Journal of Chemical Neuroanatomy - FAUvertes/ctx-sert-final-published.pdf · of Chemical...

Journal of Chemical Neuroanatomy 48–49 (2013) 29–45

Pattern of distribution of serotonergic fibers to the orbitomedial andinsular 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 Statesb 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.

� 2012 Elsevier B.V. All rights reserved.

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Journal of Chemical Neuroanatomy

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1. Introduction

The prefrontal cortex (PFC) of the rat consists of the medial(mPFC), orbital (ORB) and insular (INC) prefrontal cortices andextends 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, endopiri-

form 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, ventrolat-

eral orbital cortex; VO, ventral orbital cortex.

* Corresponding author. Tel.: +1 561 297 2362; fax: +1 561 297 2363.

E-mail address: [email protected] (R.P. Vertes).

0891-0618/$ – see front matter � 2012 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.jchemneu.2012.12.006

Each cortical region has several subdivisions, compromised ofunique chemical and morphological features. The subdivisions ofthe rat are largely functionally homologous to those of the primatePFC, and each subserves distinct affective and cognitive functions(Chudasama and Robbins, 2006; Dalley et al., 2004; Ongur andPrice, 2000; Seamans et al., 2008; Uylings et al., 2003).

The mPFC consists of four main divisions which dorsally toventrally include the medial (frontal) agranular cortex (AGm), theanterior cingulate cortex (AC), the prelimbic cortex (PL) and theinfralimbic cortex (IL) (Figs. 1 and 2). The dorsal (AGm and AC) andventral (PL and IL) mPFC reportedly serve separate and distinctfunctions, with the dorsal mPFC primarily involved in motorbehavior and the ventral mPFC in diverse emotional, cognitive andmnemonic processes (Heidbreder and Groenewegen, 2003;Hoover and Vertes, 2007; Vertes, 2006). More specifically, theAGm, or secondary motor area, mainly participates in motorplanning and execution, and the AC in motor initiation/impulsivityand attention (Chudasama et al., 2003; Dalley et al., 2004; Kesnerand Churchwell, 2011; Muir et al., 1996). By contrast, the PL is


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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.

S.B. Linley et al. / Journal of Chemical Neuroanatomy 48–49 (2013) 29–4530

involved in executive functions including decision making andworking memory, while the IL modulates autonomic and visceralactivity and fear extinction (Dalley et al., 2004; Sotres-Bayon andQuirk, 2010; Vertes, 2006).

The ORB of the rat extends medial-laterally across the ventralsurface of the rostral pole of the PFC and consists (medial tolateral) of the medial orbital (MO), ventral orbital (VO),ventrolateral orbital (VLO), lateral orbital (LO) and dorsolateralorbital (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 inbehavioral flexibility, as commonly demonstrated by orbital-associated deficits in reversal learning, impulsivity and compul-sive behaviors (Fineberg et al., 2010; Kesner and Churchwell,2011; Schoenbaum et al., 2009; Winstanley et al., 2010). The INCsurrounds the rhinal fissure and includes the dorsal (AId), ventral

(AIv) and posterior (AIp) agranular insular cortices, and thedysgranular (DI) and granular (GI) insular cortices (Figs. 1 and 2).The INC is reciprocally connected with the gustatory and visceralcortices and among its various functions is thought to serve as avisceral or multisensory receptive zone (Kobayashi, 2011; Saper,1982).

Serotonin (5-HT) innervates the entire neuroaxis including thePFC. The dorsal (DR) and median (MR) raphe nuclei are the primaryorigins of 5-HT projections to the forebrain, with the DR the mainsource of 5-HT projections to the PFC (Morin and Meyer-Bernstein,1999; Vertes, 1991; Vertes and Linley, 2008). 5-HT is a keymodulator of various homeostatic, affective and cognitive func-tions (Cools et al., 2008; Homberg, 2012; Monti, 2011; Robbins andRoberts, 2007; Sanford et al., 2008). This control, in part, ismediated by 5-HT DR projections to the PFC and return PFCmodulatory influences on the DR and MR (Goncalves et al., 2009;

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.

