B R A I N R E S E A R C H R E V I E W S 5 5 ( 2 0 0 7 ) 3 2 9 – 3 4 2

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Review

The dorsal raphe nucleus—From silver stainings to a rolein depression

Kimmo A. Michelsen, Christoph Schmitz, Harry W.M. Steinbusch⁎

Department of Neuroscience, Faculty of Health, Medicine and Life Sciences, Maastricht University, PO Box 616, 6200 MD Maastricht,The Netherlands1

A R T I C L E I N F O

⁎ Corresponding author. Fax: +31 43 3671096.E-mail address: h.steinbusch@np.unimaa

1 European Graduate School of Neuroscien

0165-0173/$ – see front matter © 2007 Elsevidoi:10.1016/j.brainresrev.2007.01.002

A B S T R A C T

Article history:Accepted 10 January 2007Available online 17 January 2007

Over a hundred years ago, Santiago Ramón y Cajal used a new stainingmethod developed byCamillo Golgi to visualize, among many other structures, what we today call the dorsalraphe nucleus (DRN) of the midbrain. Over the years, the DRN has emerged as amultifunctional and multitransmitter nucleus, which modulates or influences many CNSprocesses. It is a phylogenetically old brain area, whose projections reach out to a largenumber of regions and nuclei of the CNS, particularly in the forebrain. Several DRN-relateddiscoveries are tightly connected with important events in the history of neuroscience, forexample the invention of new histological methods, the discovery of new neurotransmittersystems and the link between neurotransmitter function and mood disorders. One of themain reasons for the wide current interest in the DRN is the nucleus' involvement indepression. This involvement is particularly attributable to themain transmitter of the DRN,serotonin. Starting with a historical perspective, this essay describes the morphology,ascending projections andmultitransmitter nature of the DRN, and stresses its role as a keytarget for depression research.

© 2007 Elsevier B.V. All rights reserved.

Keywords:Dorsal raphe nucleusSerotoninDepressionCajal

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3302. Cajal and Golgi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3303. The discovery of neurotransmitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

3.1. Serotonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3314. The dawn of neurochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

4.1. New histochemical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3314.2. Radioactive labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3324.3. Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

5. Transmitters of the DRN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3325.1. Dopamine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3325.2. GABA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3325.3. Peptide transmitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

s.nl (H.W.M. Steinbusch).ce (EURON).

er B.V. All rights reserved.

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5.4. Glutamate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3335.5. Nitric oxide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3335.6. Transient presence of additional transmitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

6. DRN morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3346.1. Cell types. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3346.2. Efferent projections of the DRN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3346.3. Fiber morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3356.4. Pathway overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3356.5. Dorsal ascending pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3356.6. Medial ascending pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3366.7. Ventral ascending pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

6.7.1. Hypothalamus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3376.7.2. Thalamus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3376.7.3. Habenula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3376.7.4. Septum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3376.7.5. Amygdaloid complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3376.7.6. Cortex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3376.7.7. Hippocampus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3376.7.8. Olfactory bulb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3376.7.9. Supra-ependymal plexus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

7. Functional neuroanatomy of the DRN with emphasis on depression . . . . . . . . . . . . . . . . . . . . . . . . . . . 3388. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338

1. Introduction

The dorsal raphe nucleus (DRN) is a bilateral, heterogenousbrainstem nucleus, located mainly in the ventral part of theperiaqueductal gray matter of the midbrain. A majority ofthe nucleus' neurons utilize its major neurotransmitter,serotonin, but several other transmitters are also present.It comes as no surprise that the first detailed outline of whatwas later to be called DRN was presented by Santiago Ramóny Cajal in his famous work on the texture of the nervoussystem (Ramón Cajal, 1904). By skillful use of the silverchromate-impregnation method developed by Camillo Golgi,Cajal was able to reveal details about DRN morphology,which are still valid.

The DRN is an interesting area in twoways. Firstly, becauseit innervates a multitude of targets throughout the brain andspinal cord via its ascending and descending pathways.Secondly, because the story of DRN research nicely illustratesseveral major breakthroughs, paradigm shifts and the emer-gence of new fields of research within neuroscience. In thisessay we outline the discoveries and technical advances of thepast century, which have taught us what we have learnedabout the DRN from the times of Cajal and Golgi to the presentday.

2. Cajal and Golgi

Golgi's silver chromate-impregnation method was undoubt-edly of crucial importance to Cajal's success in describing thetexture of the mammalian nervous system. One of the areas

he studied in newborn rabbit and kitten, was the raphe area.Cajal observed that the DRN contained four types of neurons,which he described as being voluminous, fusiform, triangularand stellate. His description is in accordance with modernreports on other mammals, which also identify four mor-phologically distinct types of neurons in the DRN. They havebeen described as round, ovoid, fusiform and triangular inhuman (Baker et al., 1990), and small round, medium-sizedfusiform and bipolar, large fusiform and very large multipolarin rat (Steinbusch et al., 1981; Steinbusch, 1984). Cajal alsorecognized that the neurons were equipped with “severaldivergent and strongly spiny dendrites” and that the fiberstended to “concentrate in ascending and descending dorso-ventral bundles”. However, he was not able to determinehow far the fibers continued. Today we know that the fibersof DRN target a multitude of regions, both close to and farfrom the DRN itself, throughout the brain and spinal cord.Some of the fibers collateralize and, thus, a single neuron canreach more than one target simultaneously. Cajal also wroteabout collateral fibers, but it is not entirely clear whether hereferred to single fibers or one of the ascending bundles as awhole.

Cajal did not refer to the DRN by its present name, which isbased on a much later classification of the raphe complex.Instead he designated an “intermediate or unpaired nucleus”as the “median subaqueductal nucleus of the raphe” of thekitten (Fig. 1). This nucleus probably resembles the dorsome-dial DRN, situatedmedially just below the aqueduct of Sylvius,as suggested by Pasik and Pasik in their annotated translationof Cajal's work (Ramón Cajal, 2000). What Cajal called themagnocellular central nucleus has been interpreted to include

Fig. 1 – Cajal's drawing of a transverse section through thecaudal region of the superior colliculus of a few-days-oldkitten. E, cells of the “subaqueductal nucleus of the raphe”,which probably resembles the DRN. A, cells of the trochlearnerve nucleus; B, collaterals within the same nucleus; C,medial longitudinal fasciculus; D, fibers of the superiorcerebellar peduncles; F, ventral cells of the raphe; G, radicularfibers of the trochlear nerve. Image source: the annotated andedited translation of Cajal's “Texture of the Nervous Systemof Man and the Vertebrates” by Pedro Pasik and Tauba Pasik(Ramón Cajal, 2000), used by permission.

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(at least parts of) the DRN and median raphe nucleus (MRN).He also mentioned that, in the magnocellular central nucleusof the raphe, a “thin cellular trail extends ventrally penetratingbetween both longitudinal fascicles”. This trail seems toresemble the interfascicular DRN and, possibly, rostral partsof the MRN (Ramón Cajal, 2000).

3. The discovery of neurotransmitters

When Cajal set the stage for neuromorphological work in theearly 20th century, the neuron doctrine was still heavilydebated. Although opposed by Golgi, the hypothesis wassupported by Cajal and by Charles Sherrington, who hadcoined the term “synapse” a few years earlier (in 1897).Sherrington described the synapse in 1906; the year thatCajal and Golgi received the Nobel Prize, and soon the neurondoctrine became widely accepted.

For a long time, it was debated whether the synapse ischemical or electric. Convincing evidence in favor of chemicaltransmission was presented in 1921, when Otto Loewidemonstrated that acetylcholine, which had been discoveredby Henry Dale in 1913, transmitted signals across the synapsebetween the vagus nerve and the heart muscle. The discoveryof other transmitters followed, including themain transmitterof the DRN, serotonin, about one decade later.

3.1. Serotonin

The story of serotonin began in 1933,whenVialli and Erspamer(1933) used an argantophyl reaction to demonstrate thepresence of an amine in the granules of the granulated cells

in the gastrointestinal mucosa. In 1940, Erspamer (1940)showed that the substance was biologically active andnamed it enteramine. It was soon detected also in the centralnervous system (Twarog and Page, 1953) and identified as theheterocyclic amine 5-hydroxytryptamine (Erspamer andAsero, 1952). Subsequent studies demonstrated the amine'seffect on smooth muscle contraction and it became regardedas a local tissue hormone (Kosterlitz and Robinson, 1957;Lembeck., 1958). The name serotonin was coined by Rapportwho reported that a substance isolated from ox blood was apotent vasoconstrictor (Rapport, 1949). It was soon shown thatenteramine was identical to serotonin, and the substancecame to be known under the latter name (Rand and Reid, 1951;Reid and Rand, 1952).

4. The dawn of neurochemistry

The connection between the DRN and serotonin was estab-lished when Dahlström and Fuxe (1964) described thedistribution of serotonergic neurons in the rat DRN. This,and other discoveries, such as the histochemical technique fordetection of cholinesterase activity introduced by Koelle andFriedenwald (1949) and modified by Lewis and Shute (1959)turned the focus of many neuroscientists towards theidentification and localization of neuronal groups usingspecific neurotransmitters, which led to something of aparadigm shift: neurotransmitters became a prime determi-nant of a neuron's identity and neurons were judged, andnamed, based on the neurotransmitters that they contained.The field of neurochemistry had emerged.

