5A; (C) taken from Fig. 5B and (D) taken from Fig. 9. Scale bar for (A) (D) = 100 mm. See list for abbreviations.

8 36 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) Fig. 8. Pattern of distribution of serotonergic fibers to the rostral PFC showing a massive 5-HT innervation of divisions of the medial (PL, AC), orbital (MO, VO, VLO, LO), and insular (AId, DI) cortices at this level. Note: (1) the stronger 5-HT innervation of deep and superficial layers than intermediate layers of these cortical regions, most prominent for the orbital cortex; and (2) the strong band of 5-HT fibers within layer 1 on the medial wall of the PFC which includes MO, PL and AC (arrows). Scale bar = 600 mm. See list for abbreviations. divisions. For example, as depicted in Figs , apart from a narrow band of labeling in layers 1 and 6 (see below), SERT+ fibers were quite homogenously distributed mediolaterally across the mpfc, particularly in IL and PL. This is exemplified in the higher magnification micrographs of Fig. 15 showing labeling in the caudal IL/PL (Fig. 15A), the rostral PL (Fig. 15B) and the caudal AC (Fig. 15C). As depicted, fibers spread evenly throughout the mediolateral extent of IL and PL (Fig. 15A and B) with generally no preferred orientation other than a tendency to stretch horizontally across IL/PL to possibly thereby contact cells in all layers. As further depicted at the caudal mpfc (Fig. 15A and C), a dense narrow band of dorsoventrally oriented fibers was present in layers 1 and 6 of IL/PL (Fig. 15A) and AC (Fig. 15C). This is most noticeable for the labeled fibers of layer 6 (see Fig. 15A). These bands are more prominent caudally than rostrally in the mpfc as exemplified by the greater homogeneity of labeling across layers of the rostral (Fig. 15B) than caudal PL (Fig. 15A). Finally, it appears that the dense band of 5-HT fibers in layer 1 of the mpfc thins (and ends) at the border between AC and AGm (Figs. 10, 12 14), thus serving to demarcate limbic from motor regions of the PFC. 4. Discussion The present report describes the pattern of distribution of serotonergic fibers to the PFC in the rat using an antiserum to SERT. As demonstrated, 5-HT fibers spread massively throughout the medial, orbital and insular divisions of the PFC, distributing to all layers of these cortical regions. A characteristic pattern of labeling of the PFC involved dense 5-HT labeling of superficial layers (layer 1), moderate in intermediate layers (layers 2 and 3) and pronounced in deep layers (layers 5 and 6). Although observed throughout the PFC, this pattern was most striking in the ORB. With the emergence of the granular/dysgranular INC of the caudal PFC, the granular cell layer (layer 4) was readily identifiable by an intense band of labeling localized to it clearly differentiating layer 4 from less densely labeled superficial and deep layers of GI/DI. The massive 5-HT innervation of limbic regions of the PFC suggests that serotonergic fibers, predominately originating midbrain raphe nuclei, are capable of strongly affecting the limbic prefrontal cortex. Accordingly, the serotonergic system is positioned to exert a profound, global influence on the PFC, likely serving to modulate/control a range of functions, prominently including attention and behavioral states Comparisons with early analyses of 5-HT innervation of the cortex in the rat With the development of immunohistochemical techniques for the identification of serotonergic cells/fibers (or procedures utilizing antibodies to serotonin), several early reports mapped the distribution of 5-HT processes within the brain/nervous system. In initial descriptions of patterns of 5-HT labeling throughout the CNS, Steinbusch (1981, 1984) demonstrated that 5-HT fibers distribute fairly evenly throughout the PFC, most densely concentrated in layers 1 and 5/6. In a study focused on