S.B. Linley et al. / Journal of Chemical Neuroanatomy 48–49 (2013) 29–45 31

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 becritical for decision-making, behavioral flexibility, behavioralinhibition and impulsivity (Chudasama and Robbins, 2006; Clarket al., 2004; Cools et al., 2011; Robbins and Roberts, 2007). Each ofthese behaviors is dependent upon 5-HT input to specificsubregions of the PFC. For instance, 5-HT afferents to the mPFCmodulate decision-making and impulsivity, while those to the ORBare involved in behavioral flexibility (Chudasama and Robbins,2006; Clark et al., 2004; Robbins and Roberts, 2007). Intracer-ebroventricular or local injections of the 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT) into the mPFC markedly en-hanced impulsive motor choices in the 5-choice serial reactiontime test, as well as impulsive behavior in delay discountingparadigms (Harrison et al., 1997; Mobini et al., 2000; Winstanleyet al., 2004). In addition, several reports using in vivo microdialysisdemonstrated increases in 5-HT in the mPFC during performanceof these tasks (Chudasama and Robbins, 2006; Clark et al., 2004;

Dalley et al., 2004, 2008; Robbins and Roberts, 2007; Winstanleyet al., 2006). Van der Plasse et al. (2007) reported that serotonergicdenervation of the mPFC produced impairments in decision-making which were dependent on the salience of an appetitivereward.

Serotonergic afferents to the ORB are crucial for cognitiveflexibility. Excitotoxic lesions of the ORB in the rat significantlyaltered reversal learning in the intradimensional extradimen-sional (IED) olfactory set shifting task but left other attentionalmeasures unimpaired (McAlonan and Brown, 2003). Reversallearning was similarly disrupted following 5-HT loss in the ORBin a primate analog of the task (Clarke et al., 2004, 2005, 2007).The application of 5-HT2A, 5-HT2C, 5-HT6, 5-HT7 receptorantagonists or 5-HT1A receptor agonists to the ORB improvedreversal 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 reversallearning in the IED task or other tasks. This is unlike the


recognized effects of catecholamines (DA and NE) on attentionaltasks in other regions of the PFC (Robbins and Roberts, 2007;Roberts et al., 1994). Finally, acute tryptophan depletion inhealthy volunteers produced impairments in reversal learning(Clark et al., 2004).

No reports have comprehensively investigated the distributionof serotonergic afferents to the mPFC, ORB and INC in the rat. Someearly reports described overall patterns of distribution of 5-HTfibers to regions of the cortex but without specific attention to thePFC (Audet et al., 1989; Berger et al., 1988; Lidov et al., 1980;Steinbusch, 1981) while others have concentrated on a particularsubregion 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 mostpart 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). Arecent report, however, described the concentration/distributionof 5-HT fibers over regions of the PFC in primates (Way et al.,2007).

The present study examined, compared, and contrastedthe distribution and laminar organization of 5-HT fibers tothe medial, orbital and insular divisions of the PFC usingimmuno-procedures for the detection of the serotonin trans-porter protein (SERT). Nielsen et al. (2006) showed that SERT as abiomarker for 5-HT produces a stronger signal than antisera toserotonin. We found that both 5-HT and SERT immunohis-tochemistry produce similar patterns of serotonergic labeling,

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.

but that SERT immunostaining, by heightening signal tobackground, provided a bolder representation of labeled 5-HTfibers (Vertes et al., 2010).

2. Materials and methods

Ten (5 male, 5 female) naıve Sprague Dawley rats (Harlan, Indianapolis, IN)

weighing 275–300 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

30–50 ml of ice cold heparinized 0.1 M phosphate buffered saline (PBS) followed by

200–300 ml of chilled 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at a

pH = 7.4. The brains were removed and postfixed for 24–48 h in 4% paraformalde-

hyde 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. 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.


2.1. 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 (NaBH4) 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 24–48 h.