4.1. New histochemical techniques

In their studies on the rat DRN, Dahlström and Fuxe usedformaldehyde-induced fluorescence (FIF), which had beendeveloped by Falck et al. (1962) for visualization of mono-amines. The FIF-technique soon became themost popular toolfor visualizing serotonergic neurons in the DRN and else-where. A major drawback of the FIF-technique was that β-carboline is highly UV-sensitive, which led to rapid fading ofthe fluorescence. In addition, freeze-drying of the tissuecompromised the level of obtainable morphological detail.The latter problem was partly overcome after modifications(Hökfelt and Ljungdahl, 1972).

From the sixties to the early eighties, themorphology of theDRN was described in cat (Taber et al., 1960), man (Braak,1970), rabbit (Felten and Cummings, 1979) and rat (Steinbuschet al., 1981). Already soon after the FIF-technique wasdiscovered, data on efferent raphe projections started toaccumulate. The first DRN projections to be reported targetedthe hypothalamus (Dahlström and Fuxe, 1964). A significantimprovement in the tool palette for fiber pathway researchersresulted, when Kristensson and Olsson (1971) establishedhorseradish peroxidase (HRP) as a retrograde tracer, whichwas taken up by nerve endings of the hypoglossal nerve andtransported to the perikaryon. LaVail and co-workers firstused it in the CNS (LaVail et al., 1973; LaVail and LaVail, 1972)and soon after it was utilized for tracing fiber inputs to theDRN (Fibiger and Miller, 1977).

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4.2. Radioactive labels

Other early methods included autoradiographic detection on[3H]-serotonin uptake (Calas et al., 1974; Chan-Palay, 1977)and [3H]-radiolabeled reserpine (Richards et al., 1979). Mean-while, radioactively labeled leucine was used as an ante-rograde tracer to map DRN projections in the cat (Bobillieret al., 1976).

4.3. Immunohistochemistry

The early seventies saw the dawn of yet another newmethod:immunohistochemistry. It became a powerful tool in thehands of numerous neuroscientists, and within the nextdecade, most major neurotransmitter pathways weremappedwith high specificity and accuracy.

With respect to the DRN, first out were antibodiesagainst tryptophan hydroxylase and amino acid decarbox-ylase (Hökfelt et al., 1973; Joh et al., 1975). The developmentof antibodies against serotonin itself (Steinbusch et al.,1978) led to increased specificity and sensitivity. Similarresults to those reported by Dahlström and Fuxe (1964)were obtained, but much more fibers were distinguished(Steinbusch, 1981).

Since then, afferent and efferent fibers have been mappedwith immunohistochemistry in combination with a variety oftract tracing applications, which utilize several anterogradelyor retrogradely diffusible lipophilic compounds. Besides ofthose already mentioned, successful anterograde tracersinclude Phaseolus vulgaris leucoagglutinin (PHA-L) and HRPconjugates with wheat germ agglutinin or cholera toxin, ofwhich the latter is also a retrograde tracer. Retrograde tracersalso include propidium iodide, and compounds known bytheir commercial brands, such as fast blue, true blue andFluoro-Gold.

5. Transmitters of the DRN

After the invention of the FIF-technique, the DRN wasregarded as a more or less purely serotonergic nucleus formany years. In the mid-seventies, however, additionalneurotransmitters were discovered in the DRN, and over thenext two decades their number grew to more than ten (Fig. 2).Most of the discoveries were made in the rat.

5.1. Dopamine

Dopamine (DA) was one of the first transmitters to bedemonstrated in DRN neurons, first with histofluorescencemethods (Lindvall and Björklund, 1974; Ochi and Shimizu,1978) and later with antibodies against tyrosine hydroxy-lase (TH) and dopamine-β-hydroxylase (DβH) (Nagatsu etal., 1979). These dopaminergic neurons are located prefer-entially in ventromedial parts. They mainly target thenucleus accumbens and lateral septum, and to a lesserextent the medial prefrontal cortex. In addition, very fewfibers project to the caudate-putamen (Stratford andWirtshafter, 1990).

5.2. GABA

GABAergic neurons were first demonstrated in the DRN byradioautographic tracing and GABA-uptake (Belin et al., 1979).The observation was supported by immunohistochemistrywith an antibody against the GABA-synthesizing enzyme γ-aminobutyric acid decarboxylase, or GAD (Mugnaini andOertel, 1985) and the GABA-degrading enzyme GABA-transa-minase, or GABA-T (Nagai et al., 1983). The GABAergic neuronssynapse with serotonergic DRN neurons (Wang et al., 1992).They are markedly smaller than most serotonergic neuronsand fire spikes characterized by short width and highfrequency (Allers and Sharp, 2003).

5.3. Peptide transmitters

Immunohistochemical stainings have shown that the DRNharbors neuropeptide Y (NPY)-containing neurons, most ofwhich are medium-sized, fusiform and bipolar (de Quidt andEmson, 1986). In situ hybridization has demonstrated thepresence of NPY mRNA in the DRN (Pau et al., 1998).

Substance P has been shown to colocalize with serotonin inthe DRN in at least rat (Hökfelt et al., 1978; Chan-Palay et al.,1978), cat (Arvidsson et al., 1994; Lovick and Hunt, 1983) andhuman (Baker et al., 1990, 1991). Substance P also colocalizeswith serotonin in ascending projections, but such fibers havenot been shown to arise from the DRN (Otake, 2005).

Low levels of prepro-galanin mRNA are present in DRNneurons (Cortes et al., 1990), yet galanin itself has beendetected with immunohistochemistry only after colchicinetreatment (Skofitsch and Jacobowitz, 1985). Galanin coloca-lizes with serotonin in the DRN. In fact, it has been reportedthat a large proportion of serotonergic DRN neurons alsocontain galanin (Melander et al., 1986). Galanin is also presentin serotonergic fibers in one of the target areas of the DRN, thecortex (Skofitsch and Jacobowitz, 1985), but it has not beenconfirmed that these projections arise in the DRN.

Enkephalin (ENK)-containing neurons were first reported inthe dorsal and lateral parts of rat DRN, just adjacent to theperiventricular grey matter (Uhl et al., 1979; Hökfelt et al.,1977). Immunohistochemical studies showed that ENK ispresent throughout the cat DRN in neurons of variablemorphology (Moss et al., 1980, 1981). However, serotonergicdouble labeled neurons were predominantly small and roundand located at the midline, dorsal to the medial longitudinalfasciculus (Glazer et al., 1981).

Corticotropin-releasing factor (CRF) immunoreactivity hasbeen demonstrated in DRN neurons after colchicine-treat-ment (Commons et al., 2003). CRF-immunoreactive neuronswere mainly clustered in the dorsomedial subregion, espe-cially in the middle DRN. Scattered neurons were seen in thelateral wings, while they were largely absent from theventromedial DRN and the most caudal pole of the DRN.Most (∼96%) of CRF-immunoreactive neurons in the dorsome-dial DRN were serotonergic, as defined by immunoreactivityfor tryptophan hydroxylase. Anterograde tracing (PHA-L)indicated that neurons in the middle portion of the dorsome-dial DRN mainly target the central nucleus of the amygdala,the dorsal hypothalamic area and the bed nucleus of the striaterminalis (Commons et al., 2003).

Fig. 2 – Two of the DRN neurotransmitters, serotonin (a) and dopamine (b), visualized with DAB-immunohistochemistry incoronal rat DRN sections. Details of a and b are seen in c and d, respectively.

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Additional neuropeptides demonstrated in neurons of theDRN are vasoactive intestinal polypeptide (VIP) (Sims et al.,1980) and cholecystokinin (CKK) (Bhatnagar et al., 2000; Otake,2005).

5.4. Glutamate

Phosphate-activated glutaminase (PAG) has been demon-strated in TH-DβH- or phenylethanolamine-N-methyltrans-ferase (PNMT)-immunoreactive neurons, suggesting thatglutamate is formed from glutamine in serotonergic andcatecholaminergic neurons of the DRN (Kaneko et al., 1990).

5.5. Nitric oxide

The presence of nitric oxide (NO) in DRN was first demon-strated by immunohistochemistry against the NO synthesisreaction product citrulline (Pasqualotto et al., 1991) andagainst argininosuccinate synthetase which turns citrulline

into argininosuccinate (Nakamura et al., 1991). Subsequently,the presence of NO in both serotonergic and non-serotonergicDRN neurons was demonstrated by colocalization of seroto-nin-immunoreactivity with activity of a NO synthesizingenzyme (Johnson and Ma, 1993; Wotherspoon et al., 1994;Rodrigo et al., 1994; Dun et al., 1994). The NOS neurons arepredominantly clustered in medioventral and mediodorsalparts of DRN (Wang et al., 1995). In the medial subnuclei,between 23 and 38% of serotonergic neurons appear tosynthesize NO, whereas 60–77% of the NADPH diaphorase-containing neurons are serotonergic. In the lateral subregions,serotonin and NADPH diaphorase activity is present, but itsactivity does not overlap with serotonergic neurons (Wother-spoon et al., 1994).

5.6. Transient presence of additional transmitters

At least two additional neurotransmitters have been reportedin the developing, but not adult, DRN. Histamine is present in

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neurons of rat and mouse DRN during embryonic develop-ment, but disappears before birth, as demonstrated by thepresence of histamine immunoreactivity and histidine dec-arboxylase (the histamine-synthesizing enzyme) mRNA (Nis-sinen and Panula, 1995; Nissinen et al., 1995; Auvinen andPanula, 1988; Karlstedt et al., 2001). Recent studies have shownthat the gastro-intestinal peptide secretin is also present in theDRN duringmouse embryonic development (Lossi et al., 2004).