9 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) Fig. 9. Pattern of distribution of serotonergic fibers to the rostral PFC showing a massive 5-HT innervation of divisions of the medial (IL, PL, AC), orbital (VO, VLO, LO), and insular (AId, DI, GI) cortices at this level. Note: (1) the dense 5-HT innervation of the granular cell layer (layer 4) of GI and to a lesser extent DI; and (2) the relative absence of 5-HT fibers in the layers 1 3 of the lateral agranular (frontal) cortex (AGl) (arrows), which differs significantly from the dense 5-HT labeling of these layers for the mpfc, ORB and INC cortices. Scale bar = 600 mm. See list for abbreviations. the cortex, Lidov et al. (1980) described a uniform pattern of 5- HT labeling over the lateral neocortex which consisted of the frontal, parietal, temporal and occipital cortices. Specifically, labeling within lateral cortices was shown to be quite homogeneous across lamina with fibers showing no preferred orientation in layers 2/3 but aligned dorsoventrally in layers 1 and 6, parallel to the vertical axis of the brain. By contrast, the retrosplenial cortex was characterized by alternating patterns of high and low density labeling: layers 1, 3 and 6, high density; layers 2 and 5, low density. Of note, layer 2 was described as essentially devoid of 5-HT axons. Finally, based on their previous analyses of the noradrenergic (NE) innervation of the cortex (Grzanna et al., 1977; Morrison et al., 1978), Lidov et al. (1980) described a much denser concentration of 5-HT than NE fibers in the cortex, and concluded that the very dense 5-HT innervation of the cortex suggests that raphe neurons may contact every cell in the cortex. In a subsequent study using radiographic techniques, Descarries and colleagues (Audet et al., 1989) compared 5-HT density ([ 3 H]5-HT labeled varicosities) in seven anterior regions of the cortex that included PL, AC, AGm, AGl, AId and the prepiriform cortex. They demonstrated a significantly greater density of 5-HT fibers in limbic than in non-limbic (sensorimotor) regions of cortex, and reported that PL and rostral AId were the most densely labeled sites of the rostral cortex. They further described a much greater variation in density of 5-HT labeling across lamina than shown by Lidov et al. (1980) with the densest concentration of fibers in layer Relationship between the serotonin innervation of the PFC and midbrain raphe projections to the PFC Anterograde or retrograde examinations of midbrain raphe projections to the forebrain/cortex have, for the most part, not thoroughly examined rostral regions of the cortex or the limbic PFC. Specifically, while projections of the dorsal (DR) and median (MR) raphe nuclei to sensorimotor regions of the frontal cortex have been fairly well characterized, this has not been the case for raphe projections to the mpfc, ORB and INC (Azmitia and Segal, 1978; O Hearn and Molliver, 1984; Vertes, 1991; Vertes and Martin, 1988; Vertes et al., 1999; Waterhouse et al., 1986). Using PHA-L, Vertes (1991) demonstrated DR projections to a relatively widespread area of the dorsomedial PFC that mainly included AGm and the laterally adjacent AGl. Projections were stronger from the rostral than caudal DR, and the rostral DR also heavily targeted INC and moderately parts of the mpfc and ORB. By comparison, MR (Vertes et al., 1999) was shown to provide at best modest input to the PFC (and to the cortex in general) or considerably less dense than demonstrated for DR (for review, Hale and Lowry, 2011; Vertes and Linley, 2007, 2008).

10 38 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) Fig. 10. Pattern of distribution of serotonergic fibers to a mid-rostrocaudal level of the PFC showing a massive 5-HT innervation of divisions of the medial (IL, PL, AC), orbital (VLO, LO), and insular (AId, DI, GI) cortices at this level. As shown, 5-HT labeling is denser in superficial and deep layers than in intermediate layers (layers 2/3) of the mpfc, ORB and INC, most prominently seen in ORB and INC. Scale bar = 600 mm. See list for abbreviations. In a comprehensive examination of DR/MR projections, as well as the distribution of 5-HT fibers, throughout the brain in hamsters, Morin and Meyer-Bernstein (1999) showed that: (1) 5-HT fibers distribute heavily to the PFC; (2) DR projects significantly to both motor and to limbic regions of the PFC; and (3) MR distributes sparsely throughout the cortex. Interestingly, they also reported for the posterior cingulate and piriform cortices that 5-HT fibers were densely concentrated in layer 1, moderately in layers 4 6, and sparsely in layers 2/3 a pattern comparable to that presently shown for the PFC. The seeming disparity between the robust 5-HT innervation of the PFC and moderate DR/MR projections to the PFC suggests additional sources of 5-HT fibers to the PFC. It is also possible that anterograde tracers fail to capture the degree of terminal distribution/branching of raphe fibers in the PFC as produced by immunostaining for 5-HT or SERT. Regarding, however, extra DR/MR 5-HT afferents to the PFC, O Hearn and Molliver (1984), using retrograde tracers, examined raphe input to frontal (motor), parietal and occipital cortices, and demonstrated considerably stronger raphe projections to PFC than to the other regions and further importantly showed that raphe afferents to cortex not only originated from DR and MR but also from the B9 area (Vertes and Crane, 1997). This indicates an additional significant source of 5-HT projections to the cortex, or to the PFC. In general accord with the foregoing findings, Waterhouse et al. (1986) demonstrated: (1) relatively pronounced DR projections, mainly originating from the rostral DR, to the frontal cortex, but considerably fewer projections to sensorimotor or occipital cortices; and (2) minor (or sparse) MR projections to each of the cortical regions. In addition, they reported that approximately 30% of DR cells distributing to the cortex sent collateral projections to the cerebellum, and proposed that this population of DR neurons may coordinate the activity of cortical and cerebellar regions processing similar information. In like manner, the DR has been shown to give rise to collateral projections to motor and limbic regions of the PFC (mpfc) (Sarter and Markowitsch, 1984), and to nucleus accumbens and to the PFC (Van Bockstaele et al., 1993) Distribution of 5-HT fibers to the cortex in other species As discussed, Morin and Meyer-Bernstein (1999) reported that 5-HT fibers distribute heavily and fairly uniformly throughout the cortex in hamsters, most densely concentrated in the insular, parietal and occipital cortices. Without subdividing regions of the PFC, the 5-HT innervation of the PFC was described as dense but less so than for the above-mentioned cortical sites. In a recent description of the distribution of SERT+ fibers to the orbitofrontal cortex (OFC) of primates (vervet monkey) using autoradiographic techniques, Way et al. (2007) demonstrated a caudal to rostral gradient of labeling such that 5-HT fibers were heavily concentrated in the agranular OFC and progressively less so in the dysgranular and granular OFC. The authors further indicated that the heterogeneous distribution of 5-HT fibers to the OFC