Following further washes, sections were placed in a secondary antibody,

biotinylated goat anti-rabbit immunoglobulin (Vector Labs, Burlingame, CA) in

Fig. 5. (A and B) Pattern of distribution of serotonergic fibers to the rostral pole of the PFC

VO, VLO, LO, DLO) prefrontal cortices at these levels. Scale bar for (A) and (B) = 600 mm

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% 3,30-diamino-

benzidine (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).

showing a massive 5-HT innervation of divisions of the medial (PL) and orbital (MO,

. See list for abbreviations.


2.2. Photomicroscopy

Lightfield photomicrographs at 100� magnification were taken for visuali-

zation 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 majorparts, the medial (mPFC), orbital (ORB) and insular (INC) cortices,each containing various subdivisions. Figs. 1 and 2 consist of arostral to caudally aligned series of transverse sections throughthe forebrain depicting divisions/subdivisions of the PFC for arepresentative case (case 14B). At the rostral pole of cortex, theprelimbic cortex (PL) is located dorsally and medial orbital cortex(MO) ventrally on the medial wall of the PFC. (Fig. 1A and B). Theanterior cingulate cortex (AC), dorsal to PL, joins these tworegions slightly caudally (Fig. 1C). At the beginning of the anteriorforceps (Fig. 1E), the infralimbic cortex (IL) replaces MO. Fromthere (Fig. 1E) to caudally through the PFC (Fig. 2D), the medialwall of the PFC consists of the dorsoventrally aligned AC, PL and ILcortices (Fig. 2A–D). As depicted, PL is the largest of the threeregions at these levels. The dorsomedially located AC extendscaudally to the level of the rostral pole of the thalamus, where it

Fig. 6. Pattern of distribution of serotonergic fibers to the rostral PFC showing a massive 5

prefrontal cortices at this level. Scale bar = 600 mm. See list for abbreviations.

subdivides into dorsal (ACd) and ventral (ACv) divisions (notshown in Figs. 1 and 2).

The ORB extends medio-laterally along the ventral surface ofthe 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 dorsalagranular insular cortex (AId), and slightly caudal to this (Fig. 1E)MO gives way to IL. At the level of nucleus accumbens (Fig. 2A), VOis no longer present and just caudally (Fig. 2B) LO is replaced by theventral agranular insular cortex (AIv). VLO continues to approxi-mately the end of the PFC (Fig. 2C), thus occupying a considerablelongitudinal extent of the PFC.

The INC is located along the lateral convexity of cortexbordering the rhinal fissure and consists of AId, AIv, thedysgranular (DI) and granular (GI) insular divisions (Figs. 1D, Eand 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 andGI at the level of nucleus accumbens (Fig. 2B). All four divisionsextend to the caudal pole of the PFC. The posterior agranularinsular cortex (AIp) emerges at the level of the septum and extendsto mid-levels of the thalamus (not shown in Figs. 1 and 2). AId is thelargest of the insular divisions extending virtually rostrocaudallythroughout the PFC.

The Nissl-stained sections of Figs. 3 and 4 show the laminarorganization of the three divisions of the PFC: orbital (Fig. 3A),insular (Fig. 3B) and medial (Fig. 4) prefrontal cortex.

3.2. Pattern of distribution of SERT immunoreactive fibers in the PFC

The patterns of distribution of SERT immunoreactive fibersthroughout the PFC are depicted in the low and higher magnificationplates of Figs. 5–15. As demonstrated, all divisions/subdivisions of

-HT innervation of divisions of the medial (PL, AC) and orbital (MO, VO, VLO, LO, DLO)


the medial, orbital and insular cortices contain a very denseconcentration of SERT immunoreactive (SERT+) fibers, with varia-tions across divisions and layers of the PFC. As shown rostrally in thePFC (Figs. 5A and B, and 6), a characteristic pattern of labeling withineach division consisted of a dense concentration of SERT+ fibers inlayer 1, a sparser distribution in layers 2/3 and intense labeling inlayers 5/6. This pattern of labeling is most evident in ORB,particularly in VO, VLO and LO, but also present in the otherdivisions. Specifically, as depicted in the higher magnificationmicrographs of ORB (Fig. 7A–D), the moderately dense labeling oflayers 2/3 was bordered by considerably stronger labeling in layers 1and 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 thevery intense labeling of layers 1 and 5 and the less robust labeling oflayers 2/3.