6. DRN morphology

Over the past decades, several newmethodologies have led tonew discoveries about the morphology of the DRN and itsprojections.

The DRN is a bilateral, heterogenous brainstem nucleus,located in the ventral part of the periaqueductal gray matterof the midbrain. Its rostral end is at the level of theoculomotor nucleus and its caudal subdivision reaches wellinto the periventricular gray matter of the rostral pons. Ithas been estimated that the human DRN contains onaverage approximately 235.000 neurons (Baker et al., 1990),of which on average approximately 165,000 (or 70%) containthe nucleus' major neurotransmitter serotonin (Baker et al.,1991).

Together with the caudal linear and median raphenucleus, the DRN forms the rostral, or superior, division ofthe raphe complex. The caudal, or inferior, division encom-passes the raphe obscurus, raphe pallidus and raphe magnusnuclei and parts of the lateral reticular formation, located inthe medulla and caudal pons (Steinbusch, 1981; Jacobs andAzmitia, 1992).

According to the original nomenclature by Dahlström andFuxe (1964), the rat raphe nuclei (including the brainstemreticular formation) are divided into nine subdivisions, B1–B9.The subdivisions were later renamed and slightly redefinedwhen the nuclei were re-examined using an antibodyagainst serotonin, and what is now considered as the DRNcorresponds to the original subdivisions B6–B7, B6 being thecaudal extension. In most species, the DRN can be dividedinto five subregions, namely the interfascicular, ventral (orventromedial), ventrolateral (or lateral), dorsal and caudalsubregions (see Baker et al., 1990). The DRN is also oftendivided along the rostrocaudal axis into a rostral, middle andcaudal portion. All the five subregions extend from therostral to the middle part of the nucleus, except for thecaudal subregion, which is located in the caudal portion ofthe DRN. The boundaries have not been precisely defined,which has made it difficult to make accurate comparisonsbetween publications. However, Abrams et al. (2004) recentlyproposed detailed stereotaxic coordinates to be used forsubsequent work. Accordingly, the rat and mouse DRN weredivided in three equally long parts along the rostrocaudalaxis, and labeled rostral, middle and caudal. For bothspecies, values are based on stereotaxic atlases (Paxinosand Watson, 1997; Paxinos and Franklin, 2001) and immu-nostainings with tryptophan hydroxylase (Abrams et al.,2004). These coordinates deal with the rostrocaudal axisonly, but division into the five subregions is fairly easy tomake on a morphological basis.

6.1. Cell types

The fourmainmorphologically different types of DRNneuronsare differentially distributed within the DRN, which seems toreflect neurochemical and functional specialization. Indeed,an increasing number of studies have supported this notion.Electrophysiological studies in the eighties led to a division ofrat serotonergic DRN neurons into two types, which werenamed Type I and Type II (or typical and atypical serotonergicneurons, respectively). Type I neurons exhibited a rhythmicfiring-pattern and were called clock-like neurons, whereasType II neurons fired irregularly and were called non-clock-like (Nakahama et al., 1981). More recently, each type wasdivided into three distinct classes based on firing patternsduring the sleep-wake cycle as measured by single-unitrecordings in cats. In addition, non-serotonergic DRN neuronswere divided into three groups as well (Sakai and Crochet,2001).

6.2. Efferent projections of the DRN

Serotonergic neurons of the DRN display a topographicorganization along the rostrocaudal axis, with respect toefferent projections (see Abrams et al., 2004). Thus, neuronslocatedmore rostrally project tomore rostral areas of the brainthan neurons located more caudally in the DRN.

Yet, individual neurons seem to project to several distinctbut functionally related targets through branched fibers (seeLowry, 2002). The first branched projections to be discoveredrun from the dorsal DRN along the dorsal raphe cortical tractto the substantia nigra and caudate-putamen (van der Kooyand Hattori, 1980a; Imai et al., 1986). Also, single neurons havebeen observed to target hippocampus and entorhinal cortex(Kohler and Steinbusch, 1982), prefrontal cortex and nucleusaccumbens (Van Bockstaele et al., 1993), the paraventricularnucleus of the thalamus (PVN) and the lateral parabrachialnucleus (PBN) (Petrov et al., 1992), the central nucleus of theamygdala (CeA) and the PVN (Petrov et al., 1994), distinct sitesin the trigeminal somatosensory pathway (Kirifides et al.,2001) and the vestibular nuclei and CeA (Halberstadt andBalaban, 2006).

This could be a key to understanding the role of the DRN asamodulator of complex autonomic functions with anatomicalcorrelates in several parts of the brain. For instance, both theCeA and the PVN, which are targeted by the same branchedfibers, are involved in anxiety and conditioned fear (Petrov etal., 1992, 1994). These fibers emerge from well-definedsubpopulations of neurons in the medial part of the middleDRN as well as more caudal clusters.

However, only a part of the neurons with branchedaxons contain serotonin, the reported range being between8% (Petrov et al., 1992) and 64% (Halberstadt and Balaban,2006) depending on the targets. This serves as a reminderthat serotonin is not the only transmitter utilized by theDRN. For example, the CeA-PVN projecting subpopulationsmentioned above (where about half the neurons areserotonergic) also contain corticotropin-releasing factor(CRF), which has been associated with anxiety and stress-related behavior. Anxiety-related behavioral changesinduced by serotonergic activity, such as development of

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learned helplessness, seem to be CRF-dependent (see Maierand Watkins, 2005). However, it has not been shown,whether the CRF-containing neurons themselves, or theserotonergic DRN neurons they target, send collaterals toCeA and PVN.

Early studies showed that most DRN neurons projectipsilaterally, and few contralaterally (Miller et al., 1975).Retrograde labeling studies of DRN efferents to the entorhinalcortex indicated that, when present, contralateral terminalsare preferentially located close to the midline (Kohler andSteinbusch, 1982). Similar results were obtained recently in astudy by Waselus and co-workers, in which all DRN neurons,which sent collaterals to lateral septum and striatum, werelocated ventromedially near the midline or slightly lateral toit. Notably, all such collateral neurons were serotonergic(Waselus et al., 2006). However, single neurons do not seemto project collaterally to both hemispheres (van der Kooy andHattori, 1980b; Kohler and Steinbusch, 1982). Besides of theirtopographic organization, different cell types also seem todisplay different projections. This has, however, not beenextensively studied but is reflected in the distribution patternsof different cell types vs. the projections emerging fromdifferent areas.

6.3. Fiber morphology

Fibers arising from the DRN are characteristically very fine andhave small varicosities, which are granular or fusiform inshape (so-called type D axons). This is in contrast to fibersarising from the MRN, which display large, spherical varicos-ities (so-called type M axons) and variations in fiber thickness(Kosofsky and Molliver, 1987). Serotonin-immunoreactivefibers display similar variation, which may help to give anindication of the origin of serotonergic fibers in purelyimmunocytochemical preparations (Kosofsky and Molliver,1987; Mulligan and Tork, 1988). Whereas the thinner DRNfibers branch frequently and target large, often diffuse areas,the thicker fibers branch infrequently, and are often seen tosurround the somata of single neurons (Mulligan and Tork,1988). On an electronmicroscopic level, the DRN fibers displaysmall, fusiform boutons and are believed to signal predomi-nantly via volume transmission, whereas the MRN fibers

Fig. 3 – The three ascending pathways (AP:s) of the ra

contact their target via large round boutons, often in largenumbers (see Tork, 1990). The morphology and origin of thefibers have also been linked to differential drug-sensitivity,first demonstrated in the forebrain, where the neurotoxicamphetamine derivatives methylenedioxyamphetamine(MDA) and p-chloroamphetamine (PCA) induce denervationof the fine axons, whereas the thick ones are unaffected by thedrugs (Mamounas and Molliver, 1988; O'Hearn et al., 1988;Mamounas et al., 1991). A suggested reason for this differenceis SERT expression, which, in amygdala, is present in the thickMRdrug-insensitive fibers but lacking from the thin DRNdrug-sensitive ones (Brown and Molliver, 2000).

Thus, functionally the serotonergic fibers seem to beorganized into two main subsystems, of which the DRNsystem has a more widespread influence via its highlydivergent branches and volume transmission, while theMRN system has extensive and direct synaptic contacts withneuronal somata.

6.4. Pathway overview

The DRN projects along several ascending and descendingpathways, most of which it shares with one or more of theother raphe nuclei. With regard to the focus of this article, theascending pathways, which target forebrain areas, are ofparticular interest. There are three ascending pathways: thedorsal, medial and ventral ascending pathways (Fig. 3). Thedorsal and ventral ascending pathways are the two mostimportant efferent projections of the DRN. They reach amultitude of targets throughout the forebrain, the mostimportant one being the caudate-putamen. In addition, fourdescending projections leave the DRN: the bulbospinal path-way, cerebellar pathway, propriobulbar pathway and one thatinnervates the locus coeruleus, dorsal tegmental nucleus andpontine raphe nucleus. The main targets of the descendingpathways are cerebellum, the lower brainstem and the spinalchord.

6.5. Dorsal ascending pathway

The dorsal ascending pathway rises from the medial androstral DRN and innervates the striatum and globus

t DRN and their main targets. See text for details.