11 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) Fig. 11. Photomicrographs showing patterns of distribution of 5-HT fibers at three rostral to caudal (A C) levels of the insular cortex. Note: (1) denser labeling in superficial and deep layers than in intermediate layers of the INC; (2) very dense labeling in the granular cell layer of GI and to some extent DI, setting it apart from bordering deep and superficial layers; and (3) the shift in the dense band of labeling in the granular cell layer (of GI) to layer 6 of the posterior agranular insular cortex (AIp). (A) Taken from Fig. 9; (B) taken from Fig. 12. Scale bar for (A) = 600 mm; for (B) = 500 mm; for (C) = 700 mm. See list for abbreviations.

12 40 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) Fig. 12. Pattern of distribution of serotonergic fibers at a mid rostrocaudal level of the PFC showing a massive 5-HT innervation of divisions of the medial (IL, PL, AC), orbital (VLO), and insular (AId, AIv, DI, GI) cortices at this level. Note: (1) the dense labeling in layer 4 of GI and DI; and (2) the considerably lighter labeling in intermediate layers (layers 2/3) than in superficial and deep layers of AId, AIv and VLO that continued medially into the piriform cortex. Scale bar = 600 mm. See list for abbreviations. suggests that serotonin should not be viewed as exerting a diffuse, non-specific action on the cortex, but rather differential effects reflecting marked variations in 5-HT densities across cortical structures. In a recent comparison of the distribution of 5-HT fibers to select regions of the frontal cortex of chimpanzees, macaques and humans using immunostaining for SERT, Raghanti et al. (2008) described significant differences in patterns of 5-HT labeling across species in cognitively associated areas (areas 9 and 32) of the cortex but not in motor regions (area 4) of the PFC. Specifically, layers 5/6 of areas 9 and 32 contained a dense concentration of 5-HT fibers in humans and chimpanzees compared to macaques, but no differences were observed in area 4 across species HT influence on thalamocortical systems Studies examining the 5-HT innervation of thalamus using immunostaining for 5-HT or SERT have shown that serotonergic axons are much more heavily concentrated in limbic (nonspecific) than in principal (relay) nuclei of the thalamus (Cropper et al., 1984; Lavoie and Parent, 1991; Vertes et al., 2010). For instance, using antisera to SERT, we demonstrated that 5-HT fibers are densely distributed in the anterior, midline, intralaminar and mediodorsal nuclei of thalamus (Vertes et al., 2010). By contrast, non-limbic areas of the thalamus, largely consisting of relay nuclei, contained few 5-HT fibers the only exception being the lateral geniculate complex. Lavoie and Parent (1991) described similar findings in the monkey leading them to state that: The densest 5-HT innervation of the thalamus was observed in nuclei located directly on the midline. These findings, together with present (and previous) demonstrations of a dense 5-HT innervation of the limbic PFC indicate that 5-HT fibers strongly target limbic prefrontal cortices together with their main thalamic inputs. This suggests a dual (direct and indirect) 5-HT mediated influence on PFC activity HT influence on prefrontal function Serotonin has been shown to play a role in the modulation of a number of affective and emotional behaviors, and 5-HT enhancement is widely recognized as a successful treatment for several mental conditions including depression, schizophrenia,