A similar pattern of labeling was observed further caudally asIL emerges and replaces MO (Fig. 9), and INC begins to occupy thelateral convexity of the PFC (Figs. 8–10). As shown, labeling wasdense in layer 1, especially dorsoventrally along the medial wallof the PFC (Figs. 8–10), about as equally pronounced in layers 5/6and less marked in intermediate layers (layers 2/3). Asdemonstrated rostrally, laminar differences were most promi-nent in ORB.

Fig. 7. High magnification photomicrographs showing patterns of distribution of seroton

caudal (D) regions of the ventrolateral orbital cortex. Demonstrates a massive 5-HT inner

innervation of superficial (layer 1) and deep layers (layer 5) than intermediate layers (la

from Fig. 5A; (C) taken from Fig. 5B and (D) taken from Fig. 9. Scale bar for (A)–(D) = 1

With the appearance of GI (and to some extent DI), labelingbecame intensified in layer 4 – or the granular cell layer. Asdepicted, 5-HT fibers were densely packed in the granule cell layerin GI as it emerged on the lateral convexity of cortex dorsal to therhinal fissure (Fig. 11A) and this pattern persisted caudallythroughout 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 ‘limbic-associated’ (mPFC, ORB, INC) cortices than in sensorimotorregions of the cortex. This is perhaps best exemplified by thestriking reduction in SERT+ fibers proceeding dorsally from theINC to the primary motor cortex (or AGl) (Fig. 9), and furthercaudally transitioning from INC to the primary somatosensorycortex (SSI) (Figs. 10, 12–14). In effect, labeling was very light inlayers 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), thepattern and density of labeling of the secondary motor cortex(AGm) was generally comparable to that of limbic regions ofthe PFC.

Although, as discussed above, labeling was less intense in layers2/3 than in bordering deep or superficial layers, this difference wasnot as prominent in the mPFC (IL, PL and AC) as for other PFC

ergic fibers to the medial orbital (A), the dorsolateral orbital (B) and rostral (C) and

vation of these divisions of the ORB and further shows a considerably stronger 5-HT

yers 2/3) of MO, VLO and DLO, most prominent for VLO (C and D). (A) and (B) taken

00 mm. See list for abbreviations.

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. 12–14, apart from anarrow band of labeling in layers 1 and 6 (see below), SERT+ fiberswere quite homogenously distributed mediolaterally across themPFC, particularly in IL and PL. This is exemplified in the highermagnification micrographs of Fig. 15 showing labeling in thecaudal IL/PL (Fig. 15A), the rostral PL (Fig. 15B) and the caudal AC(Fig. 15C). As depicted, fibers spread evenly throughout themediolateral extent of IL and PL (Fig. 15A and B) with generally nopreferred orientation other than a tendency to stretch horizontallyacross IL/PL – to possibly thereby contact cells in all layers. Asfurther depicted at the caudal mPFC (Fig. 15A and C), a densenarrow band of dorsoventrally oriented fibers was present in layers1 and 6 of IL/PL (Fig. 15A) and AC (Fig. 15C). This is most noticeablefor the labeled fibers of layer 6 (see Fig. 15A). These bands are moreprominent caudally than rostrally in the mPFC as exemplified bythe greater homogeneity of labeling across layers of the rostral(Fig. 15B) than caudal PL (Fig. 15A). Finally, it appears that thedense band of 5-HT fibers in layer 1 of the mPFC thins (and ends) atthe border between AC and AGm (Figs. 10, 12–14), thus serving todemarcate limbic from motor regions of the PFC.