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pallidus (GP). The striatum is the most important target forDRN innervation and one of the first to be extensivelystudied. The earliest anatomical indications for DRNprojections to caudate-putamen (CP) of the dorsal striatum(Anden et al., 1965) were subsequently supported by lesionstudies, which showed a drop in striatal tryptophanhydroxylase (TPH) activity (Geyer et al., 1976) as well asa decrease in [3H]5-HT uptake (Kellar et al., 1977) afterDRN lesions. Approximately a third of all serotonergic DRNneurons project to the CP. This is, however, region-spe-cific: in a cluster in the dorsomedial DRN, 80–90% of seroto-nergic neurons were found to project to the CP (Steinbuschet al., 1981). Twenty percent of DRN neurons that projectto the CP are non-serotonergic. Nucleus accumbens of theventral striatum, and particularly its core, are alsoextensively innervated by DRN fibers (Van Bockstaele andPickel, 1993; Brown and Molliver, 2000). DRN efferentstarget the striatum at caudal to midlevels (Vertes, 1991).Approximately half of the neurons are located in therostral third of the DRN, fewer in the middle third andvery few in the caudal third (Steinbusch et al., 1981;Waselus et al., 2006).

Pallidal afferents from the DRN have been demonstratedby tracing studies (Vertes, 1991; DeVito et al., 1980). Theinnervation of the GP is mainly serotonergic, as confirmed

Fig. 4 – Coronal sections through rat hypothalamus processed fodiffuse innervation pattern of serotonergic fibers (a) projecting acontaining tyrosine hydroxylase; TH (b), noradrenalin; NA (c) andnucleus, of the upper row images.

by micro-dialysis studies in the rat (McQuade and Sharp,1997).

6.6. Medial ascending pathway

The main target of the medial ascending pathway is thesubstantia nigra (SN). The projections seem to arise from therostral DRN (Imai et al., 1986) and they target the parscompacta division in particular (Fibiger and Miller, 1977;Bobillier et al., 1976). However, a study using the retrogradetracer PHA-L failed to demonstrate DRN innervation of thepars reticulata (Vertes, 1991). To a lesser extent, the pathwayalso innervates the CP. Some of the fibers branch, and targetboth the SN and CP (van der Kooy and Hattori, 1980a; Imai etal., 1986). Thus, single DRN neurons exert control over boththe SN and the CP.

6.7. Ventral ascending pathway

Via the ventral ascending pathway, the DRN innervates manyareas. The bilateral pathway ascends ventrolaterally and thenturns rostrally to enter the medial forebrain bundle. Thepathway also contains fibers from other raphe nuclei,especially median raphe. The main targets are thalamic andhypothalamic nuclei, habenula, septum, amygdala, cortex, the

r DAB-immunohistochemistry illustrate the widespread,long the ventral ascending pathway, as compared to fibersdopamine; DA (d). Images e–h show a detail, paraventricular

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olfactory bulb, hippocampus, interpeduncular nucleus andgeniculate body (Fig. 4).

6.7.1. HypothalamusVarying degrees of DRN innervation have been observed inmost hypothalamic nuclei (Vertes, 1991; van de Kar andLorens, 1979; Bobillier et al., 1976; Yoshida et al., 2006). Thisincludes many of the transmitters-specific hypothalamicneuronal systems: for instance, one out of four to five of theorexinergic neurons of the lateral hypothalamus is innervatedby neurons in the central portion of the rostral DRN (Yoshidaet al., 2006). The preoptic area and anterior hypothalamicareas including the suprachiasmatic nuclei do not seem to beinnervated (Bobillier et al., 1976; van de Kar and Lorens, 1979;Meyer-Bernstein and Morin, 1996).

6.7.2. ThalamusSeveral of the thalamic nuclei receive moderate to denseinnervation from the DRN (Bobillier et al., 1976; Vertes, 1991;Conrad et al., 1974).

6.7.3. HabenulaTheDRN innervates the lateral habenula to amoderate extent,whereas themedial habenula does not seem to receive little orno innervation (Sim and Joseph, 1993; Bobillier et al., 1976;Morin and Meyer-Bernstein, 1999). Input from DRN is mainlynon-serotonergic.

6.7.4. SeptumThe DRN sends strong innervation to the lateral septum, 80%of which is serotonergic. The innervation predominantlytargets the medial portions of the lateral septum (Waselus etal., 2006; Vertes, 1991; Kohler et al., 1982). The medial septumis not generally considered a target of DRN innervation,although micro-dialysis studies have shown that stimulationof the DRN can increase serotonin dialysate in the medialseptum by more than 55% (McQuade and Sharp, 1997).

6.7.5. Amygdaloid complexStudies using neuronal tracers, PHA-L in particular, havedemonstrated that the basolateral and lateral amygdaloidnuclei, as well as the extended amygdala receive denseinnervation from the DRN (Grove, 1988; Vertes, 1991). Also,immunohistochemical techniques in rat have shown that thebasolateral amygdaloid nuclei receive strong serotonergicinnervation. In the centromedial nuclei innervation is verylow, except for the posterior part of the medial amygdaloidnucleus and in the medial and lateral parts of the posteriornucleus (Steinbusch, 1981). The serotonin-immunoreactivityhas not been directly correlated to DRN efferents, but thefiber morphology in squirrel monkeys suggests that seroto-nergic innervation emerges predominantly from the DRN(Sadikot and Parent, 1990). In macaque monkeys, the relativefiber density in amygdaloid subnuclei does not seem tocorrespond to the rat data, probably due to species differ-ences. The highest levels were present in lateral subregionsof the central amygdala and the dorsolateral bed nucleus ofthe stria terminalis. Levels were high in basal amygdala andmoderate in centromedial amygdaloid nuclei (Freedman andShi, 2001).

6.7.6. CortexSeveral studies have dealt with cortical projections of the DRN(see e.g. Bobillier et al., 1976; O'Hearn and Molliver, 1984;Vertes, 1991). Anterograde labelingwith PHA-L has shown thatmany cortical regions receive dense (the piriform, insular andfrontal cortices), or moderately dense (occipital, entorhinal,perirhinal, frontal orbital, anterior cingulate and infralimbiccortices) projections from the DRN (Vertes, 1991). The densityis highest in the dorsal frontal cortex and low in caudalregions, with intermediate densities in areas in between(Steinbusch, 1981). The frontal cortex receives projectionsfrom nearly twice as many DRN neurons as either the parietalor occipital cortex (O'Hearn and Molliver, 1984). The corticalprojections of the rat DRN emerge predominantly from theventral subnucleus, in particular from immediately dorsal ormedial to the medial longitudinal fasciculi. These areasaccount for three fourths of the DRN innervation of the cortex,whereas the dorsal subnucleus contributes one fourth. Alongthe rostro-caudal axis, most neurons are located in themiddleDRN, and the lateral areas of the DRN do not seem to project tothe cerebral cortex at all. More than 80% of the projections areserotonergic (O'Hearn and Molliver, 1984). The ratio ofcontralateral fibers is 26–35%, and different between thesubnuclei. At least in the entorhinal cortex, the contralateralfibers seem to preferentially target medial areas (Kohler andSteinbusch, 1982; O'Hearn and Molliver, 1984).

6.7.7. HippocampusThe DRN projects to the hippocampus (Segal and Landis, 1974;Azmitia and Segal, 1978;Mamounas et al., 1991). DRN efferentsto the hippocampus emerge predominantly from the mostcaudal parts of the nucleus, close to the midline, and are bothserotonergic and non-serotonergic (Wyss et al., 1979; Kohlerand Steinbusch, 1982).

6.7.8. Olfactory bulbTracing studies with radioactively labeled amino acids in rat(Halaris et al., 1976) and cat (Bobillier et al., 1976) havedemonstrated DRN projections to the olfactory bulb. TheDRN is the primary source of serotonin in the olfactory bulb, asshown by retrograde transport of [3H]serotonin (Araneda et al.,1980a,b). Immunohistochemical stainings have demonstratedserotonergic innervation of all layers of the olfactory bulb,especially the glomerular lamina (Steinbusch, 1981).

6.7.9. Supra-ependymal plexusThe supraependymal plexus is a network of serotonergicfibers, which covers nearly all ventricular surfaces withmoderate or high density (Richards et al., 1973; Lorez andRichards, 1982; Chan-Palay, 1976). Several studies haveindicated that the supraependymal serotonergic fibers ascendfrom the medial and, in particular, dorsal raphe (Aghajanianand Gallager, 1975; Chan-Palay, 1976; Richards, 1978; Stein-busch et al., 1981; Derer, 1981; Pierce et al., 1976).

Studies on the rat lateral ventricles indicate that seroto-nergic fibers do not penetrate the ependyma, but insteadenter the ventricles from their rostral poles. These fiberstravel through the median forebrain bundle and turndorsocaudally between the caudate-putamen and corpuscallosum. Also, they do not form synaptic contacts with

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ependymal cells. They are not found between ependyma andsubependyma, but only in the lateral ventricles (Dinopouloset al., 1995).

7. Functional neuroanatomy of the DRN withemphasis on depression

Major depression is one of the most common psychiatricdiseases. It has an incidence of about 4% and a life-timeprevalence of 12–20% in Europe (Alonso et al., 2004; Paykel etal., 2005) and, thus, a deeper understanding of its mechanismsis of high clinical importance. Dysfunction of the serotonergicsystem has been linked to depression, and although adysfunctional serotonin system alone cannot explain the fullpathophysiology, it is considered a key factor in depressionand othermood disorders: for example, levels of serotonin andits metabolites are decreased and responsivity to serotoninreceptor agonists is reduced in depressed patients.

