Lateral habenula and the rostromedial tegmental nucleus innervate neurochemically distinct subdivisions of the dorsal raphe nucleus in the rat.

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

Lateral Habenula and the Rostromedial Tegmental Nucleus Innervate Neurochemically Distinct Subdivisions of the Dorsal Raphe Nucleus in the Rat Chemutai Sego,1,2 Luciano Gonçalves,1,3 Leandro Lima,1 Isadora C. Furigo,1 Jose Donato Jr,1 and Martin Metzger1* 1

Department of Physiology & Biophysics, Institute of Biomedical Sciences, University of S~ao Paulo, 05508-900 S~ao Paulo, Brazil Department of Anatomy, Institute of Biomedical Sciences, University of S~ao Paulo, 05508-900 S~ao Paulo, Brazil 3 Department of Human Anatomy, Federal University of the Triângulo Mineiro, 38025-180 Uberaba, Brazil 2

ABSTRACT The lateral habenula (LHb) is an epithalamic structure differentiated in a medial (LHbM) and a lateral division (LHbL). Together with the rostromedial tegmental nucleus (RMTg), the LHb has been implicated in the processing of aversive stimuli and inhibitory control of monoamine nuclei. The inhibitory LHb influence on midbrain dopamine neurons has been shown to be mainly mediated by the RMTg, a mostly GABAergic nucleus that receives a dominant input from the LHbL. Interestingly, the RMTg also projects to the dorsal raphe nucleus (DR), which also receives direct LHb projections. To compare the organization and transmitter phenotype of LHb projections to the DR, direct and indirect via the RMTg, we first placed injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin into the LHb or the RMTg. We then confirmed our findings by retrograde tracing

and investigated a possible GABAergic phenotype of DRprojecting RMTg neurons by combining retrograde tracing with in situ hybridization for GAD67. We found only moderate direct LHb projections to the DR, which mainly emerged from the LHbM and were predominantly directed to the serotonin-rich caudal DR. In contrast, RMTg projections to the DR were more robust, emerged from RMTg neurons enriched in GAD67 mRNA, and were focally directed to a distinctive DR subdivision immunohistochemically characterized as poor in serotonin and enriched in presumptive glutamatergic neurons. Thus, besides its well-acknowledged role as a GABAergic control center for the ventral tegmental area (VTA)–nigra complex, our findings indicate that the RMTg is also a major GABAergic relay between the LHb and the DR. J. Comp. Neurol. 522:1454–1484, 2014. C 2013 Wiley Periodicals, Inc. V

INDEXING TERMS: Raphe nuclei; serotonin, glutamate, anxiety; reward; stress

The lateral habenula (LHb) and the newly discovered rostromedial tegmental nucleus (RMTg; Jhou et al., 2009a,b), also known as the tail of the ventral tegmental area (tVTA; Kaufling et al., 2009), are now considered key components of a brain circuit implicated in the inhibitory control of monoamine nuclei (Hikosaka, 2010; Lavezzi and Zahm, 2011; Bourdy and Barrot, 2012). The LHb is an evolutionarily conserved epithalamic structure (Stephenson-Jones, 2012) composed of 10 distinct subnuclei (Andres et al., 1999; Geisler et al., 2003). Nowadays, it is well established that LHb neurons encode disappointment and expectation of negative conditions (Ullsperger and von Cramon, 2003; Shepard et al., 2006) and are primarily excited by reward omission and aversive stimuli and outcomes (Matsumoto and Hikosaka, 2007, 2009). Consistent C 2013 Wiley Periodicals, Inc. V

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with a role in encoding disappointments, the LHb has been implicated in a broad array of functions and pathologic conditions (for reviews, see Lecourtier and Kelly, 2007; Hikosaka et al., 2008; Hikosaka, 2010; Shelton et al., 2012). Notably, several studies point to a prominent role of the LHb in mechanisms of stress and pain, as well as in major depressive disorder (Caldecott-

Grant sponsor: FAPESP; Grant numbers: 2012/02388-3; 2011/ 03292-7; 2010/18086-0; Grant sponsor: CNPq; Grant numbers: 14199/2012-7; 143177/2008-7. *CORRESPONDENCE TO: Martin Metzger, PhD, Dept. of Physiology & Biophysics, Institute of Biomedical Sciences I, University of S~ao Paulo, USP, Av. Prof. Lineu Prestes 1524, 05508-900 S~ao Paulo, SP, Brazil. E-mail: [email protected] Received May 7, 2013; Revised December 23, 2013; Accepted December 23, 2013. DOI 10.1002/cne.23533 Published online December 28, 2013 in Wiley (wileyonlinelibrary.com)

The Journal of Comparative Neurology | Research in Systems Neuroscience 522:1454–1484 (2014)

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Habenular inputs to the dorsal raphe nucleus

Hazard et al., 1988; Morris et al., 1999; Amat et al., 2001; Shumake et al., 2003; Sartorius et al., 2010; Li et al., 2011; Meng et al., 2011). In rats, the LHb receives major inputs from the entopeduncular nucleus, lateral preoptic area, lateral hypothalamus, and prefrontal cortex (Herkenham and Nauta, 1977; Groenewegen et al., 1993; Kowski et al., 2008; Li et al., 2011; Kim et al., 2012) and sends direct projections to monoaminergic and cholinergic cell groups in the mid- and hindbrain (Herkenham and Nauta, 1979; Araki et al., 1988; Cornwall et al., 1990; Kim, 2009; Brinschwitz et al., 2010; Bernard and Veh, 2012; Gonçalves et al., 2012). Electrical stimulation of the LHb powerfully suppresses dopamine (DA) neurons in the ventral tegmental area (VTA; Christoph et al., 1986; Ji and Shepard, 2007), as well as serotonin (5-HT) neurons in the dorsal raphe nucleus (DR; Wang and Aghajanian, 1977; but see Ferraro et al., 1996). However, since most LHb projection neurons are glutamatergic (Geisler et al., 2007; Brinschwitz et al., 2010) and direct LHb inputs to the VTA and DR are comparatively sparse (Olmechenko et al., 2010; Gonc ¸alves et al., 2012), it seems unlikely that this suppressive effect is

Abbreviations 4V Aq DR DRC DRD DRDC DRDCe DRDSh DRL DRR DRV DTgP fr IGP IP isRt LDTg lH LHb LHbL LHbLMc LHbM LHbMC LHbMPc LPAG Me5 ml mlf MnR MT PMnR Pn PTg PVP RLi RMTg RRF Rt sm SNC SNR VLPAG xscp

Fourth ventricle Aqueduct Dorsal raphe nucleus Dorsal raphe nucleus, caudal part Dorsal raphe nucleus, dorsal part DRD, core DRD, central part DRD, shell Dorsal raphe nucleus, lateral part Dorsal raphe nucleus, rostral part Dorsal raphe nucleus, ventral part Dorsal tegmental nucleus, pericentral part Fasciculus retroflexus Globus pallidus, internal part Interpeduncular nucleus Isthmic reticular formation Laterodorsal tegmental nucleus Lateral hypothalamic region Lateral habenular nucleus LHb, lateral part Magnocellular subnucleus of the lateral part of the LHb LHb, medial part Central subnucleus of the medial part of the LHb Parvocellular subnucleus of the medial part of the LHb Lateral periaqueductal gray Mesencephalic trigeminal nucleus Medial lemniscus Medial longitudinal fasciculus Median raphe nucleus Medial terminal nucleus of the accessory optic tract Paramedian raphe nucleus Pontine nuclei Pedunculopontine tegmental nucleus Paraventricular thalamic nucleus, posterior part Rostral linear nucleus of the raphe Rostromedial tegmental nucleus Retrorubral field Reticular thalamic nucleus Stria medullaris thalamus Substantia nigra, compact part Substantia nigra, reticular part Ventrolateral periaqueductal gray Decussation superior cerebellar peduncle

mediated by monosynaptic LHb inputs to DA and 5-HT neurons, suggesting the involvement of a gaminobutyric acid (GABA)ergic relay. In the case of midbrain DA cells, there is now anatomical and electrophysiological evidence in several species including primates (Hong et al., 2011) and mice (Stamatakis and Stuber, 2012) that the inhibitory LHb influence on these cells is mainly mediated by the RMTg, a mostly GABAergic structure (Perrotti et al., 2005; Olson and Nestler, 2007) that stretches from the caudal pole of the VTA deeply into the mesopontine tegmentum (Jhou et al., 2009a,b; Bourdy and Barrot, 2012). The RMTg receives a dominant glutamatergic input emerging from the lateral LHb (Herkenham and Nauta, 1979; Geisler et al., 2007; Kim, 2009; Gonc ¸alves et al., 2012). Major GABAergic outputs of the RMTg are in turn directed to all midbrain DA cell groups (Geisler and Zahm, 2005; Ferreira et al., 2008; Jhou et al., 2009b; Balcita-Pedicino et al., 2011; Zahm et al., 2011). Electrophysiological studies revealed that RMTg neurons are, similar to LHb neurons, excited by omitted rewards (Hong et al., 2011) and aversive stimuli (Jhou et al., 2009a; Lecca et al., 2011, 2012). Furthermore, mu-opioid receptors are highly enriched in the RMTg (Jhou et al., 2009b) and there is now considerable evidence that GABAergic RMTg neurons, and not, as formerly assumed, local GABAergic VTA interneurons (Johnson and North, 1992), are a key site in the muopioid receptor-dependent regulation of VTA DA neurons (Lecca et al., 2011, 2012; Jalabert et al., 2011; Matsui and Williams, 2011; Jhou et al., 2012; Margolis et al., 2012). So far, the RMTg has predominantly been explored as an inhibitory control center of the VTA (Lavezzi and Zahm, 2011; Bourdy and Barrot, 2012). However, there is clear evidence that the RMTg also projects to major cholinergic and serotonergic cell groups including the pedunculopontine tegmental nucleus (PTg) and the DR (Jhou et al., 2009b; Lavezzi et al., 2012). The DR is located on the midline beneath the cerebellar aqueduct and fourth ventricle and forms one of the most prominent 5-HT brain cell groups (Steinbusch, 1981; Jacobs and Azimita, 1992; Abrams et al., 2004; Vasudeva et al., 2011). The DR is the major source of 5-HT projections to the fore- and midbrain (Vertes, 1991) and also sends descending projections to the hindbrain (Vertes and Kosics, 1994). Among an extremely broad range of physiological functions, the DR 5-HT system has been traditionally implicated in the behavioral responses to stress and punishments as well as the processing of aversive events (Deakin and Graeff, 1991; Maier and Watkins, 2005; Cools et al., 2008, 2011; Dayan and Huys, 2009). Interestingly, as

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demonstrated for distinct LHb divisions (Wirtshafter et al., 1994), specific DR subregions have been described to be involved in different stress responses and defensive behaviors (Grahn et al., 1999; Spiacci et al., 2012), as well as in specific circuits relevant to anxiety and other affective disorders (Commons et al., 2003; Abrams et al., 2004; Lowry et al., 2008; Hale and Lowry, 2011; Hale et al., 2012). Moreover, there is increasing evidence in several species that the DR is a neurochemically heterogeneous structure containing, besides distinct clusters of 5-HT neurons, several other differentially distributed major transmitters and neuropeptides (Charara and Parent, 1998; Michelsen et al., 2007; Fu et al., 2010; Calizo et al., 2011). Notably, as recently evidenced by the robust expression of the type 3 vesicular glutamate transporter (VGLUT3; Gras et al., 2002), some DR subregions display large proportions (up to 80%) of neurons with a mixed serotonergic/glutamatergic or purely glutamatergic phenotype (Hioki et al., 2010; Calizo et al., 2011; Soiza-Reilly and Commons, 2011). As outlined above, the DR is anatomically and functionally related to the LHb (Wang and Aghajanian, 1977; Peyron et al., 1998; Amat et al., 2001; Gonc ¸alves et al., 2012) and RMTg (Jhou et al., 2009b, Lavezzi et al., 2012). However, compared to the detailed knowledge about direct and indirect pathways between the LHb, VTA, and RMTg (Gonçalves et al., 2012), information about the topography of LHb and RMTg projections to the DR remains incomplete. Thus, we herein investigated by anterograde and retrograde tracing techniques the organization of LHb projections to the DR, direct and indirect via the RMTg, in a subnuclear context. Furthermore, we examined a possible GABAergic phenotype of DR-projecting RMTg neurons by combining retrograde tracing from the DR with in situ hybridization for glutamate decarboxylase 67 kDA (GAD67). We additionally investigated a possible glutamatergic and/or serotonergic transmitter phenotype of DR neurons in the principal target regions of the RMTg and LHb by combining double immunofluorescence staining for PHA-L and either 5-HT or VGLUT3.