4. Discussion

The present report describes the pattern of distribution ofserotonergic fibers to the PFC in the rat using an antiserum to SERT.As demonstrated, 5-HT fibers spread massively throughout themedial, orbital and insular divisions of the PFC, distributing to all

layers of these cortical regions. A characteristic pattern of labeling ofthe PFC involved dense 5-HT labeling of superficial layers (layer 1),moderate in intermediate layers (layers 2 and 3) and pronounced indeep layers (layers 5 and 6). Although observed throughout the PFC,this pattern was most striking in the ORB. With the emergence of thegranular/dysgranular INC of the caudal PFC, the granular cell layer(layer 4) was readily identifiable by an intense band of labelinglocalized to it clearly differentiating layer 4 from less densely labeledsuperficial 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 ofstrongly affecting the limbic prefrontal cortex. Accordingly, theserotonergic system is positioned to exert a profound, globalinfluence on the PFC, likely serving to modulate/control a range offunctions, prominently including attention and behavioral states.

4.1. Comparisons with early analyses of 5-HT innervation of the cortex

in the rat

With the development of immunohistochemical techniquesfor the identification of serotonergic cells/fibers (or proceduresutilizing antibodies to serotonin), several early reports mappedthe distribution of 5-HT processes within the brain/nervoussystem. In initial descriptions of patterns of 5-HT labelingthroughout the CNS, Steinbusch (1981, 1984) demonstrated that5-HT fibers distribute fairly evenly throughout the PFC, mostdensely concentrated in layers 1 and 5/6. In a study focused on

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 thefrontal, parietal, temporal and occipital cortices. Specifically,labeling within lateral cortices was shown to be quite homoge-neous across lamina with fibers showing no preferred orienta-tion in layers 2/3 but aligned dorsoventrally in layers 1 and 6,parallel to the vertical axis of the brain. By contrast, theretrosplenial cortex was characterized by alternating patterns ofhigh and low density labeling: layers 1, 3 and 6, high density;layers 2 and 5, low density. Of note, layer 2 was described asessentially devoid of 5-HT axons. Finally, based on their previousanalyses 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 inthe cortex, and concluded that the very dense 5-HT innervationof the cortex suggests that ‘‘raphe neurons may contact everycell in the cortex’’.

In a subsequent study using radiographic techniques, Descar-ries and colleagues (Audet et al., 1989) compared 5-HT density([3H]5-HT labeled varicosities) in seven anterior regions of thecortex that included PL, AC, AGm, AGl, AId and the prepiriformcortex. They demonstrated a significantly greater density of 5-HTfibers in ‘limbic’ than in non-limbic (sensorimotor) regions ofcortex, and reported that PL and rostral AId were the most denselylabeled sites of the rostral cortex. They further described a much

greater variation in density of 5-HT labeling across lamina thanshown by Lidov et al. (1980) with the densest concentration offibers in layer 1.

4.2. Relationship between the serotonin innervation of the PFC and

midbrain raphe projections to the PFC

Anterograde or retrograde examinations of midbrain rapheprojections to the forebrain/cortex have, for the most part, notthoroughly examined rostral regions of the cortex – or the limbicPFC. Specifically, while projections of the dorsal (DR) and median(MR) raphe nuclei to sensorimotor regions of the frontal cortexhave been fairly well characterized, this has not been the case forraphe projections to the mPFC, ORB and INC (Azmitia and Segal,1978; O’Hearn and Molliver, 1984; Vertes, 1991; Vertes andMartin, 1988; Vertes et al., 1999; Waterhouse et al., 1986). UsingPHA-L, Vertes (1991) demonstrated DR projections to a relativelywidespread area of the dorsomedial PFC that mainly includedAGm and the laterally adjacent AGl. Projections were strongerfrom the rostral than caudal DR, and the rostral DR alsoheavily targeted INC and moderately parts of the mPFC andORB. By comparison, MR (Vertes et al., 1999) was shown toprovide at best modest input to the PFC (and to the cortex ingeneral) – or considerably less dense than demonstrated for DR(for review, Hale and Lowry, 2011; Vertes and Linley, 2007, 2008).