The first implication of a connection between serotoninand depression was made in the early sixties, when the firstantidepressant, iproniazid, was found to inhibit monoamineoxidase B, which degrades serotonin and other monoamines.Subsequently, the search for drugs, which would selectivelyenhance the transmission of one monoamine only, led to thedevelopment of selective serotonin reuptake inhibitors (SSRI).SSRI enhance serotonergic signaling by inhibiting the reup-take of the transmitter from the synaptic cleft and constitutethe most successful antidepressants today.

As amajor source of serotonergic input to the forebrain, theDRN has naturally received much attention in depressionresearch. Recent evidence includes a post-mortem study,which found a 31% decrease in overall neuron number in theDRN of depressed patients with a mean age of 50 years(Baumann et al., 2002). On the other hand, another studyfound no decrease in DRN neuron number and pathology inelderly people who had suffered from depression (Hendrick-sen et al., 2004). This may reflect differences in the etiologybetween depression among middle-aged and elderly. Inaddition, tryptophan hydroxylase immunoreactivity andmRNA levels in the DRN are higher in depressed suicidevictims than in controls (Boldrini et al., 2005; Bach-Mizrachiet al., 2006).

Given the heterogenicity of the DRN and its large number ofneurotransmitters, depression research has lately expandedits realms to focus on other transmitter systems as well. Alltransmitters of the DRN are found in close proximity ofserotonergic neurons, and several of them have been identi-fied in the same neurons. Thus, they are likely to interact withthe serotonergic system.

For instance, CRF seems to modulate serotonergic neuronactivity via GABA-ergic neurons in the DRN. It has beensuggested that CRF receptors 1 and 2 upregulate and down-regulate, respectively, GABA-ergic neurons, which, in turn,inhibit serotonergic DRN neurons. CRF also acts directly viaCRF2 receptors on serotonergic neurons (Valentino andCommons, 2005). Galanin and galanin-agonists have alsobeen shown to have antidepressant-like effects, probably byupregulating serotonergic transmission via galanin receptorson serotonergic DRN neurons (see Kuteeva et al., 2007;

Karlsson and Holmes, 2006; Lu et al., 2005). Thus, subtype-specific galanin receptor agonists and/or antagonists couldprove to be useful tools in the development of antidepressants(see Ögren et al., 2006). In addition, it has been proposed thatsubstance P (the endogenous ligand for neurokinin 1 receptor)activates glutamatergic input to the serotonergic system. Thenet effect would differ topographically within the DRN,leading to a decrease in serotonergic activity in the ventralDRN and an increase in the dorsal DRN (Valentino andCommons, 2005). Neurokinin receptor antagonists mighthave antidepressant effects, but currently the availableevidence is contradictory (see Keller et al., 2006; Krameret al., 2004).

8. Summary

During the past century starting from Cajal's silver stainings,the DRNhas developed from an object of purelymorphologicalstudies towards being recognized as a complex multifunc-tional and multitransmitter nucleus and an important targetfor depression research. During the next decades, under-standing the interactions between the many transmittersystems of the DRN will be of crucial importance for thedevelopment of new and better treatments for depression. Inaddition, the DRN's involvement in neurogenesis and neuro-degeneration may open up new aspects of its function andinfluence future treatment strategies.

The DRN, situated in the dorsal part of themesencephalon,is a phylogenetically old part of the brain, and modulates orinfluences a wide variety of CNS processes. A hundred yearsafter Cajal's description, it is still a highly interesting brainarea due to its involvement in serious neurological andpsychiatric disease, but also in cognitive, locomotive andanxiety-related functions.

Acknowledgments

K.A.M. is supported by European Union Framework 6 Inte-grated Project NEWMOODGrant LSHM-CT-2004-503474 and bygrants from Helsingin Sanomain 100-vuotissäätiö, AlfredKordelinin yleinen edistys- ja sivistysrahasto, Orionin tutki-mussäätiö and K. Albin Johanssons stiftelse.

R E F E R E N C E S

Abrams, J.K., Johnson, P.L., Hollis, J.H., Lowry, C.A., 2004. Anatomicand functional topography of the dorsal raphe nucleus. Ann.N Y Acad. Sci. 1018, 46–57.

Aghajanian, G.K., Gallager, D.W., 1975. Raphe origin ofserotonergic nerves terminating in the cerebral ventricles.Brain Res. 88, 221–231.

Allers, K.A., Sharp, T., 2003. Neurochemical and anatomicalidentification of fast- and slow-firing neurones in the rat dorsalraphe nucleus using juxtacellular labelling methods in vivo.Neuroscience 122, 193–204.

Alonso, J., Angermeyer, M.C., Bernert, S., Bruffaerts, R., Brugha,T.S., Bryson, H., de Girolamo, G., Graaf, R., Demyttenaere, K.,Gasquet, I., Haro, J.M., Katz, S.J., Kessler, R.C., Kovess, V.,Lepine, J.P., Ormel, J., Polidori, G., Russo, L.J., Vilagut, G.,

339B R A I N R E S E A R C H R E V I E W S 5 5 ( 2 0 0 7 ) 3 2 9 – 3 4 2

Almansa, J., Arbabzadeh-Bouchez, S., Autonell, J., Bernal, M.,Buist-Bouwman, M.A., Codony, M., Domingo-Salvany, A.,Ferrer, M., Joo, S.S., Martinez-Alonso, M., Matschinger, H.,Mazzi, F., Morgan, Z., Morosini, P., Palacin, C., Romera, B., Taub,N., Vollebergh, W.A., 2004. Prevalence of mental disorders inEurope: results from the European Study of the Epidemiologyof Mental Disorders (ESEMeD) project. Acta Psychiatr. Scand.21–27.

Anden, N.E., Dahlström, A., Fuxe, K., Larsson, K., 1965. Mappingout of catecholamine and 5-hydroxytryptamine neuronsinnervating the telencephalon and diencephalon. Life Sci. 4,1275–1279.

Araneda, S., Bobillier, P., Buda, M., Pujol, J.F., 1980a. Retrogradeaxonal transport following injection of [3H]serotonin in theolfactory bulb: I. Biochemical study. Brain Res. 196,405–415.

Araneda, S., Gamrani, H., Font, C., Calas, A., Pujol, J.F., Bobillier, P.,1980b. Retrograde axonal transport following injection of[3H]-serotonin into the olfactory bulb: II. Radioautographicstudy. Brain Res. 196, 417–427.

Arvidsson, U., Cullheim, S., Ulfhake, B., Luppi, P.H., Kitahama, K.,Jouvet, M., Hökfelt, T., 1994. Quantitative and qualitativeaspects on the distribution of 5-HT and its coexistence withsubstance P and TRH in cat ventral medullary neurons.J. Chem. Neuroanat. 7, 3–12.

Auvinen, S., Panula, P., 1988. Development ofhistamine-immunoreactive neurons in the rat brain. J. Comp.Neurol. 276, 289–303.

Azmitia, E.C., Segal, M., 1978. An autoradiographic analysis ofthe differential ascending projections of the dorsal andmedian raphe nuclei in the rat. J. Comp. Neurol. 179,641–667.

Bach-Mizrachi, H., Underwood, M.D., Kassir, S.A., Bakalian, M.J.,Sibille, E., Tamir, H., Mann, J.J., Arango, V., 2006. Neuronaltryptophan hydroxylase mRNA expression in the humandorsal andmedian raphe nuclei: major depression and suicide.Neuropsychopharmacology 31, 814–824.

Baker, K.G., Halliday, G.M., Tork, I., 1990. Cytoarchitecture ofthe human dorsal raphe nucleus. J. Comp. Neurol. 301,147–161.

Baker, K.G., Halliday, G.M., Hornung, J.P., Geffen, L.B., Cotton, R.G.,Tork, I., 1991. Distribution, morphology and number ofmonoamine-synthesizing and substance P-containingneurons in the human dorsal raphe nucleus. Neuroscience 42,757–775.

Baumann, B., Bielau, H., Krell, D., Agelink, M.W., Diekmann, S.,Wurthmann, C., Trubner, K., Bernstein, H.G., Danos, P., Bogerts,B., 2002. Circumscribed numerical deficit of dorsal rapheneurons in mood disorders. Psychol. Med. 32, 93–103.

Belin, M.F., Aguera, M., Tappaz, M., McRae-Degueurce, A., Bobillier,P., Pujol, J.F., 1979. GABA-accumulating neurons in the nucleusraphe dorsalis and periaqueductal gray in the rat: abiochemical and radioautographic study. Brain Res. 170,279–297.

Bhatnagar, S., Viau, V., Chu, A., Soriano, L., Meijer, O.C., Dallman,M.F., 2000. A cholecystokinin-mediated pathway to theparaventricular thalamus is recruited in chronically stressedrats and regulates hypothalamic–pituitary–adrenal function.J. Neurosci. 20, 5564–5573.

Bobillier, P., Seguin, S., Petitjean, F., Salvert, D., Touret, M., Jouvet,M., 1976. The raphe nuclei of the cat brain stem: atopographical atlas of their efferent projections as revealed byautoradiography. Brain Res. 113, 449–486.

Boldrini, M., Underwood, M.D., Mann, J.J., Arango, V., 2005. Moretryptophan hydroxylase in the brainstem dorsal raphe nucleusin depressed suicides. Brain Res. 1041, 19–28.

Braak, H., 1970. Über die Kerngebiete de menschlichenHirnstammes. II Die Raphekerne. Z. Zellforsch. Mikrosk. Anat.107, 123–141.