MATERIALS AND METHODS Animals and surgery Adult male Wistar rats (180–220 g) obtained from a local breeding facility at the Institute of Biomedical Sciences, University of S~ao Paulo, were used in all experiments. The animals were kept in group cages under controlled temperature (23 C) and illumination (12-hour cycle), with water and food ad libitum. All experiments were carried out in compliance with the National Insti-

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TABLE 1. Cases Used for Analysis Tracers injected Injections sites

Total n 5 24

PHA-L – LHb

7

PHA-L – RMTg CTb – DR

3 9

CTb – RMTg; FG – DR CTb – RMTg; FG – VTA

3 2

Analyzed cases *R71, *R77, *R81, *R82, R113, R114, R118 R85, R86, R87 R51, R66, R90, R95, R96, R97, R114, RL13, RL23 *R63, *R64, *R65 RL21, RL28

Asterisks indicate that these cases were prepared for an earlier study (Gonc ¸alves et al., 2012).

tutes of Health (NIH) guidelines for the care and use of laboratory animals (NIH publication No. 80-23, revised 1996) and were approved by the institutional Animal Ethics Committees of the Institute for Biomedical Sciences at the University of S~ao Paulo (Protocols 8/2012 and 153/2012). All efforts were made to minimize the number of animals used and their suffering. Rats were anesthetized by subcutaneous injections with a cocktail of ketamine (Syntec, Hortolândia, Brazil; 5 mg/100 g body weight), acepromazine (Univet, S~ao Paulo, Brazil) 0.04 mg/100 g bw), and xylazine (Syntec; 1 mg/100 g bw) diluted in distilled water. Rats were placed into a Kopf stereotaxic instrument, their skulls exposed, and small bore holes drilled with a dental burr to allow selected brain structures to be targeted by borosilicate glass pipettes. The anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L; 2.5% in 0.1 M phosphate buffer, Vector Laboratories, Burlingame, CA,) and the retrograde tracers cholera toxin subunit b (CTb; low salt, 1% in distilled water, List Biological Laboratories, Campbell, CA) and FluoroGold (FG; 2% in PB, pH 7.4, Fluorochrome, Englewood, CO) were used in the present study. Cases with tracer injections and their respective injection sites are listed in Table 1. All tracers were injected iontophoretically using a positive pulsed current (7 seconds on, 7 seconds off) set at 5 lA for CTb and FG injections (15 minutes into the DR), and at 4 lA for PHA-L injections (15 minutes into the LHb and RMTg) through glass micropipettes (10–15 lm internal tip diameter for PHA-L, 15–20 lm for CTb and FG). Before withdrawal, the pipettes were left in position for at least 3 minutes and a negative continuous current, set at 3 lA, was applied to minimize tracer backflow along the pipette tract. The stereotaxic coordinates were obtained from the rat brain atlas of Paxinos and Watson (2007) and subsequently refined empirically. The following coordinates were used: for the LHb, 3.7 mm posterior to bregma, 5.0 mm ventral to the dura, and 0.6–1.0 mm from the



TABLE 2. Primary Antibodies Used Dilution Antigen 5-HT CTb FG NeuN PHA-L PHA-L Som VGLUT3

Immunogen Serotonin coupled to bovine serum albumin (BSA) with paraformaldehyde Purified toxin from Vibrio cholera FluoroGold (hydroxystilbamidine) Purified cell nuclei from mouse brain Purified lectin from Phaseolus vulgaris Purified lectin from Phaseolus vulgaris Peptide of somatostatin of human origin (aa 1–106) Synthetic peptide from rat VGLUT3 (aa 569–588)

Source

IPe

IF

Immunostar (Hudson, WI), #200800, rabbit polyclonal

1:80,000

1:40,000

List Laboratories (Burlingame, CA), #104, goat polyclonal Millipore (Temecula, CA), #AB153, rabbit polyclonal Millipore (Temecula, CA), #MAB377, mouse monoclonal DAKO (Carpinteria, CA), #B275, rabbit polyclonal Vector (Burlingame, CA), #AS2224, goat polyclonal Santa Cruz (Santa Cruz, CA) #sc25262, mouse monoclonal Millipore (Temecula, CA), #AB5421, guinea pig polyclonal

1:10,000 1:5,000

1:800

1:10,000 1:2,500 1:5,000 1:2,500 1:2,500 1:400

1:3,000

1:1,500

1:5,000

Abbreviations: 5-HT, serotonin; CTb, cholera toxin, subunit b; FG, FluoroGold; IF, immunofluorescence staining; IPe, immunoperoxidase staining; NeuN, neuron-specific nuclear protein; PHA-L, Phaseolus vulgaris leucoagglutinin; Som, somatostatin; VGLUT3, vesicular glutamate transporter 3.

midline; for the RMTg, 7.1 mm posterior to bregma, 8.6 mm ventral to the dura, and 6.2 mm from the midline; for the DR, 7.0–8.6 mm posterior to bregma, 5.6 mm ventral to the dura, and 0.0 from the midline.

Perfusion and tissue processing Following a survival period of 7 days for CTb or FG injections and 10 days for PHA-L injections, the rats were deeply anesthetized and perfused transcardially with 90 ml of 0.9% saline solution followed by 500 ml 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.3) at 4 C. The brains were postfixed for 4 hours at 4 C and cryoprotected with 20% sucrose solution in PB at 4 C. Brains were sectioned in the coronal plane at 40 lm into four series on a sliding microtome. One series of sections was processed for immunoperoxidase staining for CTb, FG, or PHA-L. In the case of rats, which received PHA-L injections into the LHb or RMTg, sections through the DR of another series were processed for PHA-L/5-HT or PHA-L/VGLUT3 using a double immunofluorescence protocol. Double immunofluorescence staining was also employed in rats, which received injections of CTb into the RMTg together with FG injections into the DR. An additional series of sections was stained with thionin to serve as a reference for cytoarchitecture.

Antibody characterization and immunohistochemical controls The primary antibodies used in this study (Table 2) have been fully characterized and tested for specificity. The goat antibody against CTb (List, #104) was raised against the purified toxin from Vibrio cholerae (List datasheet), which is not physiologically present in the organism. The rabbit polyclonal antibody raised against FG (hydroxystilbamidine) was purchased as antibody-

containing serum without preservative (#AB153) from Millipore (Temecula, CA). Two different polyclonal antibodies against PHA-L were used in the present study. The rabbit antibody against PHA-L (DAKO, Carpinteria, CA, #B275) was produced by hyperimmunizing rabbits with the purified lectin, which is absent in mammalian brain (Dako datasheet). The goat antibody raised against Phaseolus vulgaris agglutinin (Vector, #AS2224) reacts strongly with both P. vulgaris erythroagglutinin (PHA-E) and P. vulgaris leucoagglutinin (PHA-L) and was produced by hyperimmunizing goats with the purified lectins (Vector datasheet). Importantly, sections from brains that did not receive a PHA-L, FG, or CTb injection were devoid of reaction product when immunoreacted with the respective primary antibodies at optimal dilutions. The rabbit antibody against 5-HT (Immunostar, Hudson, WI, #200800) was raised against 5-HT derived from rat brain, coupled to bovine serum albumin (BSA) with paraformaldehyde. Preabsorption of the diluted antibody with 25 lg/ml of 5-HT/BSA complex completely eliminated the reaction, whereas pretreatment with BSA did not affect the immunostaining (manufacturer’s technical information). Furthermore, the 5-HT immunoreactions obtained in our rat hindbrain tissue revealed a virtually identical staining pattern as the Pet1 riboprobe, which is a specific marker of the serotonergic neuronal phenotype (Hendricks et al., 1999). The guinea pig antibody against VGLUT3 (Millipore, #AB5421) was raised against a synthetic peptide afegeeplsyqneedfsets corresponding to amino acids 569– 588 of rat VGLUT3. According to manufacturer’s specification, the guinea pig anti-VGLUT3 polyclonal antiserum recognizes a single band of 65 kDa on western blots from lysate of rat-derived PC12 cells. Preabsorption with the cognate peptide eliminates immunostaining on rat brain tissue, as reported by the manufacturer


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as well as in the literature (e.g., Gabellec et al., 2007). Importantly, the VGLUT3 antibody led to robust immunolabeling not only of axons but also of numerous cell bodies, whose distribution was consistent with that described in previous immunohistochemical studies using different anti-VGLUT3 antibodies, as well as the known distribution of cells expressing VGLUT3 mRNA (Gras et al., 2002; Fremeau et al., 2002; Hioki et al., 2010). The monoclonal mouse antibody against somatostatin (Som; Santa Cruz Biotechnology, Santa Cruz, CA, #sc-25262) was raised against Som of human origin. The antibody detects a principal band of 17 kDA on western blots from human Som transfected 293 whole cell lysates (Santa Cruz Antibody Data). Moreover, the labeling pattern obtained with this antiserum in the present study was similar to the immunostaining pattern reported by other authors using different anti-Som antibodies (Johansson et al., 1984). The monoclonal mouse antibody against the neuron-specific nuclear protein called NeuN (Millipore, #MAB377) was raised against purified cell nuclei from mouse brain. This antibody recognizes the nuclei and cell bodies of most neuronal cell types but not glial fibrillary acidic proteinpositive cells throughout the central nervous system of rodents (Mullen et al., 1992). The antibody detects several bands at 46–48 kDa on western blots with isolated mouse brain nuclei (Millipore Datasheet). Optimal antibody concentrations were individually determined for each antibody. Specificity of the secondary antibodies was indicated by the observed absence of specific labeling in tissue sections collected from naive animals and animals that had received a tracer injection, to which the immunohistochemical method was applied with omission of the primary antibody step.