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 wellas the distribution of 5-HT fibers, throughout the brain in hamsters,Morin and Meyer-Bernstein (1999) showed that: (1) 5-HT fibersdistribute heavily to the PFC; (2) DR projects significantly to bothmotor and to ‘limbic’ regions of the PFC; and (3) MR distributessparsely throughout the cortex. Interestingly, they also reportedfor the posterior cingulate and piriform cortices that 5-HT fiberswere densely concentrated in layer 1, moderately in layers 4–6,and sparsely in layers 2/3 – a pattern comparable to that presentlyshown for the PFC.

The seeming disparity between the robust 5-HT innervationof the PFC and moderate DR/MR projections to the PFC suggestsadditional sources of 5-HT fibers to the PFC. It is also possible thatanterograde tracers fail to capture the degree of terminaldistribution/branching of raphe fibers in the PFC as producedby 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 demonstratedconsiderably stronger raphe projections to PFC than to the otherregions and further importantly showed that raphe afferents tocortex not only originated from DR and MR but also from the B9area (Vertes and Crane, 1997). This indicates an additionalsignificant 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 occipitalcortices; and (2) minor (or sparse) MR projections to each ofthe cortical regions. In addition, they reported that approximately30% of DR cells distributing to the cortex sent collateralprojections to the cerebellum, and proposed that this populationof DR neurons may coordinate the activity of cortical andcerebellar regions processing similar information. In like manner,the DR has been shown to give rise to collateral projections tomotor and ‘limbic’ regions of the PFC (mPFC) (Sarter andMarkowitsch, 1984), and to nucleus accumbens and to the PFC(Van Bockstaele et al., 1993).

4.3. Distribution of 5-HT fibers to the cortex in other species

As discussed, Morin and Meyer-Bernstein (1999) reported that5-HT fibers distribute heavily and fairly uniformly throughout thecortex in hamsters, most densely concentrated in the insular,parietal and occipital cortices. Without subdividing regions of thePFC, the 5-HT innervation of the PFC was described as dense – butless so than for the above-mentioned cortical sites.

In a recent description of the distribution of SERT+ fibers to theorbitofrontal cortex (OFC) of primates (vervet monkey) usingautoradiographic techniques, Way et al. (2007) demonstrated acaudal to rostral gradient of labeling such that 5-HT fibers wereheavily concentrated in the agranular OFC and progressively less soin the dysgranular and granular OFC. The authors further indicatedthat the heterogeneous distribution of 5-HT fibers to the OFC

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.


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 effectsreflecting marked variations in 5-HT densities across corticalstructures.

In a recent comparison of the distribution of 5-HT fibers toselect regions of the frontal cortex of chimpanzees, macaquesand humans using immunostaining for SERT, Raghanti et al.(2008) described significant differences in patterns of 5-HTlabeling across species in ‘cognitively associated’ areas (areas 9and 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 denseconcentration of 5-HT fibers in humans and chimpanzeescompared to macaques, but no differences were observed inarea 4 across species.

4.4. 5-HT influence on thalamocortical systems

Studies examining the 5-HT innervation of thalamus usingimmunostaining for 5-HT or SERT have shown that serotonergicaxons are much more heavily concentrated in ‘limbic’ (non-specific) 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 demonstratedthat 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, largelyconsisting of relay nuclei, contained few 5-HT fibers – the onlyexception being the lateral geniculate complex. Lavoie andParent (1991) described similar findings in the monkey leadingthem to state that: ‘‘The densest 5-HT innervation of thethalamus was observed in nuclei located directly on themidline’’. These findings, together with present (and previous)demonstrations of a dense 5-HT innervation of the limbic PFCindicate that 5-HT fibers strongly target limbic prefrontalcortices together with their main thalamic inputs. This suggestsa dual (direct and indirect) 5-HT mediated influence on PFCactivity.