13 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) Fig. 13. Pattern of distribution of serotonergic fibers at the caudal PFC showing a massive 5-HT innervation of divisions of the medial (IL, PL, AC), orbital (VLO), and insular (AId, AIv, DI, GI) cortices at this level. Note: (1) the dense labeling in layer 4 (granular cell layer) of GI and DI that continued dorsally into layer 4 of the primary somatosensory cortex (arrows); (2) the considerably lighter labeling of layers 2/3 than in bordering layers of INC and ORB as well as a similar pattern in the medially adjacent piriform cortex; and (3) the narrow dense, dorsoventrally oriented bands of 5-HT fibers in layers 1 and 6 of IL, PL and AC. Scale bar = 600 mm. See list for abbreviations. and obsessive compulsive disorder (Cools et al., 2008; Geyer and Vollenweider, 2008; Goddard et al., 2008; Remington, 2008). Both depression and obsessive compulsive disorders appear to be associated with low levels of 5-HT in the PFC, and the compulsivity produced by OFC in rats is attenuated by the administration of selective serotonin reuptake inhibitors (Goddard et al., 2008; Joel et al., 2005). Serotonin in the PFC also modulates a number of cognitive processes including attention, behavioral inhibition, flexibility, and decision-making (Chudasama and Robbins, 2006; Dalley et al., 2004; Homberg, 2012). Moreover, 5-HT exerts dissociable effects on the mpfc and ORB (Robbins and Roberts, 2007). For example, 5-HT modulates behavioral flexibility in the ORB, or the ability to adapt behavior to changes in rules and reward values (Clark et al., 2004). Serotonergic lesions of the ORB produce perseverative responding a mark of behavioral inflexibility (Clarke et al., 2004, 2005, 2007; Walker et al., 2009). By contrast, 5-HT is critically involved in behavioral inhibition and decisionmaking in the mpfc. Specifically, the depletion of 5-HT in the mpfc increases impulsivity in the 5-choice reaction time test and impulsive behavior in delay discounting paradigms, and increases in extracellular 5-HT has been described during the performance of delayed discounting tasks (Chudasama and Robbins, 2006; Clark et al., 2004; Dalley et al., 2004, 2008; Winstanley et al., 2006). In human imaging studies, ORB activity correlates with response inhibition on a go/no-go task (Homberg, 2012). It is well recognized that the actions of 5-HT on the PFC (as well as on other parts of the brain) involve a diverse set of 5-HT receptors. While it may be premature to ascribe distinct functional roles to specific types of 5-HT receptors or their interactions in the PFC, recent studies have described the unique involvement of certain classes of 5-HT receptors in PFC function (for review, Puig and Gulledge, 2011). Specifically, it has been shown that: (1) 5-HT 1A, 5-HT 2A and to some extent 5-HT 3A receptors are highly expressed in the PFC localized to both pyramidal cells (PCs) and interneurons (INs); (2) 5-HT 1A receptors exert inhibitory effects, 5-HT 2A receptors excitatory effects on PCs and INs and 5HT 3A receptors excite INs; (3) 5-HT 1A receptors are primarily located on the soma and initial segments