Immunoperoxidase staining Immunohistochemical procedures were carried out on free-floating sections at room temperature unless otherwise stated. CTb (Luppi et al., 1990), PHA-L (Gerfen and Sawchenko, 1984), and FG (Chang et al., 1990) were processed immunohistochemically. Sections destined for incubation with antibodies to FG, Som, or 5-HT were pretreated with 1% sodium borohydrate (Sigma, Deisenhofen, Germany) in PB for 10 minutes, then washed in PB and preincubated in 1% H2O2 and PB containing 10% methanol for 10–15 minutes. These sections were then incubated with a polyclonal anti-FG raised in rabbit (Millipore) diluted 1:5,000, a monoclonal mouse anti-Som diluted 1:800 (Santa Cruz), or a polyclonal anti-5-HT raised in rabbit (Immunostar) diluted 1:80,000–1:120,000, all in PB containing 0.3% Triton X-100 for 24–48 hours at 4 C. In the case of CTb, sections were preincubated for 30 minutes in 2% normal goat serum (NGS, Jackson Immu-

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noresearch Laboratories, West Grove, PA) and then incubated with a polyclonal anti-CTb raised in goat (List) diluted 1:10,000 in PB containing 0.3% Triton X-100 for 24–48 hours at 4 C. In the case of PHA-L, the preincubation was omitted, sections being incubated directly with a polyclonal anti-PHA-L raised in rabbit (DAKO) diluted 1:5,000 in PB containing 0.3% Triton X-100 and 10% skimmed milk for 24–48 hours at 4 C. Sections were subsequently rinsed in PB and incubated for 2 hours in the appropriate affinity purified biotinylated secondary antibodies (goat antirabbit IgG, #BA-1000, Vector, dilution: 1:200 for PHA-L, FG, and 5-HT; horse antimouse IgG, #BA-2000, Vector, dilution 1:200 for Som; donkey antigoat, #705-066-147, Jackson; dilution: 1:4.000 for CTb). Sections were rinsed again followed by a 2-hour incubation in an ABC Kit (ABC Elite Kit, Vector). After thorough rinsing in PB the peroxidase reaction product was visualized using the glucose oxidase procedure (Itoh et al., 1979) and the metal-free 3,30 -diamenobenzidine (DAB) tetrahydrochloride as chromogen. In selected cases, the DAB reaction was intensified with 0.5% nickel sulfate. Finally, sections were rinsed again in PB, mounted on gelatin-coated slides, dipped for 20 seconds in a 0.05% aqueous solution of osmium tetroxide, dehydrated through a series of ascending concentrations of ethanol, transferred into xylene, and coverslipped with DPX (Sigma) mounting medium.

Immunofluorescence staining Sections were pretreated as described above and then incubated for 48–72 hours at 4 C in mouse antiNeuN diluted 1:5,000 or cocktails of either a rabbit anti-FG diluted 1:2,500 and a goat anti-CTb diluted 1:10,000, or a goat anti-PHA-L diluted 1:2,500 and a rabbit anti-5-HT diluted 1:40,000, or a rabbit anti-PHA-L diluted 1:2,500 and a guinea pig anti-VGLUT3 diluted 1:1,500, all diluted in PB containing 1% normal donkey serum (NDS, Jackson) and 0.3% Triton X-100. Sections were subsequently rinsed in PB and incubated for 1 hour in DyLight 488-conjugated donkey antimouse (for NeuN immunostaining) or a cocktail consisting of either DyLight 488-conjugated donkey antirabbit and DyLight 594-conjugated donkey antigoat, or DyLight 488conjugated donkey antirabbit and Cy3-conjugated donkey antiguinea pig (all from Jackson Immunoresearch and diluted 1:500 in PB containing 1% NDS). The sections were finally thoroughly rinsed in 0.05 M Tris-HCl, mounted on gelatin-coated slides, coverslipped with slow-fade-medium (Invitrogen, S~ao Paulo, Brazil), and sealed with nail polish. Several sets of controls were performed, including the omission of one or both primary antibodies, omission of one of the secondary antibodies, and switching of the fluorophores related to the



different markers. All these control procedures resulted in the expected single fluorescence labeling or, in the case of omission of both primary antisera, in the absence of fluorescence labeling.

Hybridizations with 35S-radiolabeled GAD67 probe combined with immunohistochemical detection of CTb Free-floating brain sections of rats with CTb injections into the DR were rinsed with DEPC-treated PBS, pH 7.0 for 1 hour and in 0.1% sodium borohydride (Sigma) in DEPC-PBS for 15 minutes. Sections were then incubated for 10 minutes in 0.25% acetic anhydride (Merck, Darmstadt, Germany) in 0.1 M triethanolamine. GAD67 35Slabeled riboprobe was generated from cDNA templates as described previously (Elias et al., 2001). The GAD67 plasmids were kindly provided by Drs. N. Tillakaratne and J.C. Bittencourt (University of California, Los Angeles, CA, and University of S~ao Paulo, S~ao Paulo, Brazil). cRNA probes were diluted to 106 cpm/ml in hybridization buffer and applied to brain sections. The solution consisted of 50% formamide, 10 mM Tris-HCl (Fisher Scientific, Fair Lawn, NY), 0.01% sheared salmon sperm DNA (Sigma), 0.01% yeast tRNA (Sigma), 0.05% total yeast RNA (Sigma), 10 mM dithiothreitol (Amresco, Solon, OH), 10% dextran sulfate, 0.3 M NaCl, 1 mM EDTA (pH 8.0), and 13 Denhardt’s solution (Amresco). Sections were hybridized overnight at 50 C. The following day, tissue was rinsed four times in 43 sodium chloride/sodium citrate (SSC) and incubated in 0.002% RNase A (Roche Diagnostics, Mannheim, Germany) diluted in 0.5M NaCl, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA for 30 minutes at 37 C. Sections were submitted to stringency wash in 0.13 SSC for 60 minutes at 55 C. Subsequently, sections were incubated for 48 hours in anti-CTb antisera (1:5,000) and processed for immunoperoxidase staining as previously described using DAB as chromogen. Tissue was mounted onto SuperFrost Plus slides (Fisher Scientific) and dehydrated in increasing concentrations of ethanol. After air drying, slides were placed in X-ray film cassettes with BMR-2 film (Kodak, Rochester, NY) for 2 days. Slides were then dipped in NTB2 photographic emulsion (Kodak), dried, and stored in desiccant-containing, foil-wrapped slide boxes at 4 C for 16 days. Slides were developed with D19 developer (Kodak), dehydrated in increasing concentration of ethanol, cleared in xylenes, and coverslipped.

Data analysis Immunoperoxidase stained sections were examined under both bright and darkfield illumination and immunofluorescence stained sections were examined under epi-

fluorescence illumination using a Zeiss Axioimager A1 microscope (Zeiss, Muenchen, Germany). Digital color photomicrographs were acquired using a Zeiss Axiocam HRc camera. Images of double immunofluorescencestained sections were acquired and analyzed with the Axiovision software (Zeiss), which permits the acquisition of images from several separate fluorescence channels, as well as the subsequent superposition of these images. Selected double immunofluorescence-stained sections were analyzed with a Zeiss LSM 510 confocal laser scanning microscope (Zeiss, Goettingen, Germany) using step intervals of about 200 nm along the z axis. The overall distribution of the anterograde and retrograde labeling of representative cases were mapped with the aid of a computer drawing program (AutoCad, Release 13) combined with a microscope (Leitz, Diaplan, Wetzlar, Germany) and camera lucida aimed at a flat-screen computer monitor. Quantification of 5-HT-only, VGLUT3-only, and 5-HT/ VGLUT3 cell bodies in DR sections stained by double immunofluorescence techniques for 5-HT and VGLUT3 was performed with the aid of the Axiovision software (Zeiss, v. 4.8.2). Identification of single- or double-labeled neurons was performed on images captured in a 1388 3 1040 pixel format with a 203 objective centered on distinct DR subregions, which, if necessary, were circumscribed on the images. All neurons within the respective subregions displaying a well-stained soma in sharp focus were considered for analysis, plotted electronically, and classified as either single- or double-labeled. This analysis was performed on two sections each spaced at 160 lm at midrostrocaudal (7.4 to 7.8 posterior to bregma) and caudal (8.5 to 8.9 mm posterior to bregma) DR levels in three rats. The averages were used to provide means and standard errors of the mean (SEMs). Tissue sections processed for in situ hybridization followed by immunohistochemical detection of CTb were examined under both bright and darkfield illumination with a Zeiss Axioimager A1 microscope. Quantification of CTb-labeled cells in the RMTg containing GAD67 mRNA was performed in two rats (RL13, RL23) similar to that described in Jhou et al. (2009b). In brief, photomicrographs were centered on the RMTg at two rostrocaudal levels corresponding to 6.60 and 6.84 mm posterior to bregma and captured under brightfield illumination at two focal planes with a 203 objective. The RMTg was circumscribed on the images and CTb-only and CTb/ GAD67 cells were plotted with the Axiovision software. Importantly, silver grains indicative of GAD67 mRNA occupied preferentially the exposed surface of the sections, whereas the CTb antibodies showed a far greater penetration. Thus, only CTb-labeled cells in sharp focus at the tissue surface were considered for analysis. CTb cells were defined as double-labeled if the density of


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Figure 1. Photomicrographs illustrating dorsal raphe (DR) subregions at a midrostrocaudal level. A,B: Adjacent sections immunostained for serotonin (5-HT) and NeuN. C,D: Double immunofluorescence staining for 5-HT (green) and VGLUT3 (magenta) illustrating the separate channel for the type 3 vesicular glutamate transporter (VGLUT3, C) and the merged image (D). Note that a central subregion of the dorsal part of the DR (DRDCe) may be characterized as poor in 5-HT and enriched in VGLUT-3-only neurons. For abbreviations, see list. Scale bars 5 200 lm in A (applies to A,B); 100 lm in C (applies to C,D).

overlying silver grains was more than three times greater (10 silver grains per cell) than background levels observed over non-GABAergic tissue regions in the same section. Image processing and lettering was carried out with Adobe Photoshop software (v. 7.0; Adobe Systems, Mountain View, CA). Color balance, contrast, and brightness of the images were adjusted to a variable extent. The Canvas software (ACD Systems, Victoria, Canada, v. 9.0) was used for line drawings. The nomenclature used in the present study is based on the atlas of Paxinos and Watson (2007) unless otherwise specified. The term RMTg was adopted as first defined by Jhou et al. (2009a,b). We adhered to the nomenclature of Andres et al. (1999) for the different subnuclei within the LHb complex (see also Geisler et al., 2003). According to these authors, the LHb complex may be subdivided in a medial (LHbM) and a lateral (LHbL) division, each one further subdivided on morphologic and cytochemical grounds into five distinct subnuclei. With regard to DR subdivisions, several attempts were made in the past to subdivide the DR into distinct cell groups or subregions (Dahlstr€om and Fuxe, 1964; Steinbusch, 1981; Baker et al., 1990; Abrams et al., 2004; Jensen et al., 2008; Hioki et al.,

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2010; Hale and Lowry, 2011; Alonso et al., 2013). In the present study, we mostly adopted the nomenclature used in a recent publication by Hioki et al. (2010). Thus, we distinguished within the DR a rostral (DRR), a ventral (DRV), a lateral (DRL, in the text also referred as lateral wings), a dorsal (DRD), and a caudal (DRC) subregion. Based on connectional and immunohistochemical grounds (see Results), the DRD was divided into three subregions. Thus, beside the DRD core (DRDC) and DRD shell (DRDSh; Abrams et al., 2005; Lowry et al., 2008; Hioki et al., 2010) we distinguished a separate central DRD subregion (DRDCe). Located just ventral to the DRDC at midrostrocaudal DR levels (from about 7.4 to 7.8 posterior to bregma), the DRDCe can be characterized by its poor 5-HT content allied to densely packed VGLUT3-only neurons (Fig. 1).