4.5. 5-HT influence on prefrontal function

Serotonin has been shown to play a role in the modulation ofa number of affective and emotional behaviors, and 5-HTenhancement is widely recognized as a successful treatment forseveral mental conditions including depression, schizophrenia,

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 andVollenweider, 2008; Goddard et al., 2008; Remington, 2008). Bothdepression and obsessive–compulsive disorders appear to beassociated with low levels of 5-HT in the PFC, and the compulsivityproduced by OFC in rats is attenuated by the administration ofselective serotonin reuptake inhibitors (Goddard et al., 2008; Joelet al., 2005).

Serotonin in the PFC also modulates a number of cognitiveprocesses including attention, behavioral inhibition, flexibility,and decision-making (Chudasama and Robbins, 2006; Dalleyet al., 2004; Homberg, 2012). Moreover, 5-HT exerts dissociableeffects on the mPFC and ORB (Robbins and Roberts, 2007). Forexample, 5-HT modulates behavioral flexibility in the ORB, or theability to adapt behavior to changes in rules and reward values(Clark et al., 2004). Serotonergic lesions of the ORB produceperseverative 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 decision-making in the mPFC. Specifically, the depletion of 5-HT in themPFC increases impulsivity in the 5-choice reaction time test

and impulsive behavior in delay discounting paradigms, andincreases in extracellular 5-HT has been described during theperformance of delayed discounting tasks (Chudasama andRobbins, 2006; Clark et al., 2004; Dalley et al., 2004, 2008;Winstanley et al., 2006). In human imaging studies, ORB activitycorrelates with response inhibition on a go/no-go task (Hom-berg, 2012).

It is well recognized that the actions of 5-HT on the PFC (aswell as on other parts of the brain) involve a diverse set of 5-HTreceptors. While it may be premature to ascribe distinctfunctional roles to specific types of 5-HT receptors or theirinteractions in the PFC, recent studies have described the uniqueinvolvement of certain classes of 5-HT receptors in PFC function(for review, Puig and Gulledge, 2011). Specifically, it has beenshown that: (1) 5-HT1A, 5-HT2A and to some extent 5-HT3A

receptors are highly expressed in the PFC localized to bothpyramidal cells (PCs) and interneurons (INs); (2) 5-HT1A

receptors exert inhibitory effects, 5-HT2A receptors excitatoryeffects on PCs and INs and 5HT3A receptors excite INs; (3) 5-HT1A

receptors are primarily located on the soma and initial segments

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-HT2A receptors are highly expressed on the apicaldendrites of PCs; and finally (4) 5-HT1A and 5-HT2A receptor-containing interneurons are primarily localized to deep layers(5/6) of the PFC and 5-HT3A containing INs to superficial layers ofthe PFC (Santana et al., 2004; Puig et al., 2010; Puig and Gulledge,2011). Regarding the possible functional implications of thisorganization, it has been proposed that 5-HT1A receptors exertmarked inhibitory actions on the soma of PCs to suppress actionpotential generation, whereas 5-HT2A receptors excite (depolar-ize) distal apical dendrites of PCs to produce oscillatorymembrane potential changes in large numbers of PCs givingrise to cortical rhythms of sleep-waking states (Puig andGulledge, 2011). In accord with the foregoing, the presentdemonstration of a denser 5-HT innervation of deep (layers 5/6)than superficial (layer 2) layers of the PFC suggests that 5-HTmay exert its greatest effect on PCs (and INs) of the deep layers of

the PFC in the modulation of the cortical EEG and associatedbehavioral states.

4.6. Conclusions

Serotonergic fibers distribute massively throughout themPFC, ORB, and INC and to each of their subdivisions. Whilethe entire PFC receives a dense 5-HT innervation, a distinctlaminar organization was observed such that labeling was moreintense in superficial (layer 1) and deep layers (layers 5/6) thanin intermediate layers (layers 2/3). This pattern was mostprominent in the ORB. In the granular divisions of the INC(GI and DI), 5-HT fibers were densely concentrated in thegranule cell layer (layer 4). The serotonergic input to the PFC,which much more strongly targets ‘limbic’ than sensorimotorregions of the PFC, likely plays a critical role in the modulation of

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 prefrontalcortex.

Acknowledgment

This research was supported by National Science Foundationgrant IOS 0820639 to RPV.

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