Brown, P., Molliver, M.E., 2000. Dual serotonin (5-HT) projections tothe nucleus accumbens core and shell: relation of the 5-HTtransporter to amphetamine-induced neurotoxicity.J. Neurosci. 20, 1952–1963.

Calas, A., Alonso, G., Arnauld, E., Vincent, J.D., 1974.Demonstration of indolaminergic fibres in themedia eminenceof the duck, rat and monkey. Nature 250, 241–243.

Chan-Palay, V., 1976. Serotonin axons in the supra- andsubependymal plexuses and in the leptomeninges; their rolesin local alterations of cerebrospinal fluid and vasomotoractivity. Brain Res. 102, 103–130.

Chan-Palay, V., 1977. Indoleamine neurons and their processes inthe normal rat brain and in chronic diet-induced thiaminedeficiency demonstrated by uptake of 3H-serotonin. J. Comp.Neurol. 176, 467–493.

Chan-Palay, V., Jonsson, G., Palay, S.L., 1978. Serotonin andsubstance P coexist in neurons of the rat's central nervoussystem. Proc. Natl. Acad. Sci. U. S. A. 75, 1582–1586.

Commons, K.G., Connolley, K.R., Valentino, R.J., 2003. Aneurochemically distinct dorsal raphe-limbic circuit with apotential role in affective disorders.Neuropsychopharmacology 28, 206–215.

Conrad, L.C., Leonard, C.M., Pfaff, D.W., 1974. Connections of themedian and dorsal raphe nuclei in the rat: an autoradiographicand degeneration study. J. Comp. Neurol. 156, 179–205.

Cortes, R., Ceccatelli, S., Schalling, M., Hökfelt, T., 1990. Differentialeffects of intracerebroventricular colchicine administration onthe expression of mRNAs for neuropeptides andneurotransmitter enzymes, with special emphasis on galanin:an in situ hybridization study. Synapse 6, 369–391.

Dahlström, A., Fuxe, K., 1964. Evidence for the existence ofmonoamine-containing neurons in the central nervoussystem. I. Demonstration of monoamines in the cell bodies ofbrain stem neurons. Acta Physiol. Scand. 62 (SUPPL-55).

de Quidt, M.E., Emson, P.C., 1986. Distribution of neuropeptideY-like immunoreactivity in the rat central nervoussystem—II. Immunohistochemical analysis. Neuroscience18, 545–618.

Derer, P., 1981. The supraependymal fibres (SEF) of the mousebrain. as visualized by the Golgi method. J. Physiol. (Paris) 77,211–218.

DeVito, J.L., Anderson, M.E., Walsh, K.E., 1980. A horseradishperoxidase study of afferent connections of the globus pallidusin Macaca mulatta. Exp. Brain Res. 38, 65–73.

Dinopoulos, A., Dori, I., Parnavelas, J.G., 1995. Serotonergicinnervation of the lateral geniculate nucleus of the rat duringpostnatal development: a light and electron microscopicimmunocytochemical analysis. J. Comp. Neurol. 363, 532–544.

Dun, N.J., Dun, S.L., Forstermann, U., 1994. Nitric oxide synthaseimmunoreactivity in rat pontine medullary neurons. Neu-roscience 59, 429–445.

Erspamer, V., 1940. Pharmacologische Studien über Enteramin. I.Naunyn-Schmiedeberg's Arch. Pharmacol. 196, 343–365.

Erspamer, V., Asero, B., 1952. Identification of enteramine, thespecific hormone of the enterochromaffin cell system, as5-hydroxytryptamine. Nature 169, 800–801.

Falck, B., Hillarp, N.A., Thieme, G., Torp, A., 1962. Fluorescence ofcatecholamines and related compounds with formaldehyde.J. Histochem. Cytochem. 10, 348–354.

Felten, D.L., Cummings, J.P., 1979. The raphe nuclei of the rabbitbrain stem. J. Comp. Neurol. 187, 199–243.

Fibiger, H.C., Miller, J.J., 1977. An anatomical andelectrophysiological investigation of the serotonergicprojection from the dorsal raphe nucleus to the substantianigra in the rat. Neuroscience 2, 975–987.

Freedman, L.J., Shi, C., 2001. Monoaminergic innervation of themacaque extended amygdala. Neuroscience 104, 1067–1084.

Geyer, M.A., Puerto, A., Dawsey, W.J., Knapp, S., Bullard, W.P.,Mandell, A.J., 1976. Histologic and enzymatic studies of the

340 B R A I N R E S E A R C H R E V I E W S 5 5 ( 2 0 0 7 ) 3 2 9 – 3 4 2

mesolimbic and mesostriatal serotonergic pathways. BrainRes. 106, 241–256.

Glazer, E.J., Steinbusch, H., Verhofstad, A., Basbaum, A.I., 1981.Serotonin neurons in nucleus raphe dorsalis andparagigantocellularis of the cat contain enkephalin. J. Physiol.(Paris) 77, 241–245.

Grove, E.A., 1988. Neural associations of the substantiainnominata in the rat: afferent connections. J. Comp. Neurol.277, 315–346.

Halaris, A.E., Jones, B.E., Moore, R.Y., 1976. Axonal transport inserotonin neurons of the midbrain raphe. Brain Res. 107,555–574.

Halberstadt, A.L., Balaban, C.D., 2006. Serotonergic andnonserotonergic neurons in the dorsal raphe nucleus sendcollateralized projections to both the vestibular nuclei and thecentral amygdaloid nucleus. Neuroscience 143, 641–654.

Hendricksen, M., Thomas, A.J., Ferrier, I.N., Ince, P., O'Brien, J.T.,2004. Neuropathological study of the dorsal raphe nuclei inlate-life depression and Alzheimer's disease with and withoutdepression. Am. J. Psychiatry 161, 1096–1102.

Hökfelt, T., Ljungdahl, A., 1972. Modification of the Falck–Hillarpformaldehyde fluorescence method using the Vibratome:simple, rapid and sensitive localization of catecholamines insections of unfixed or formalin fixed brain tissue. Histochemie29, 325–339.

Hökfelt, T., Fuxe, K., Goldstein, M., 1973. Immunohistochemicallocalization of aromatic L-amino acid decarboxylase (DOPAdecarboxylase) in central dopamine and 5-hydroxytryptaminenerve cell bodies of the rat. Brain Res. 53, 175–180.

Hökfelt, T., Ljungdahl, A., Terenius, L., Elde, R., Nilsson, G., 1977.Immunohistochemical analysis of peptide pathways possiblyrelated to pain and analgesia: enkephalin and substance P.Proc. Natl. Acad. Sci. U. S. A. 74, 3081–3085.

Hökfelt, T., Ljungdahl, A., Steinbusch, H., Verhofstad, A., Nilsson,G., Brodin, E., Pernow, B., Goldstein, M., 1978.Immunohistochemical evidence of substance P-likeimmunoreactivity in some 5-hydroxytryptamine-containingneurons in the rat central nervous system. Neuroscience 3,517–538.

Imai, H., Steindler, D.A., Kitai, S.T., 1986. The organization ofdivergent axonal projections from themidbrain raphe nuclei inthe rat. J. Comp. Neurol. 243, 363–380.

Jacobs, B.L., Azmitia, E.C., 1992. Structure and function of the brainserotonin system. Physiol. Rev. 72, 165–229.

Joh, T.H., Shikimi, T., Pickel, V.M., Reis, D.J., 1975. Brain tryptophanhydroxylase: purification of, production of antibodies to, andcellular and ultrastructural localization in serotonergicneurons of rat midbrain. Proc. Natl. Acad. Sci. U. S. A. 72,3575–3579.

Johnson, M.D., Ma, P.M., 1993. Localization of NADPH diaphoraseactivity in monoaminergic neurons of the rat brain. J. Comp.Neurol. 332, 391–406.

Kaneko, T., Akiyama, H., Nagatsu, I., Mizuno, N., 1990.Immunohistochemical demonstration of glutaminase incatecholaminergic and serotoninergic neurons of rat brain.Brain Res. 507, 151–154.

Karlsson, R.M., Holmes, A., 2006. Galanin as amodulator of anxietyand depression and a therapeutic target for affective disease.Amino Acids 31, 231–239.

Karlstedt, K., Nissinen, M., Michelsen, K.A., Panula, P., 2001.Multiple sites of L-histidine decarboxylase expression inmousesuggest novel developmental functions for histamine. Dev.Dyn. 221, 81–91.

Kellar, K.J., Brown, P.A., Madrid, J., Bernstein, M.,Vernikos-Danellis, J., Mehler, W.R., 1977. Origins of serotonininnervation of forebrain structures. Exp. Neurol. 56, 52–62.

Keller, M., Montgomery, S., Ball, W., Morrison, M., Snavely, D., Liu,G., Hargreaves, R., Hietala, J., Lines, C., Beebe, K., Reines, S.,2006. Lack of efficacy of the substance p (neurokinin1 receptor)

antagonist aprepitant in the treatment of major depressivedisorder. Biol. Psychiatry 59, 216–223.

Kirifides, M.L., Simpson, K.L., Lin, R.C., Waterhouse, B.D., 2001.Topographic organization and neurochemical identity ofdorsal raphe neurons that project to the trigeminalsomatosensory pathway in the rat. J. Comp. Neurol. 435,325–340.

Koelle, G.B., Friedenwald, J.S., 1949. A histochemical method forlocalizing cholinesterase activity. Proc. Soc. Exp. Biol. Med. 70,617–622.