RESULTS Anterograde tracing experiments: LHb inputs to the DR Since there is increasing evidence that the LHb contains distinct subdivisions (Andres et al., 1999; Geisler et al., 2003) that project in a topographic manner to



Figure 2. Photomicrographs illustrating PHA-L injections centered in the medial division (LHbM; case R81; A), in the central part of the ventral lateral habenula (LHb; C), and in the magnocellular subnucleus of the lateral division (LHbLMc; case R82; D) of the LHb, as well as the resulting anterograde labeling in the DR. B: Low- and high-power photomicrographs of anterograde labeling in the caudal part of the DR (DRC) resulting from the injection shown in A. Note the dense terminal-like PHA-L labeling in the DRC with many labeled axons exhibiting bouton-like swellings and terminal ramifications (indicated by arrowheads in the inset). E: Sparse anterograde labeling at a level through the midrostrocaudal part of the dorsal raphe nucleus resulting from the large injection depicted in B. White dots indicate the positions of PHA-L impregnated neurons marking the core region of the injection sites. For abbreviations, see list. Scale bars 5 200 lm in A (applies to A,C,D); 150 lm in B (applies to B,E); 10 lm in the inset in B.

the midbrain and mesopontine tegmentum (Kim, 2009; Gonçalves et al., 2012), our injections into the LHb were aimed at different locations along the mediolateral axis of the LHb complex. All of the seven PHA-L injections analyzed in the present study (R71, R77, R81, R82, R113, R114, R118) were mostly confined to the LHb and did not encroach onto the medial habenula. They had a similar appearance with a dense core containing a variable small number of darkly stained neu-

rons, surrounded by a diffuse background labeling. Four of the cases (R71, R77, R81, R82) were the same as those used in an earlier study (Gonçalves et al., 2012). Three cases with injections confined to the LHb were chosen as prototypes to illustrate PHA-L labeling in the DR (Fig. 2). Case R81 had a small injection centered in the medialmost portion of the LHbM (Fig. 2A), case R118 a small ventral injection in the central part of the LHb (Fig. 2C), and case R82 had a larger injection


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involving most of the LHbL (Fig. 2D). Since the resulting anterograde labeling in the RMTg and other pontomesencephalic structures has been reported previously (Gonçalves et al., 2012), our description will be mostly confined to the DR. In all analyzed cases, PHA-L labeling in the DR was bilateral, with only slightly fewer fibers found in the contralateral hemisphere. The PHA-L injection centered in the medialmost portion of the LHb complex and involving the marginal, parvocellular (LHbMPc), and central (LHbMC) subnuclei of the LHbM (case R81; Figs. 2A, 3A) produced only very sparse anterograde labeling in the DRR and light to moderate labeling at midrostrocaudal levels through the DR. At these levels, few PHA-L labeled axons were typically scattered throughout all DR subregions (Fig. 3B). Many of these LHb axons were poorly branched and mostly nonvaricose. However, some displayed varicosities, originating modest terminal fields in the DRV and lateral wings. Starting at about 8.3 mm posterior to bregma the density of PHA-L1 axons gradually increased near the midline (Fig. 3C). Slightly more caudally, extending from about 8.5 till 9.2 mm posterior to bregma, a prominent terminal field (Figs. 2B, 3D) with many axons exhibiting bouton-like swellings and terminal ramifications (inset in Fig. 2B) occupied the entire DRC. In addition, several patchy terminal fields were located throughout the whole rostrocaudal extent of the median raphe nucleus (Figs. 2B, 3B–D). Case R118 had a small PHA-L injection in the central one-third of the ventral LHb involving mostly the basal subnucleus and the lateral part of the magnocellular subnucleus (LHbLMc) of the LHbL (Figs. 2C, 3E). This injection resulted in sparse anterograde labeling in the rostral and midrostrocaudal DR (Fig. 3F). At about 8.3 mm posterior to bregma the density of PHA-L1 axons increased (Fig. 3G), whereas more caudally, the DRC was only very sparsely innervated (Fig. 3H). A comparatively large PHA-L injection in case R82 (Figs. 2D, 3I), centered in the magnocellular subnucleus of the LHbL was chosen as representative for LHbL injections. In this case, as in other cases with injections that did not encroach onto the LHbM, few scattered poorly branched and in their majority sparsely beaded PHA-L labeled fibers were detected throughout all DR subdivisions (Figs. 2E, 3J–L). They were extremely sparse in the DRR, slightly more common at midrostrocaudal DR levels (Fig. 3J), and progressively thinned out caudalwards (Figs. 3K,L). Importantly, in sharp contrast to the injection in case R81, the DRC (Fig. 3L) was almost devoid of PHA-L labeling in cases with LHbL injections. Moreover, after all LHb injections, caudal raphe cell groups like the pontine raphe nucleus and the raphe magnus nucleus were devoid of any significant PHA-L labeling.

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Retrograde tracing experiments: LHb inputs to the DR and RMTg To confirm that the DR receives topographically organized inputs from the LHb complex, in another group of rats deposits of CTb or FG were placed along the rostrocaudal axis of the DR. Out of a total of 18 CTb and 3 FG injections into the DR, 12 (9 CTb, 3 FG, see Table 1) were largely restricted to the DR and resulted in robust bilateral retrograde labeling in the LHb complex. CTb and FG injections in the DR had a similar appearance, with a darkly stained core region surrounded by a peripheral halo largely confined to the DR. In some cases the periphery of the injections slightly encroached into the VLPAG. Comparable injections of the two tracers resulted in a similar general distribution of retrograde labeling in the LHb and other brain structures. However, CTb injections tended to be better defined than FG injections, while the latter produced a higher number of retrogradely labeled neurons in corresponding brain regions. All DR injections led to robust bilateral retrograde labeling in the LHb, whereas the medial habenula was devoid of labeled neurons. Two cases (R95, R64) were chosen to illustrate our findings (Fig. 4). Case 95 had a CTb injection centered in the dorsal part of the DRC (Fig. 4A) and is representative of injections into the DRC. Confirming a focal projection from the LHbM to the DRC, as indicated by our anterograde tracing findings, the resulting retrograde labeling in this case was mostly confined to a prominent cell cluster mostly occupying the LHbMPc and LHbMC of the LHbM, which were indistinguishable for the purposes of this study. In contrast, the entire LHbL was almost devoid of labeled cells (Fig. 4B). Case 64 had an FG injection centered at a midrostrocaudal DR level encompassing primarily the DRDCe and DRV (Fig. 4C). As in case R95, a prominent cluster of FGlabeled neurons was found in the LHbMPc/LHbMC nuclei. However, besides this focal labeling in the LHbM, a considerable number of retrogradely labeled neurons was found in the LHbL, particularly in the LHbLMc and at the outer rim of the LHbL (Fig. 4D). To confirm our anterograde tracing findings that the RMTg receives most of its LHB input from the LHbL, CTb was deposited into the RMTg of 10 animals. In five cases the injection substantially involved the RMTg without encroaching to any degree into the adjacent interpeduncular nucleus. Three of these cases (R63, 64, 65) had an additional FG injection in the DR and two other cases (see Table 1) an additional FG injection into the VTA (RL21, RL28). Case RL21 was chosen to illustrate our findings (Fig. 5). This case had a relatively large CTb injection that, as revealed in sections doubleimmunostained for CTb and Som, covered most of the



Figure 3. Semischematic drawings illustrating three different injection sites along the mediolateral axis of the LHb (A,E,I) and the resulting anterograde labeling in the hypothalamus, as well as in the DR and adjacent mesopontine structures. PHA-L labeling results from injections into the LHbM (A–D; case R81), central LHb (E–H; case R118), and LHbL (I–L; case R82), respectively. Sections are presented in a rostrocaudal sequence. For photographs of the injection sites, see Figure 2. Rostral levels through the DR are not shown, since PHA-L labeling was almost absent at these levels. Note that the DRC is robustly labeled following injections into the LHbM, but not the central LHb and LHbL. Note also that the RMTg is robustly labeled following injections into the LHbL, but not the LHbM and central LHB. Finally, note that sections through the injection sites (A,E,I) are represented in a minor scale than the rest of the drawings. For abbreviations, see list.


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Figure 4. Photomicrographs of representative CTb (A) and FG (C) injection sites centered in the DRC (A; case R95), DRDCe/DRV (C; case R64) and the resulting retrograde labeling in the LHb (B,D). Note in B that following DRC injections retrograde labeling in the LHb was mostly restricted to the LHbM. Scale bars 5 150 lm in A (applies to A,C); 200 lm in B (applies to B,D).

Som-rich (Jhou et al., 2009b; Lavezzi and Zahm, 2011) RMTg (Fig. 5A,B). Confirming a fairly strict topographical organization of LHb projections to the RMTg (Gonçalves et al., 2012), the bulk of retrograde labeling in this, and all other cases that received CTb injections into the RMTg, was found in the magnocellular and oval subnucleus of the LHbL (Fig. 5C). CTb injections into the RMTg also confirmed a robust DR projection to the RMTg (Kaufling et al., 2009). Interestingly, within the DR we found many retrogradely labeled neurons and anterogradely labeled axons (Fig. 5D) concentrated in the VGLUT3-rich DRDCe subregion. Furthermore, as revealed by double immunofluorescence labeling for CTb and VGLUT3, a substantial part of the RMTg-projecting neurons in the DR contained VGLUT3 (Fig. 5E). In line with this observation, the RMTg was outlined by a dense network of VGLUT31 axonal processes (Fig. 5F).