14 42 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) Fig. 14. Pattern of distribution of serotonergic fibers at the caudal PFC showing a massive 5-HT innervation of divisions of the medial (IL, PL, AC) and insular (AId, AIv, DI, GI) cortices at this level. Note: (1) the continuous dense band of 5-HT fibers in layer 4 stretching from INC dorsally through the primary somatosensory cortex; (2) the considerably lighter labeling in intermediate layers (layers 23) than in deeper layers of INC as well as dorsally in SSI and AGl; and (3) the quite homogenous distribution of fibers throughout all lamina of IL, PL and AC with the exception of narrow dense bands of 5-HT fibers in outer layer 1 and inner layer 6. Scale bar = 600 mm. See list for abbreviations. of PCs, while 5-HT 2A receptors are highly expressed on the apical dendrites of PCs; and finally (4) 5-HT 1A and 5-HT 2A receptorcontaining interneurons are primarily localized to deep layers (5/6) of the PFC and 5-HT 3A containing INs to superficial layers of the PFC (Santana et al., 2004; Puig et al., 2010; Puig and Gulledge, 2011). Regarding the possible functional implications of this organization, it has been proposed that 5-HT 1A receptors exert marked inhibitory actions on the soma of PCs to suppress action potential generation, whereas 5-HT 2A receptors excite (depolarize) distal apical dendrites of PCs to produce oscillatory membrane potential changes in large numbers of PCs giving rise to cortical rhythms of sleep-waking states (Puig and Gulledge, 2011). In accord with the foregoing, the present demonstration of a denser 5-HT innervation of deep (layers 5/6) than superficial (layer 2) layers of the PFC suggests that 5-HT may exert its greatest effect on PCs (and INs) of the deep layers of the PFC in the modulation of the cortical EEG and associated behavioral states Conclusions Serotonergic fibers distribute massively throughout the mpfc, ORB, and INC and to each of their subdivisions. While the entire PFC receives a dense 5-HT innervation, a distinct laminar organization was observed such that labeling was more intense in superficial (layer 1) and deep layers (layers 5/6) than in intermediate layers (layers 2/3). This pattern was most prominent in the ORB. In the granular divisions of the INC (GI and DI), 5-HT fibers were densely concentrated in the granule cell layer (layer 4). The serotonergic input to the PFC, which much more strongly targets limbic than sensorimotor regions of the PFC, likely plays a critical role in the modulation of

15 S.B. Linley et al. / Journal of Chemical Neuroanatomy (2013) Fig. 15. High magnification photomicrographs showing patterns of distribution of 5-HT fibers at a caudal level of IL/PL (A), a rostral level of PL (B), and a caudal level of AC (C). (A) Shows that aside from narrow dorsoventrally oriented bands of 5-HT fibers in layers 1/6, fibers were quite evenly distributed throughout all lamina of IL/PL with a preferred horizontal orientation. (B) Shows that 5-HT fibers spread homogenously throughout all lamina of the rostral PL including layer 6. (C) Shows dense aggregates of 5- HT fibers in layers 1 and 6 of the dorsal and ventral AC, with considerably lighter labeling in intervening lamina including layer 5. (A) Taken from Fig. 13; (B) taken from Fig. 5. Scale bar for (A) = 250 mm; for (B) = 175 mm; for (C) = 150 mm. See list for abbreviations. affective and cognitive behaviors associated with the prefrontal cortex. Acknowledgment This research was supported by National Science Foundation grant IOS to RPV. References Audet, M.A., Descarries, L., Doucet, G., Quantified regional and laminar distribution of the serotonin innervation in the anterior half of adult rat cerebral cortex. Journal of Chemical Neuroanatomy 2, Azmitia, E.C., Segal, M., An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. Journal of Comparative Neurology 179, Berger, B., Trottier, S., Verney, C., Gaspar, P., Alvarez, C., Regional and laminar distribution of the dopamine and serotonin innervation in the macaque cerebral cortex: a radioautographic study. Journal of Comparative Neurology 273, Boulougouris, V., Glennon, J.C., Robbins, T.W., Dissociable effects of selective 5-HT2A and 5-HT2C receptor antagonists on serial spatial reversal learning in rats. Neuropsychopharmacology 33, Boulougouris, V., Robbins, T.W., Enhancement of spatial reversal learning by 5-HT2C receptor antagonism is neuroanatomically specific. Journal of Neuroscience 30, Chudasama, Y., Passetti, F., Rhodes, S.E., Lopian, D., Desai, A., Robbins, T.W., Dissociable aspects of performance on the 5-choice serial reaction time task following lesions of the dorsal anterior cingulate, infralimbic and orbitofrontal cortex in the rat: differential effects on selectivity, impulsivity and compulsivity. Behavioural Brain Research 146, Chudasama, Y., Robbins, T.W., Functions of frontostriatal systems in cognition: comparative neuropsychopharmacological studies in rats, monkeys and humans. Biological Psychology 73, Clark, L., Cools, R., Robbins, T.W., The neuropsychology of ventral prefrontal cortex: decision-making and reversal learning. Brain and Cognition 55, Clarke, H.F., Dalley, J.W., Crofts, H.S., Robbins, T.W., Roberts, A.C., Cognitive inflexibility after prefrontal serotonin depletion. Science 304,

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