Kohler, C., Steinbusch, H., 1982. Identification of serotonin andnon-serotonin-containing neurons of the mid-brain rapheprojecting to the entorhinal area and the hippocampalformation. A combined immunohistochemical and fluorescentretrograde tracing study in the rat brain. Neuroscience 7,951–975.

Kohler, C., Chan-Palay, V., Steinbusch, H., 1982. The distributionand origin of serotonin-containing fibers in the septal area: acombined immunohistochemical and fluorescent retrogradetracing study in the rat. J. Comp. Neurol. 209, 91–111.

Kosofsky, B.E., Molliver, M.E., 1987. The serotoninergic innerva-tion of cerebral cortex: different classes of axon terminalsarise from dorsal and median raphe nuclei. Synapse 1,153–168.

Kosterlitz, H., Robinson, J., 1957. Inhibition of the peristaltic reflexof the isolated guinea-pig ileum. J. Physiol. 136, 249–262.

Kramer, M.S., Winokur, A., Kelsey, J., Preskorn, S.H., Rothschild, A.J., Snavely, D., Ghosh, K., Ball, W.A., Reines, S.A., Munjack, D.,Apter, J.T., Cunningham, L., Kling, M., Bari, M., Getson, A., Lee,Y., 2004. Demonstration of the efficacy and safety of a novelsubstance P (NK1) receptor antagonist in major depression.Neuropsychopharmacology 29, 385–392.

Kristensson, K., Olsson, Y., 1971. Uptake and retrograde axonaltransport of peroxidase in hypoglossal neurons. Electronmicroscopical localization in the neuronal perikaryon. ActaNeuropathol. (Berl) 19, 1–9.

Kuteeva, E., Wardi, T., Hökfelt, T., Ögren, S.O., 2007. Galaninenhances and a galanin antagonist attenuates depression-likebehaviour in the rat. Eur. Neuropsychopharmacol. 17, 64–69.

LaVail, J.H., LaVail, M.M., 1972. Retrograde axonal transport in thecentral nervous system. Science 176, 1416–1417.

LaVail, J.H., Winston, K.R., Tish, A., 1973. A method based onretrograde intraaxonal transport of protein for identification ofcell bodies of origin of axons terminating within the CNS. BrainRes. 58, 470–477.

Die Beeinflüssung der Darmmotilität durch Hydroxytryptamin.Pflugers Arch. 265, 567–574.

Lewis, P.R., Shute, C., 1959. Selective staining of visceral efferentsin the rat brain stem by a modified Koelle technique. Nature183, 1743–1744.

Lindvall, O., Björklund, A., 1974. The organization of the ascendingcatecholamine neuron systems in the rat brain as revealed bythe glyoxylic acid fluorescence method. Acta Physiol. Scand.,Suppl. 412, 1–48.

Lorez, H.P., Richards, J.G., 1982. Supra-ependymal serotoninergicnerves in mammalian brain: morphological, pharmacologicaland functional studies. Brain Res. Bull. 9, 727–741.

Lossi, L., Bottarelli, L., Candusso, M.E., Leiter, A.B., Rindi, G.,Merighi, A., 2004. Transient expression of secretin inserotoninergic neurons of mouse brain during development.Eur. J. Neurosci. 20, 3259–3269.

Lovick, T.A., Hunt, S.P., 1983. Substance P-immunoreactive andserotonin-containing neurones in the ventral brainstem of thecat. Neurosci. Lett. 36, 223–228.

Lowry, C.A., 2002. Functional subsets of serotonergic neurones:implications for control of the hypothalamic–pituitary–adrenalaxis. J. Neuroendocrinol. 14, 911–923.

Lu, X., Barr, A.M., Kinney, J.W., Sanna, P., Conti, B., Behrens, M.M.,Bartfai, T., 2005. A role for galanin in antidepressant actions

341B R A I N R E S E A R C H R E V I E W S 5 5 ( 2 0 0 7 ) 3 2 9 – 3 4 2

with a focus on the dorsal raphe nucleus. Proc. Natl. Acad. Sci.U. S. A. 102, 874–879.

Maier, S.F., Watkins, L.R., 2005. Stressor controllability and learnedhelplessness: the roles of the dorsal raphe nucleus, serotonin,and corticotropin-releasing factor. Neurosci. Biobehav. Rev. 29,829–841.

Mamounas, L.A., Molliver, M.E., 1988. Evidence for dual serotonergicprojections to neocortex: axons from the dorsal and medianraphe nuclei are differentially vulnerable to the neurotoxinp-chloroamphetamine (PCA). Exp. Neurol. 102, 23–36.

Mamounas, L.A., Mullen, C.A., O'Hearn, E., Molliver, M.E., 1991.Dual serotoninergic projections to forebrain in the rat:morphologically distinct 5-HT axon terminals exhibitdifferential vulnerability to neurotoxic amphetaminederivatives. J. Comp. Neurol. 314, 558–586.

McQuade, R., Sharp, T., 1997. Functional mapping of dorsal andmedian raphe 5-hydroxytryptamine pathways in forebrain ofthe rat using microdialysis. J. Neurochem. 69, 791–796.

Melander, T., Hökfelt, T., Rokaeus, A., Cuello, A.C., Oertel, W.H.,Verhofstad, A., Goldstein, M., 1986. Coexistence of galanin-likeimmunoreactivity with catecholamines,5-hydroxytryptamine, GABA and neuropeptides in the rat CNS.J. Neurosci. 6, 3640–3654.

Meyer-Bernstein, E.L., Morin, L.P., 1996. Differential serotonergicinnervation of the suprachiasmatic nucleus and theintergeniculate leaflet and its role in circadian rhythmmodulation. J. Neurosci. 16, 2097–2111.

Miller, J.J., Richardson, T.L., Fibiger, H.C., McLennan, H., 1975.Anatomical and electrophysiological identification of aprojection from the mesencephalic raphe to thecaudate-putamen in the rat. Brain Res. 97, 133–136.

Morin, L.P., Meyer-Bernstein, E.L., 1999. The ascendingserotonergic system in the hamster: comparison withprojections of the dorsal and median raphe nuclei.Neuroscience 91, 81–105.

Moss, M.S., Glazer, E.J., Basbaum, A.I., 1980. Enkephalin neurons inthe raphe dorsalis and periaqueductal grey of the cat: acomparison with substance P. Anat. Rec. 196.

Moss, M.S., Glazer, E.J., Basbaum, A.I., 1981.Enkephalin-immunoreactive perikarya in the cat raphedorsalis. Neurosci. Lett. 21, 33–37.

Mugnaini, E., Oertel, W.H., 1985. An atlas of the distribution ofGABAergic neurons and terminals in the rat CNS as revealed byGAD immunohistochemistry. In: Björklund, A., Hökfelt, T.(Eds.), Handbook of Chemical Neuroanatomy, vol. 4. GABA andNeuropeptides in the CNS. Part I. Elsevier Science Publishers,B.V., Amsterdam, pp. 436–608.

Mulligan, K.A., Tork, I., 1988. Serotoninergic innervation of the catcerebral cortex. J. Comp. Neurol. 270, 86–110.

Nagai, T., McGeer, P.L., McGeer, E.G., 1983. Distribution ofGABA-T-intensive neurons in the rat forebrain and midbrain.J. Comp. Neurol. 218, 220–238.

Nagatsu, I., Inagaki, S., Kondo, Y., Karasawa, N., Nagatsu, T.,1979. Immunofluorescent studies on the localization oftyrosine hydroxylase and dopamine-â-hydroxylase in themes-, di-, and telencephalon of the rat using unperfusedfresh frozen sections. Acta Histochem. Cytochem. 12,20–37.

Nakahama, H., Shima, K., Yamamoto, M., Aya, K., 1981. Regularityof the spontaneous discharge of neurons in the nucleus raphedorsalis of the cat. Neurosci. Lett. 23, 161–165.

Nakamura, H., Saheki, T., Ichiki, H., Nakata, K., Nakagawa, S., 1991.Immunocytochemical localization of argininosuccinatesynthetase in the rat brain. J. Comp. Neurol. 312, 652–679.

Nissinen, M.J., Panula, P., 1995. Developmental patterns ofhistamine-like immunoreactivity in the mouse. J. Histochem.Cytochem. 43, 211–227.

Nissinen, M.J., Karlstedt, K., Castren, E., Panula, P., 1995.Expression of histidine decarboxylase and cellular

histamine-like immunoreactivity in rat embryogenesis.J. Histochem. Cytochem. 43, 1241–1252.

Ochi, J., Shimizu, K., 1978. Occurence of dopamine-containingneurons in the midbrain raphe nuclei of the rat. Neurosci. Lett.8, 317–320.

Ögren, S.O., Kuteeva, E., Hökfelt, T., Kehr, J., 2006. Galanin receptorantagonists: a potential novel pharmacological treatment formood disorders. CNS Drugs 20, 633–654.

O'Hearn, E., Molliver, M.E., 1984. Organization of raphe-corticalprojections in rat: a quantitative retrograde study. Brain Res.Bull. 13, 709–726.

O'Hearn, E., Battaglia, G., De Souza, E.B., Kuhar, M.J., Molliver, M.E.,1988. Methylenedioxyamphetamine (MDA) andmethylenedioxymethamphetamine (MDMA) cause selectiveablation of serotonergic axon terminals in forebrain:immunocytochemical evidence for neurotoxicity. J. Neurosci.8, 2788–2803.

Otake, K., 2005. Cholecystokinin and substance P immunoreactiveprojections to the paraventricular thalamic nucleus in the rat.Neurosci. Res. 51, 383–394.