Retrograde tracing experiments: RMTg and other pontomesencephalic inputs to the DR Sections from rats that had CTb injections into the DR were also analyzed in relation to retrograde labeling in the RMTg and adjacent structures of the mesopontine tegmentum. Specifically, we wanted to evaluate whether retrogradely neurons labeled from the DR out-

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line the RMTg in its rostrocaudal extent, as recently described for VTA-projecting RMTg neurons (Gonçalves et al., 2012). The distribution of retrograde labeling from a representative CTb injection centered on the DRDCe at about 7.4 mm posterior to bregma (case R90; Fig. 6A) is shown in Figure 7. This injection was restricted to the midrostrocaudal segment of the DR and did not encroach into the DRR, DRC, or VLPAG. Substantial bilateral retrograde labeling was observed in the RMTg and numerous other pontomesencephalic structures, including the VTA, substantia nigra, pars compacta (SNC) and reticulata, the retrorubral field (RRF), the lateral and ventrolateral periaqueductal gray, the isthmic reticular formation, as well as the PTg and laterodorsal tegmental nucleus (LDTg). The RMTg was virtually outlined throughout its rostrocaudal extent by CTb-labeled neurons (Fig. 6B,C). Cluster-like CTb labeling in the RMTg extended from about 6.4 mm to about 7.6 mm posterior to bregma and was at all rostrocaudal levels closely associated with fibers of the decussation of the superior cerebellar peduncle. At the caudal pole of the RMTg, CTb-labeled neurons thinned out and progressively merged with retrogradely labeled neurons in the laterally adjacent PTg. Importantly, all our DR injections (n 5 9) that did not involve to a substantial degree the DRDCe subregion resulted in a less distinct pattern of retrograde labeling



Figure 5. Photomicrographs of a representative large CTb injection centered in the RMTg in case RL21 (A,B), the resulting retrograde labeling in the LHb (C), and DR (D,E), as well as a section through the RMTg of a na€ıve animal immunolabeled for VGLUT3 (F). Note in B that the injection is centered in the somatostatin (Som)-rich RMTg. Arrowheads indicate the same blood vessels. Note in C that the bulk of retrogradely labeled cells in the LHb is found in the LHbL. Note in D the strong retrograde and also anterograde CTb-labeling in the DRDCe (shown enlarged in the inset) and in E that many of the RMTg-projecting neurons in the DRDCe are double-labeled for VGLUT3 (examples are indicated by arrows, see also the inset). Note in F the dense innervation of the RMTg by VGLUT31 axons (inset). Asterisks indicate the same position in the low-power micrographs and the enlargements (insets). Scale bars 5 250 lm in A (applies to A,B,F); 200 lm in C; 100 lm in D (applies to D,E); 20 lm in the inset in D (applies to the insets in D,E); 50 lm in the inset in F.

in the caudal RMTg. As exemplified here for a CTb injection into the DRR (Fig. 6D, case R51), such injections typically led to robust retrograde labeling in the rostral RMTg (Fig. 6E). In the caudal RMTg, however, resulting CTb labeling was often confined to its medial part (Fig. 6F) and sometimes almost indistinguishable from that in adjacent areas.

Combination of retrograde tracing from the DR with in situ hybridization for GAD67 In order to verify whether RMTg projections to the DR are predominantly GABAergic, as described for RMTg projections to the VTA (Jhou et al., 2009b; Kaufling et al., 2009), we analyzed sections double-labeled for CTb by immunohistochemistry and for GAD67 mRNA by in situ


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Figure 6. Photomicrographs of representative CTb injection sites centered in the central subdivision of the DRDCe (A, case R90) and in the rostral part of the DR (DRR; D, case R51) and the resulting retrograde labeling at a rostral (B,C) and a caudal level (E,F) through the RMTg. Note that injections centered in the DRDCe resulted in robust retrograde labeling that outlined the RMTg core at rostral (B) and caudal (C) levels, whereas injections centered outside the DRDCe resulted in comparatively less distinct labeling in the caudal RMTg (F). Scale bars 5 300 lm in A (applies to A,D); 250 lm in B (applies to B,C,E,F).

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Figure 7. A–HSemischematic drawings of a CTb injection site in the DRDCe (case R90) and the resulting retrograde labeling in frontal sections through the mesopontine tegmentum. Sections are presented in a rostrocaudal sequence and each dot represents one retrogradely labeled neuron. The core of this injection is indicated in black and the surrounding halo by hatching. The gray shaded area indicates the RMTg core region. For abbreviations, see list.

hybridization in two rats (RL13, RL23) that had CTb injections into the midrostrocaudal DR. Under darkfield illumination, GABAergic structures such as the reticular

thalamic nucleus appeared heavily labeled (Fig. 8A), confirming the specificity of our GAD67 35S-labeled riboprobe (Elias et al., 2001). The RMTg stood out bilaterally as a


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Figure 8. Darkfield photomicrographs of GAD67 transcript in the reticular nucleus of the thalamus (Rt, A), internal part of the globus pallidus (IGP), and in the rostral part of the RMTg (B). Note in B that the RMTg stands out bilaterally as a heavily labeled cluster situated dorsolateral to the caudal half of the IP. C–E: Photomicrographs of a CTb injection in the DR (C) and the resulting retrograde CTb labeling and GAD67 transcript in the RMTg (D,E). Note that aggregates of silver grains are accumulated over almost all CTb-labeled neurons in the RMTg core region. Arrowheads point to examples of CTb-labeled RMTg neurons expressing GAD67 and the arrows to CTb-labeled neurons outside the RMTg lacking GAD67 transcript. Asterisks indicate the same blood vessel. Scale bars 5 300 lm in A; 250 lm in B; 200 lm in C; 100 lm in D; 20 lm in E.

cluster containing GAD67 transcript. This cluster was particularly prominent at rostral levels through the RMTg in a position dorsolateral to the caudal half of the interpeduncular nucleus (Fig. 8B). More caudally, it became progressively less distinct, since many neurons in adjacent structures such as the PTg also displayed GAD67 transcript. Careful examination of double-labeled sections

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under brightfield optics in two animals (RL13, RL23) revealed that more than 93% (131 of 140) of CTblabeled cells in sharp focus at the tissue surface of sections through the RMTg core region contained GAD67 mRNA. Thus, in the rostral (Fig. 8D) and caudal RMTg, most CTb-labeled cells typically exhibited agglomerations of silver grains indicative of GAD67 mRNA (Fig. 8E).



Anterograde tracing experiments: RMTg inputs to the DR To unravel the exact topography of RMTg projections to the DR, small injections of PHA-L were aimed at the RMTg in three rats (R85, R86, R87). Case R86 was chosen as prototype to illustrate our findings (Figs. 9, 10). This case had a relatively large PHA-L injection that covered most of the Som-rich RMTg region situated dorsolateral to the caudal half of the interpeduncular nucleus (Fig. 9A–D). The distribution of the resulting anterograde labeling in the DR is shown in Figure 10. PHA-L labeled axons were found in all DR subdivisions and some also extended into the dorsally adjacent VLPAG. In general, PHA-L labeling in the DR was denser near the midline than in the lateral wings. In the DRR (Fig. 9E), DRDSh, lateral wings, and DRV, poorly branched axons that often could be followed over a long distance and only occasionally exhibited varicosities were intermingled with some profusely branched and beaded axons, forming modest terminal fields. A very prominent focal terminal field with a great number of axons exhibiting bouton-like swellings and terminal ramifications occupied the DRDCe (Figs. 9F–H). This characteristic terminal field in the DRDCe extended from about 7.4 to 7.8 mm posterior to bregma. Prominent terminal-like PHA-L labeling could additionally be observed in the dorsally adjacent DRDC (Fig. 9F). At more caudal levels through the DR, PHA-L-labeled axons thinned out and only very few, mostly nonvaricose, axons could be observed in the DRC (Fig. 10D). Additional pontomesencephalic structures that presented robust PHA-L labeling included the VTA-nigra complex including the RRF (Figs. 9C,D), the LDTg (Fig. 10D), and the PTg. More caudally, very few PHA-Llabeled axons were detected in the precoeruleus area, whereas the locus ceruleus and subceruleus complex was almost devoid of PHA-L labeling. The remaining two cases had smaller PHA-L injections into the RMTg that mostly involved the lateral part of the caudal RMTg. In general, the resulting anterograde labeling in the DR was similar to that described for case R86, although, probably due to the smaller size of the injections, less expressive.

Combined immunofluorescence labeling for PHA-L and/or 5-HT/VGLUT3 Nowadays, there is increasing evidence that besides the classical neurotransmitters 5-HT, GABA, and DA, the DR also contains large populations of neurons displaying a mixed serotonergic/glutamatergic or purely glutamatergic phenotype, with all these different phenotypes intermingled in varying proportions in distinct DR

subregions (Fu et al., 2010; Hioki et al., 2010; Shikanai et al., 2012). A possible glutamatergic phenotype of DR neurons is evidenced by the expression of VGLUT3, which is the only vesicular glutamate transporter expressed by DR neurons (Gras et al., 2002, Hioki et al., 2010). It is noteworthy that Hioki et al. (2010), examining the neuronal composition of the DR, concluded that 1) VGLUT3 expression in the DR is restricted to neurons, and 2) almost all VGLUT3expressing DR neurons are negative for GAD67 and tyrosine hydroxylase. To specify, whether the prominent terminal field resulting from PHA-L injections into the RMTg is indeed mainly located in the 5-HT-poor DRDCe subregion, and in order to specify the transmitter phenotype of DR neurons targeted by LHb and RMTg axons, we revealed PHA-L labeling by immunofluorescence methods and combined it with immunofluorescence staining for 5-HT or VGLUT3. Analyzing sections double immunostained for PHA-L and 5-HT, we found that the DRC displayed a moderately dense network of mostly varicose LHb axons, some of them directed to 5-HT1 neurons and forming appositions with the latter (Fig. 11A–C). In contrast, the prominent terminal field formed by RMTg axons mainly targeted the DRDCe, which is mostly devoid of 5-HT1 neurons (Fig. 11D–G). The adjacent DRDSh contained a considerable number of 5-HT neurons but only few PHA-L labeled axons (Fig. 11D,F). We then analyzed the distribution of VGLUT31 cell bodies in DR sections immunostained for VGLUT3 alone or PHA-L/VGLUT3. Prominent but differentially distributed VGLUT3 immunoreactivity was detected in cell bodies and axons throughout the DR. The DRDCe contained numerous small, mostly round, densely packed VGLUT31 cell bodies (Fig. 12A). However, VGLUT31 cell bodies were also enriched in other midline DR subregions, such as the DRR, DRV, DRDSh, and DRC. In contrast, the lateral wings and the DRDC contained less and typically only weakly stained VGLUT31 cell bodies. Analyzing confocal microscope images of sections double immunostained for PHA-L and VGLUT3, we observed that PHAL-labeled RMTg axon terminals in the DRDCe were intermingled with VGLUT31 cell bodies, sometimes forming appositions with the latter (Fig. 12B,C). In sections double immunostained for 5-HT and VGLUT3, we then semiquantified 5-HT-only (VGLUT3-/ 5HT1), VGLUT3-only (VGLUT31/5HT-), and VGLUT3/5HT (VGLUT31/5HT1) cells in the DRC and DRDCe, the major target regions of the LHb and RMTg, respectively, as well as in two subregions adjacent to the DRDCe, the DRDSh and DRV (Table 3). Notably, the DRDCe (Fig. 12D) was unique among all DR subregions analyzed by containing the highest percentage of VGLUT3-only


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Figure 9. Photomicrographs illustrating a PHA-L injection into the RMTg (case R86; A,C,D), an adjacent section double-immunostained for PHA-L and Som (B), as well as the resulting anterograde labeling at different levels through the DR (E–H). Note that the injection is centered in the Som-rich rostral RMTg (A,B) and also involves the caudal RMTg (C,D). Arrowheads indicate the same blood vessels. E,F: Darkfield photomicrographs of anterograde labeling at a rostral (E) and midrostrocaudal (F) DR level. Note in E the focal terminal field in the DRDCe. G,H: Detail photomicrographs of the terminal field in the DRDCe. Note that PHA-L-labeled axons in the DRDCe present numerous bouton-like swellings and terminal ramifications, whereas axons in surrounding DR subregions are less varicose (examples indicated by arrows). Asterisks indicate the same position in G and H. For abbreviations, see list. Scale bars 5 200 lm in A (applies to A,B); 500 lm in C (applies to C,D); 250 lm in E (applies to E,F); 125 lm in G; 20 lm in F.