Pasqualotto, B.A., Hope, B.T., Vincent, S.R., 1991. Citrulline in therat brain: immunohistochemistry and coexistence withNADPH-diaphorase. Neurosci. Lett. 128, 155–160.

Pau, K.Y., Yu, J.H., Lee, C.J., Spies, H.G., 1998. Topographiclocalization of neuropeptide Y mRNA in the monkeybrainstem. Regul. Pept. 75–76, 145–153.

Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in StereotaxicCoordinates. Academic Press, San Diego, CA.

Paxinos, G., Watson, C., 1997. The Rat Brain in StereotaxicCoordinates. Academic Press, San Diego, CA.

Paykel, E.S., Brugha, T., Fryers, T., 2005. Size and burden ofdepressive disorders in Europe. Eur. Neuropsychopharmacol.15, 411–423.

Petrov, T., Krukoff, T.L., Jhamandas, J.H., 1992. The hypothalamicparaventricular and lateral parabrachial nuclei receivecollaterals from raphe nucleus neurons: a combined doubleretrograde and immunocytochemical study. J. Comp. Neurol.318, 18–26.

Petrov, T., Krukoff, T.L., Jhamandas, J.H., 1994. Chemically definedcollateral projections from the pons to the central nucleus ofthe amygdala and hypothalamic paraventricular nucleus inthe rat. Cell Tissue Res. 277, 289–295.

Pierce, E.T., Foote,W.E., Hobson, J.A., 1976. The efferent connectionof the nucleus raphe dorsalis. Brain Res. 107, 137–144.

Ramón Cajal, S., 1904. Textura del Sistema Nervioso del Hombre ylos Vertebrados. Madrid.

Ramón Cajal, S., 2000. In: Pasik, P., Pasik, T. (Eds.), Texture of theNervous System of Man and the Vertebrates. An Annotatedand Edited Translation. Springer-Verlag, Vienna.

Rand, M., Reid, G., 1951. Source of ‘serotonin’ in serum. Nature 168,385.

Rapport, M.M., 1949. Serum vasoconstrictor (serotonin). V. Thepresence of creatinine in the complex. A proposedstructure of the vasoconstrictor principle. J. Biol. Chem.180, 961–969.

Reid, G., Rand, M., 1952. Pharmacological actions of synthetic5-hydroxytryptamine (serotonin, thrombocytin). Nature 169,801–802.

Richards, J.G., 1978. Cytochemistry and autoradiography in thesearch for transmitter-specific neuronal pathways. In:Coupland, R.E. (Ed.), Peripheral Neuroendocrine Interaction.Springer, Heidelberg, pp. 1–14.

Richards, J.G., Lorez, H.P., Tranzer, J.P., 1973. Indolealkylaminenerve terminals in cerebral ventricles: identification byelectron microscopy and fluorescence histochemistry. BrainRes. 57, 277–288.

Richards, J.G., Da Prada, M., Wursch, J., Lorez, H.P., Pieri, L., 1979.Mapping monoaminergic neurons with [3H]reserpine byautoradiography. Neuroscience 4, 937–950.

342 B R A I N R E S E A R C H R E V I E W S 5 5 ( 2 0 0 7 ) 3 2 9 – 3 4 2

Rodrigo, J., Springall, D.R., Uttenthal, O., Bentura, M.L.,Abadia-Molina, F., Riveros-Moreno, V., Martinez-Murillo, R.,Polak, J.M., Moncada, S., 1994. Localization of nitric oxidesynthase in the adult rat brain. Philos. Trans. R. Soc. Lond., BBiol. Sci. 345, 175–221.

Sadikot, A.F., Parent, A., 1990. The monoaminergic innervation ofthe amygdala in the squirrel monkey: animmunohistochemical study. Neuroscience 36, 431–447.

Sakai, K., Crochet, S., 2001. Differentiation of presumedserotonergic dorsal raphe neurons in relation to behavior andwake-sleep states. Neuroscience 104, 1141–1155.

Segal, M., Landis, S., 1974. Afferents to the hippocampus of the ratstudied with themethod of retrograde transport of horseradishperoxidase. Brain Res. 78, 1–15.

Sim, L.J., Joseph, S.A., 1993. Dorsal raphe nucleus efferents:termination in peptidergic fields. Peptides 14, 75–83.

Sims, K.B., Hoffman, D.L., Said, S.I., Zimmerman, E.A., 1980.Vasoactive intestinal polypeptide (VIP) in mouse and rat brain:an immunocytochemical study. Brain Res. 186, 165–183.

Skofitsch, G., Jacobowitz, D.M., 1985. Immunohistochemicalmapping of galanin-like neurons in the rat central nervoussystem. Peptides 6, 509–546.

Steinbusch, H.W., 1981. Distribution ofserotonin-immunoreactivity in the central nervous system ofthe rat-cell bodies and terminals. Neuroscience 6, 557–618.

Steinbusch, H.W., 1984. Serotonin-immunoreactive neurons andtheir projections in the CNS. In: Björklund, A., Hökfelt, T. (Eds.),Handbook of Chemical Neuroanatomy, Vol. 3. Elsevier, NewYork, pp. 68–125.

Steinbusch, H.W., Verhofstad, A.A., Joosten, H.W., 1978.Localization of serotonin in the central nervous system byimmunohistochemistry: description of a specific and sensitivetechnique and some applications. Neuroscience 3, 811–819.

Steinbusch, H.W., Nieuwenhuys, R., Verhofstad, AA., van der, K.D.,1981. The nucleus raphe dorsalis of the rat and its projectionupon the caudatoputamen. A combined cytoarchitectonic,immunohistochemical and retrograde transport study.J. Physiol. (Paris) 77, 157–174.

Stratford, T.R., Wirtshafter, D., 1990. Ascending dopaminergicprojections from the dorsal raphe nucleus in the rat. Brain Res.511, 173–176.

Taber, E., Brodal, A., Walberg, F., 1960. The raphe nuclei of thebrain stem in the cat. I. Normal topography andcytoarchitecture and general discussion. J. Comp. Neurol. 114,161–187.

Tork, I., 1990. Anatomy of the serotonergic system. Ann. N Y Acad.Sci. 600, 9–34.

Twarog, B., Page, I., 1953. Serotonin content of some mammaliantissues and urine and a method for its determination. Am. J.Physiol. 175, 157–161.

Uhl, G.R., Goodman, R.R., Kuhar, M.J., Childers, S.R., Snyder, S.H.,1979. Immunohistochemical mapping of enkephalincontaining cell bodies, fibers and nerve terminals in the brainstem of the rat. Brain Res. 166, 75–94.

Valentino, R.J., Commons, K.G., 2005. Peptides that fine-tune theserotonin system. Neuropeptides 39, 1–8.

Van Bockstaele, E.J., Pickel, V.M., 1993. Ultrastructure ofserotonin-immunoreactive terminals in the core and shell ofthe rat nucleus accumbens: cellular substrates forinteractions with catecholamine afferents. J. Comp. Neurol.334, 603–617.

Van Bockstaele, E.J., Biswas, A., Pickel, V.M., 1993. Topography ofserotonin neurons in the dorsal raphe nucleus that send axoncollaterals to the rat prefrontal cortex and nucleus accumbens.Brain Res. 624, 188–198.

van de Kar, L.D., Lorens, S.A., 1979. Differential serotonergicinnervation of individual hypothalamic nuclei and otherforebrain regions by the dorsal and median midbrain raphenuclei. Brain Res. 162, 45–54.

van der Kooy, D., Hattori, T., 1980a. Dorsal raphe cells withcollateral projections to the caudate-putamen and substantianigra: a fluorescent retrograde double labeling study in the rat.Brain Res. 186, 1–7.

van der Kooy, D., Hattori, T., 1980b. Bilaterally situated dorsalraphe cell bodies have only unilateral forebrain projections inrat. Brain Res. 192, 550–554.

Vertes, R.P., 1991. A PHA-L analysis of ascending projections ofthe dorsal raphe nucleus in the rat. J. Comp. Neurol. 313,643–668.

Vialli, M., Erspamer, V., 1933. Cellule enterochromaffini e cellulebasigranulose acidofile nei vertebrati. Z. Zellforsch. 19, 743–744.

Wang, Q.P., Ochiai, H., Nakai, Y., 1992. GABAergic innervation ofserotonergic neurons in the dorsal raphe nucleus of the ratstudied by electron microscopy double immunostaining. BrainRes. Bull. 29, 943–948.

Wang, Q.P., Guan, J.L., Nakai, Y., 1995. Distribution and synapticrelations of NOS neurons in the dorsal raphe nucleus: acomparison to 5-HT neurons. Brain Res. Bull. 37, 177–187.

Waselus, M., Galvez, J.P., Valentino, R.J., Van Bockstaele, E.J., 2006.Differential projections of dorsal raphe nucleus neurons to thelateral septum and striatum. J. Chem. Neuroanat. 31, 233–242.

Wotherspoon, G., Albert, M., Rattray, M., Priestley, J.V., 1994.Serotonin and NADPH-diaphorase in the dorsal raphe nucleusof the adult rat. Neurosci. Lett. 173, 31–36.

Wyss, J.M., Swanson, L.W., Cowan, W.M., 1979. A study ofsubcortical afferents to the hippocampal formation in the rat.Neuroscience 4, 463–476.

Yoshida, K., McCormack, S., Espana, R.A., Crocker, A., Scammell,T.E., 2006. Afferents to the orexin neurons of the rat brain.J. Comp. Neurol. 494, 845–861.