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Figure 10. A–D: Semischematic drawings illustrating the distribution of anterogradely labeled axons in the DR following a PHA-L injection into the RMTg (case R86; the injection is depicted in Fig. 9A–D). Sections are presented in a rostrocaudal sequence. Note the sparse anterograde labeling in the DRC. For abbreviations, see list.

(68.2% 6 0.02%), as well as the lowest percentages of 5HT-only (6.2% 6 0.07%) and VGLUT3/5-HT cells (25.6% 6 0.05%). In contrast, the DRC (60.8% 6 0.06%) displayed besides the DRV (51 6 0.03%; Fig. 12E) the highest proportion of VGLUT3/5-HT cells, but contained much less VGLUT3-only cells (32.1 6 0.09%) than the DRDCe. Notably, in all DR subregions analyzed, 5-HTonly cells were only a small minority, with the highest proportions found in the DRDSh (11.8% 6 0.08%) and DRV (10.6% 6 0.03%).

DISCUSSION In the present study we systematically investigated by anterograde and retrograde tracing techniques the organization of LHb projections to the DR, direct and indirect via the RMTg, in a subnuclear context. We also examined a possible GABAergic phenotype of DRprojecting RMTg neurons by combining retrograde tracing from the DR with in situ hybridization for GAD67 and investigated the transmitter phenotype of DR neurons in the respective principal target regions of the LHb and RMTg by combining double immunofluorescence staining for PHA-L and either 5-HT or VGLUT3. A summary diagram of our findings is shown in Figure 13. The main findings are: 1) Moderate direct LHb projections to the DR mainly emerge from the LHbM and are predominantly directed to the DRC. 2) RMTg projections to the DR are more robust, emerge from RMTg neurons

enriched in GAD67 mRNA, and are focally directed to a central DR subdivision poor in 5-HT and enriched in VGLUT3 neurons, herein defined as DRDCe that also projects back to the RMtg. Our results will be discussed with reference to the findings of previous tracttracing studies and with reference to the functional role of the RMTg as an interface between the LHb and DR.

Methodological considerations An inherent limitation for the interpretation of tracing results is the potential uptake and transport of anterograde and retrograde tracers by injured fibers passing through the injection site (Cliffer and Giesler, 1988; Dado et al., 1990; Chen and Aston-Jones, 1995). Substantial necrosis at the injection site has been considered the principal cause of tracer uptake by injured fibers (Dado et al., 1990; Luppi et al., 1995). Thus, in the present study, PHA-L, FG, and CTb were injected iontophoretically using discontinuous currents to minimize the development of heat and consequent tissue damage near the electrode tips (Groenewegen and Wouterlood, 1990; Luppi et al., 1990). Overall, there is little evidence that uptake by fibers of passage might have significantly affected major results of the present study. For example, our anterograde data about LHb projections to the DR are in good agreement with previous anterograde studies (Herkenham and Nauta, 1979; Araki et al., 1988; Kim, 2009) and were largely


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Figure 11. Single channels and merged images of double-immunofluorescence staining for 5-HT (green), and PHA-L (magenta) in the DRC (A–C) and at a midrostrocaudal DR level (D–G). PHA-L labeling in the DRC results from an injection into the LHbM (case R81; Fig. 2A) and that in the midrostrocaudal DR from an injection into the RMTg (case R86; Fig. 9A–D). Note that in the DRC, LHbM axons are directed to 5-HT1 neurons, some of them forming appositions (indicated by arrowheads). In contrast, in the midrostrocaudal DR, RMTg axons selectively target the DRDCe subregion, which is mostly devoid of 5-HT neurons. Scale bars 5 50 lm in A (applies to A–C); 250 lm in D (applies to D–F); 50 lm in G.

supported by the pattern of retrograde labeling in the LHb resulting from CTb or FG injections into the DR. Nevertheless, we cannot exclude that some minor uptake of PHA-L, FG, or CTb occurred in damaged fibers of passage.

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Another obvious methodological limitation was the accurate assignment of our PHA-L injections to distinct subnuclei of the LHb complex. None of the PHA-L injections placed in the LHb complex was small enough to be confined to one of the 10 LHb subnuclei (Andres



Figure 12. Single channel for VGLUT3 (magenta, A) and merged images of double immunofluorescence staining for VGLUT3 and PHA-L (green, B,C) in the midrostocaudal DR. PHA-L labeling results from an injection in the RMTg (case R86; Fig. 9A–D). Note that varicose RMTg axons are mainly found in the DRDCe, where they form numerous appositions with VGLUT31 cell bodies (indicated by arrowheads). D,E: Double immunofluorescence staining for VGLUT3 (magenta) and 5-HT (green) in the DRDCe and DRDSh (D) and in the DRV (E). Note that VGLUT3-only neurons (indicated by arrows) are much more common in the DRDCe than in the DRDSh and DRV. Note also the high number of double-labeled neurons in the DRV (indicated by white fluorescence). The rectangle in A indicates the area shown enlarged in B. Scale bars 5 100 lm in A; 40 lm in B; 25 lm in C; 20 lm in D (applies to D,E).

et al., 1999; Geisler et al., 2003). Thus, it was often impossible to decide if only one or several subnuclei contribute to the resulting pattern of anterograde labeling in the mesopontine tegmentum. Similarly, we considered our attempt to assign retrogradely labeled neurons resulting from tracer injections into the RMTg or DR to distinct LHb subnuclei without counterstaining the material by immunohistochemical and/or histochemical methods not always unambiguous. Neverthe-

less, this approach rendered valuable information about which LHb subnuclei provide major afferents to the DR and RMTg and largely validated our anterograde tracing data.

Topography of LHb projections to the DR It is well established that the DR receives a direct input from the LHb (Pasquier et al., 1976; Aghajanian and Wang, 1977; Herkenham and Nauta, 1979; Araki

TABLE 3. Percentages of VGLUT3 and/or 5-HT-Expressing DR Neurons

VGLUT3-only VGLUT3/5-HT 5-HT-only

DRDCe

DRC

DRDSh

DRV

68.2 6 0.02% (n 5 297) 25.6 6 0.05% (n 5 117) 6.2 6 0.07% (n 5 20)

32.1% 6 0.09% (n 5 256) 60.8% 6 0.06% (n 5 479) 7.2% 6 0.04 (n 5 57)

44.8 6 0.12% (n 5 152) 43.5 6 0.14% (n 5 143) 11.8 6 0.08% (n 5 34)

38.4 6 0.02% (n 5 393) 51.0 6 0.03% (n 5 526) 10.6 6 0.03% (n 5 104)

The numbers indicate the mean 6 SEM of the percentages in three rats. Numbers in parentheses indicate the total of neurons analyzed in three rats.


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Figure 13. Schematic diagram summarizing our major findings. Moderate direct LHb projections to the DR mainly emerge from the LHbM and are predominantly directed to the DRC, which contains about two-thirds of neurons exhibiting either a VGLUT3/5-HT or 5-HT-only phenotype. In contrast, indirect LHb projections to the DR mainly arise from the LHbL, which densely innervates the RMTg (Gonc ¸alves et al., 2012). The RMTg then sends robust GABAergic projections to the DR that are primarily directed to the DRDCe, whose major neuronal population (about two-thirds) is composed of VGLUT3-only neurons, some of which project back to the RMTg.

et al., 1988; Peyron et al., 1998; Kim, 2009; Jhou et al., 2009b; Poller et al., 2011; Bernard and Veh, 2012). The LHb projection to the DR was formerly assumed to be GABAergic (Wang and Aghajanian, 1977). Nowadays there is overwhelming evidence for a predominant glutamatergic phenotype of LHb neurons (Aizawa et al., 2012), including those projecting to the DR and VTA (Kal en et al., 1985; Brinschwitz et al., 2010). However, LHb projections to the DR have not yet been investigated in detail by anterograde tracing. Due to the outstanding functional importance of the LHb-DR projection, we herein reexamined it using anterograde and retrograde tracing methods. In general, most of our findings on LHb projections to the DR are in good agreement with those of the aforementioned studies and extend them in several respects. Major newly discovered features revealed in the present study include a detailed description of the topography of LHb projections to the DR and the notable finding of a focal projection from the LHbM to the DRC. Regarding the subnuclear origin of LHb projections to the DR, our findings are well in line with a recent detailed retrograde tracing study (Bernard and Veh, 2012). Like in this study, we found that, in general terms, the LHbMPc/LHbMC complex and the LHbLMc contribute most to LHb projections to the DR. Our

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anterograde tracing findings further specified that the LHbMPc/LHbMC complex particularly gives rise to a focal projection to the DRC, which was well in line with our retrograde tracing data. On the other hand, our anterograde tracing findings indicate that rather modest projections emerging from the LHbM and LHbL target other DR subregions. At first glance, the latter finding might appear at odds with our own retrograde tracing data, particularly the robust retrograde labeling in the LHb resulting from retrograde tracer injections into the rostral and midrostrocaudal segments of the DR. However, this might be due to technical shortfalls. Thus, it has been reported that FG and CTb are exceedingly sensitive retrograde tracers that are taken up and transported so avidly as to limit the investigators’ capacity to distinguish between major and minor projections (for a detailed discussion, see Brog et al., 1993). Furthermore, our observations of an only moderate innervation of the rostral and midrostocaudal DR are fully supported by the results from previous anterograde tracing studies targeting the LHb (Herkenham and Nauta, 1979; Araki et al., 1988; Kim, 2009). For example, Kim (2009) already illustrated strong anterograde labeling in the DRC following injections of biotinylated dextran amines into the medial one-third of the LHb. On the other hand, he depicted only sparse



anterograde labeling in the DR following tracer injections into the central and lateral one-thirds (see Figs. 7, 8, 9 in Kim, 2009). Comparing the pattern of retrograde LHb labeling resulting from tracer injections into the DR or RMTg, we confirmed the striking topography of LHb projections to the mesopontine tegmentum described in previous anterograde tracing studies (Herkenham and Nauta, 1979; Kim, 2009, Gonçalves et al., 2012). Thus, whereas midline injections into the DR led to retrograde labeling that was mostly confined to the LHbM, injections into the RMTg mainly resulted in retrograde labeling in the LHbL. The differential distribution of retrogradely labeled neurons in the LHb clearly indicates that LHb outputs to the DR and RMTg are mostly segregated. In line with such a view of mostly segregated LHb outputs to different mesencephalic and mesopontine structures, Bernard and Veh (2012) described in a recent double retrograde tracing study only low numbers of double-labeled LHb neurons resulting from dual injections targeting the DR and VTA, or median raphe nucleus and VTA.

RMTg and other mesencephalic and mesopontine tegmental inputs to the DR There is now evidence that the RMTg not only massively innervates the VTA-nigra complex, but also sends substantial projections to the DR (Jhou et al., 2009b; Lavezzi et al., 2012). Furthermore, combining CTb retrograde tracing with GAD immunohistochemistry, Gervasoni et al. (2000) illustrated some GAD-immunoreactive DR-projecting neurons in a position that might correspond to the RMTg (see their Fig. 8C). However, the exact distribution pattern of RMTg axons in the DR has never been investigated in detail. It also remained to be clarified whether RMTg projections to the DR are predominantly GABAergic, as described for RMTg projections to the VTA (Jhou et al., 2009b; Kaufling et al., 2009; Balcita-Pedicino et al., 2011). Thus, we reinvestigated RMTg projections to the DR by retrograde and anterograde tracing and examined a possible GABAergic phenotype of DR-projecting RMTg neurons by combining retrograde tracing from the DR with in situ hybridization for GAD67. In general, our findings confirm a robust projection from the RMTg to the DR described previously (Jhou et al., 2009b; Lavezzi et al., 2012). Importantly, they specify that the RMTg input to the DR mainly arises from RMTg neurons enriched in GAD67 mRNA. Furthermore, we show here for the first time that, although RMTg axons target all DR subregions, there is an outstanding dense focal input directed to a central DRD subdivision,

defined herein as DRDCe. This latter finding is also supported by our retrograde tracing data, demonstrating that retrograde labeling in the RMTg was strongest in cases in which tracer injections into the DR substantially involved the DRDCe. Similar to the VTA-projecting RMTg neurons (Gonc ¸alves et al., 2012), DR-projecting neurons in these cases virtually outlined the RMTg core region in its entire rostrocaudal extent. Furthermore, their position undoubtedly corresponded to the position of the core region of Fos-labeled neurons activated by psychostimulant application in previous studies (Geisler et al., 2008; Kaufling et al., 2009; Jhou et al., 2009a,b). Interestingly, placing pairs of injections of two different retrograde tracers into targets including the DR/VTA and DR/PTg, Lavezzi et al. (2012) recently described that about 10% of neurons in the RMTg core region send collaterals to both the DR and VTA and about 5% to the DR and pars dissipata of the PTg. These findings suggest that a minority of RMTg neurons may simultaneously project to the DR and VTA, or to the DR and PTg. Our anterograde tracing findings confirm substantial RMTg projections to the PTg and LDTg (Jhou et al., 2009b; Lavezzi et al., 2012), but shed doubt on a robust RMTg projection to the locus ceruleus and subceruleus complex described by Jhou et al. (2009b). Furthermore, our retrograde tracer injections into the DR confirmed and extended previous tracing findings describing or depicting DR inputs from the PTg, LDTg, SNC, RRF, VTA, and ventrolateral periaqueductal gray (e.g., Steininger et al., 1992; Peyron et al., 1995, 1996; Gervasoni et al., 2000; Kirouac et al., 2004; Zahm et al., 2011). Interestingly, all of the aforementioned structures are innervated by the RMTg (present findings; Ferreira et al., 2008; Jhou et al., 2009b; Lavezzi et al., 2012) and most of them are mutually interconnected (e.g., Pasquier et al., 1977; Deutch et al., 1988; Vertes, 1991; Semba and Fibiger, 1992; Honda and Semba, 1994; Lavoie and Parent, 1994; Van Bockstaele et al., 1994; Oakman et al., 1995; Broderick and Phelix, 1997; Omelchenko and Sesack, 2005, 2006, 2010; Braz et al., 2009; Zahm et al., 2011; Lima et al., 2012). However, the transmitter phenotype of these connections between nuclei traditionally considered to be dopaminergic (SNC, VTA, RRF), serotonergic (DR), or cholinergic (PTg, LDTg) should be carefully evaluated. For example, the projections from the VTA-nigra complex to the DR have been shown to be predominantly GABAergic (Kirouac et al., 2004), whereas the bulk of DR projections to the VTA seem to display a glutamatergic or mixed glutamatergic/serotonergic phenotype (Lima et al., 2012; Wang et al., 2012). Although it has never been systematically investigated whether neuronal types other than GABA should be


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included within the RMTg, a predominant GABAergic phenotype is assumed to be one of the anatomical hallmarks of the RMTg (Lavezzi and Zahm, 2011; Bourdy and Barrot, 2012). Thus, the structure now recognized as RMTg was initially described as a bilateral cluster of GABAergic neurons at the caudal pole of the VTA that express variants of the Fos gene after psychostimulant administration (Perrotti et al., 2005). Our observation that more than 90% of the DR-projecting RMTg neurons display a GABAergic phenotype gives further evidence to the view that the RMTg represents a relatively pure GABAergic cell population. However, this view should be challenged in future by quantitative in situ hybridization studies combined with NeuN immunohistochemistry. Importantly, the predominant GABAergic phenotype of DR-projecting RMTg neurons indicates that the RMTg is not only an important GABAergic control center for the VTA but might also powerfully modulate neuronal activity in the DR, as well as in other pontomesencephalic structures such as the RRF, SNC, and PTg/LDTg complex.

Transmitter phenotype of DR neurons in the respective principal target regions of the LHb and RMTg Initial findings of DR anatomy and physiology suggested that the DR is a rather homogeneous, mostly serotonergic structure with a global mode of operation (e.g., Jacobs and Fornal, 1991). However, nowadays there is increasing evidence that 5-HT release in forebrain regions occurs in a regional-specific manner (Kirby et al., 1995) and that the DR is a highly heterogeneous structure composed of several subregions containing distinct neuronal subpopulations that differ with respect to their transmitter phenotypes, projections, and electrophysiological properties (Commons et al., 2003; Abrams et al., 2004; Day et al., 2004; Fu et al., 2010; Hioki et al., 2010; Calizo et al., 2011; Crawford et al., 2011, 2013; Hale and Lowry, 2011; Soiza-Reilly and Commons, 2011; Vasudeva et al., 2011; Waselus et al., 2011; Bang et al., 2012; Hale et al., 2012; Shikanai et al., 2012). Our findings demonstrating that LHb and RMTg preferentially innervate differential DR subregions provide further evidence that distinct DR districts receive a differential distribution of afferents (Peyron et al., 1998; Lee et al., 2003, 2007; Gonçalves et al., 2009; Hale et al., 2011). Notably, they demonstrate that RMTg inputs to the DR are preferentially directed to a neurochemically highly peculiar DR subregion herein designated the DRDCe, which is characterized by the paucity of 5-HT and the abundance of densely packed VGLUT3 neurons. Recent in situ hybridization and immunohistochemical studies (Fremeau et al., 2002; Gras et al., 2002;

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Commons, 2009; Hioki et al., 2010) revealed an abundant expression of VGLUT3 in neurons of the DR and median raphe nucleus. Furthermore, in an elegant and detailed double-fluorescence in situ hybridization study, Hioki et al. (2010) detected a high degree of colocalization of VGLUT3 and tryptophan hydroxylase 2 mRNA signals in the DR and median raphe nucleus. Notably, their findings indicate that in most DR subregions, 5HT-only neurons are a minor subpopulation. The present data about the respective proportions of 5-HT-only, VGLUT3-only, and VGLUT3/5-HT neurons in selected DR subregions are difficult to compare with the dataset of Hioki et al. (2010), since those authors also included GABA and DA neurons in their analysis. Nevertheless, many key results are in good agreement. Thus, as described by these authors, we found that in most DR subregions 5-HT-only neurons are only a small minority (about 10%) and that among all DR subregions analyzed, the DRC and DRV display the highest degree of colocalization between 5-HT and VGLUT3. Importantly, Hioki et al. (2010) described that the DRDSh stands out as the DR subregion containing the highest percentage of VGLUT3-only neurons. In the present study, we distinguished the DRDCe as a separate DR subregion located within the DRDSh and revealed that the DRDCe contained an even higher percentage of VGLUT3-only neurons than the DRDSh (68% versus 49%, respectively). Other major arguments in favor of designating the DRDCe as a separate DRD subregion are: 1) a dense focal input from the RMTg, 2) an extremely low proportion of 5-HT-only neurons, and 3) a denser cell packing compared to the DRDSh. On the other hand, the DRC herein identified as the major recipient of direct LHb inputs contained about 68% of neurons exhibiting either a VGLUT3/5-HT or 5-HT-only phenotype, indicating that two-thirds of its neurons use 5-HT as one of its neurotransmitters. Importantly, VGLUT3-only and/or VGLUT3/5-HT neurons in the DRD have meanwhile been shown to be the source of distinct projections to targets including the VTA-nigra complex, preoptic area, hippocampal CA1 region, and medial septum (Geisler et al., 2007; Jackson et al., 2009; Hioki et al., 2010). In contrast, in brain regions such as the neocortex and striatum, VGLUT3 is mostly expressed in GABAergic (Fremeau et al., 2002; Hioki et al., 2004) or cholinergic (Gras et al., 2002, 2008) interneurons, respectively. VGLUT3 has also convincingly been proven to be critically involved in fast excitatory glutamatergic transmission in the auditory system (Ruel et al., 2008; Seal et al., 2008), as well as in the DR projection to the VTA (Wang et al., 2012). Moreover, the loss of VGLUT3 expression has been shown to lead to anxiety-



associated behaviors, a decrease of 5-HT1A-mediated neurotransmission in the DR, and an increase of 5-HT transmission in the hippocampus and cerebral cortex (Amilhon et al., 2010). Overall, these findings underline a major role for glutamatergic neurotransmission in the normal functioning of the DR. However, the specific functions and connections of VGLUT-3-only and VGLUT3/5-HT neurons in the DR should clearly be challenged by further studies targeting these prominent neuronal populations. Interestingly, a great number of presumptive glutamatergic neurons, characterized by the expression of the type 2 vesicular glutamate transporter, have recently also been described in the VTA (Kawano et al., 2006; Yamaguchi et al., 2007, 2011; Hnasko et al., 2012) and PTg/LDTg complex (Wang and Morales, 2009).

General functional comments There is now overwhelming evidence that the RMTg is an intermediate structure in a pathway that connects the LHb to midbrain DA neurons (Jhou et al., 2009b; Balcita-Pedicino et al., 2011; Gonçalves et al., 2012) and that both the LHb and RMTg are central structures of an anti-reward circuit that encode disappointments and expectation of negative conditions. Thus, neurons in the LHb (Matsumoto and Hikosaka, 2007, 2009) and RMTg (Jhou et al., 2009a; Lecca et al., 2011; Hong et al., 2011) respond to the negative value of a stimulus and are primarily excited by reward omission and aversive stimuli and outcomes. Importantly, this firing pattern of LHb and RMTg neurons is inverse to that of putative VTA DA neurons which primarily respond with phasic excitations to rewarding, reward-predicting, and appetitive stimuli (see, e.g., Schultz, 1998, 2007; Bromberg-Martin et al., 2010b). The present anatomical findings strongly indicate that the RMTg is not only an important GABAergic control node for the VTA-nigra complex but might also serve as a major GABAergic relay between the LHb and the DR. A comparison of the organization of LHb projections to the VTA and DR, directly and indirectly via the RMTg, reveals similarities between direct LHb projections to the VTA and DR but also important differences in RMTg projections to these structures. Thus, both VTA (Omelchenko et al., 2009; Brinschwitz et al., 2010; Gonc ¸alves et al., 2012) and DR (present findings; Herkenham and Nauta, 1979; Araki et al., 1988; Kim, 2009) receive minor direct LHb projections, mostly emerging from segregated neuronal populations in the LHbM (Bernard and Veh, 2012). Comparing RMTg projections to the VTA and DR, important differences can be noted. The RMTg densely innervates in a rather homogeneous manner the entire VTA-nigra complex, including the RRF

(present findings; Ferreira et al., 2008; Jhou et al., 2009b). Furthermore, ultrastructural studies revealed that the vast majority of synapses formed by RMTg axons in the VTA and SNC are onto dendrites immunoreactive for tyrosine hydroxylase (Balcita-Pedicino et al., 2011; Barrot et al., 2012), strongly suggesting that the inhibitory RMTg influence is mediated directly onto DA neurons. In contrast, we here show that RMTg projections to the DR are not homogeneous, with most of them focally directed to the DRDCe, whose neurons display a predominantly glutamatergic phenotype and some of which project back to the RMTg. Due to the novelty of our findings, we can only speculate about the functional significance of the focal direct LHb input to the DRC as well as the reciprocal connection between the RMTg and DRDCe. Both the DRC as well as the DRD (often without distinguishing between its subdivisions) have been implicated as important components of stress- and anxietyrelated circuits (for reviews, see Lowry et al., 2008; Hale and Lowry, 2011; Hale et al., 2012). In line with such a view, both are selectively interconnected with key structures involved in the regulation of emotional behaviors. For example, the DRD receives major afferents from the prefrontal cortex (Gabbott et al., 2005; Gonçalves et al., 2009), bed nucleus of the stria terminalis, medial and central nucleus of the amygdala (Lee et al., 2007), medial and lateral preoptic area, lateral hypothalamus and VLPAG (Peyron et al., 1998), with most of these projections being reciprocal (e.g., Vertes, 1991; Commons et al., 2003). The DRC has a similar but slightly reduced set of connections with the abovecited structures and additionally receives a focal input from the LHbM (present findings) and sends massive outputs to the lateral septum (Vertes, 1991; Waselus et al., 2006) and ventricular system (Mikkelsen et al., 1997). Interestingly, a series of studies indicates that the DRC and LHb jointly are critically involved in the neurochemical and behavioral response to uncontrollable stress, notably in the behavioral phenomenon called behavioral depression (Weiss, 1968) or learned helplessness (Maier and Seligman, 1976; Maier and Watkins, 2005). Thus, inescapable but not escapable shock induces C-Fos expression preferentially in the DRC (Grahn et al., 1999) and augments 5-HT efflux in the DR (Maswood et al., 1998), with the latter effect abolished by habenular lesions (Amat et al., 2001). It is thus tempting to speculate that the here demonstrated direct LHbM input to the DRC might be responsible for these effects. Given that the LHbM is preferentially activated by different forms of stress (Chastrette et al., 1991; Wirtshafter et al., 1994; Brown and Shepard, 2013) and that LHb neurons are predominantly


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glutamatergic (e.g., Aizawa et al., 2012), the activation of this pathway might directly stimulate 5-HT neurons in the DRC and thus lead to 5-HT release. On the other hand, it has been repeatedly shown that high-frequency local electrical stimulation of the LHb results in powerful suppression of presumptive 5HT DR neurons (Wang and Aghajanian, 1977; Stern et al., 1979; Ferraro et al., 1996). Here it is tempting to speculate that such inhibition is mediated by the GABAergic RMTg, which receives glutamatergic efferents from the LHbL (Kim, 2009; Balcita-Pedicino et al., 2011; Gonçalves et al., 2012). However, given that a great part of the GABAergic RMTg efferents are directed to VGLUT31 neurons in the DRDCe, it remains unclear how inhibition of 5-HT neurons is achieved in this scenario. One possibility is that VGLUT31 neurons in the DRDCe might drive 5-HT neurons in other DR subregions via axon collaterals. Thus, it has been demonstrated that VGLUT31 axons are frequently apposed to 5-HT DR neurons (Soiza-Reilly and Commons, 2011) and that most of these intrinsic projections might emanate from VGLUT31 DRD neurons that express the receptor for substance P, neurokinin 1 (Valentino et al., 2003; Commons, 2009; see also Lacoste et al., 2009). Another possibility is that the here demonstrated moderate RMTg projections to DR subregions enriched in 5HT neurons such as the lateral wings and DRDC are sufficient to promote the inhibitory LHb influence onto 5-HT DR neurons. Meanwhile, it has been demonstrated in 5-HTdepleted rats that many VGLUT31 neurons in the DRD are projection neurons that innervate targets like the preoptic area, anterior hypothalamus, VTA, and SNC (Hioki et al., 2010; see also Halberstadt and Balaban, 2008). Our own preliminary retrograde tracing findings indicate that VGLUT3-only and VGLUT3/5-HT neurons in the DRDCe substantially contribute to DR projections to the VTA (Lima et al., 2012) and send a very robust projection to the SNC (our unpublished findings). Thus, given that the DRD is richly interconnected with structures involved in the regulation of emotional behaviors (see above), the DRDCe might form an important modulatory link between these structures and the VTA-nigra complex. Moreover, the here described projection from the DRDCe to the RMTg might represent a feedback pathway (see also Good et al., 2013) that is involved in the fine tuning and perhaps coordination of RMTg outputs to the DR and other monoaminergic centers. However, these assumptions clearly await testing by electrophysiological methods. As indicated by recent theoretical accounts as well as behavioral studies in animals and humans, 5-HT and DA may have unifying affective, activational, and deci-

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sion functions (Boureau and Dayan, 2011; Cools et al., 2011). Thus, whereas DA primarily serves to promote behavioral activation to seek rewards (Salamone and Correa, 2012), 5-HT might be critical for punishmentinduced inhibition, promoting withdrawal in the face of aversive predictions (Crockett et al., 2009, 2012; Robinson et al., 2012). A slightly alternative view is that the 5-HT system is critically involved in "waiting to avoid future punishments" and also in "waiting to obtain future rewards" (Miyazaki et al., 2012). Consistent with this, recording studies in presumptive 5-HT DR neurons have indicated that some of them encode behavioral tasks in a systematic manner and encode participation in a behavioral task primarily in terms of its future motivational outcomes (Nakamura et al., 2008; BrombergMartin et al., 2010a). As convincingly outlined in a recent review (Hikosaka, 2010), motor suppression under adverse conditions might be the key mechanism that underlies the various functions of the different circuits involving the LHb and the RMTg. In agreement with such a view, it has been recently demonstrated in an elegant optogenetic study in mice that activation of LHb terminals in the RMTg promotes active, passive, and conditioned behavioral avoidance (Stamatakis and Stuber, 2012). Importantly, the present findings in conjunction with previous data (Lavezzi et al., 2012) indicate that the LHb-RMTg circuit is critical in the inhibitory control of both DA neurons in the VTA-nigra complex and 5-HT, as well as non-5-HT DR neurons. Consistent with a possible integrative role of the LHb-RMTg pathway in regulating both DA and 5HT functions, the findings of a recent combined behavioral/manganese-enhanced magnetic resonance imaging study indicate that the LHb couples the DA and 5HT systems (Sourani et al., 2012). A functional coupling of DA and 5-HT systems via the LHb-RMTg and other circuits discussed here may be relevant to explain the involvement of DA in psychomotor symptoms of depression (Sobin and Sackheim, 1997; Buyukdura et al., 2011), as well as the role of 5-HT in nonmotor symptoms in Parkinson’s disease such as depression and anxiety (Tan et al., 2011; Lindgren and Dunnett, 2012).

ACKNOWLEDGMENTS The authors thank Ana M.P. Campos for expert technical assistance, Roberto Cabado Modia Junior for help with confocal microscopy procedures, and Prof. Sara Shammah-Lagnado for many fruitful discussions and critical reading of the article.

CONFLICT OF INTEREST The authors declare that they have no conflict of interest.



ROLE OF AUTHORS All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: C.S., J.D. Jr, M.M. Acquisition of data: C.S., L.G., L.L., I.C.F, J.D. Jr., M.M. Analysis and interpretation of data: C.S., L.G., M.M. Drafting of the article: C.S., M.M. Critical revision of the article for important intellectual content: C.S., L.G., L.L., I.C.F, J.D. Jr., M.M. Obtained funding: J.D. Jr., M.M. Study supervision: M.M.

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Sources of input to the rostromedial tegmental nucleus, ventral tegmental area, and lateral habenula compared: A study in rat.

Substance P afferents from the habenula innervate the dorsal raphe nucleus.

The rostromedial tegmental nucleus and alcohol addiction.

The role of lateral habenula-dorsal raphe nucleus circuits in higher brain functions and psychiatric illness.

Cellular profile of the dorsal raphe lateral wing sub-region: relationship to the lateral dorsal tegmental nucleus.

Habenula-Induced Inhibition of Midbrain Dopamine Neurons Is Diminished by Lesions of the Rostromedial Tegmental Nucleus.

Regulation of nucleus accumbens dopamine release by the dorsal raphe nucleus in the rat.

Inhibition of lateral vestibular nucleus neurons by 5-hydroxytryptamine derived from the dorsal raphe nucleus.

Cytoarchitecture of the human dorsal raphe nucleus.

Bombesin immunoreactive neurons in the hypothalamic paraventricular nucleus innervate the dorsal vagal complex in the rat.

Fine structure of the lateral mammillary projection to the dorsal tegmental nucleus of Gudden in the rat.

Pharmacological Manipulation of the Rostromedial Tegmental Nucleus Changes Voluntary and Operant Ethanol Self-Administration in Rats.

Role for the Rostromedial Tegmental Nucleus in Signaling the Aversive Properties of Alcohol.

A PHA-L analysis of ascending projections of the dorsal raphe nucleus in the rat.

Two major network domains in the dorsal raphe nucleus.

Ascending dopaminergic projections from the dorsal raphe nucleus in the rat.

The rostromedial tegmental nucleus modulates behavioral inhibition following cocaine self-administration in rats.

Effect of sex steroid hormones on the number of serotonergic neurons in rat dorsal raphe nucleus.

Development of the dorsal lateral geniculate nucleus in the cat.

Studies of the cat's medial interlaminar nucleus: a subdivision of the dorsal lateral geniculate nucleus.

Response properties of cells in the dorsal lateral geniculate nucleus of the albino rat.

Neural circuit interactions between the dorsal raphe nucleus and the lateral hypothalamus: an experimental and computational study.

Principles of Organization of the Dorsal Lateral Geniculate Nucleus.

Neurons of the dorsal lateral geniculate nucleus of the albino rat.