Forebrain afferents to the rat dorsal raphe nucleus demonstrated by retrograde and anterograde tracing methods

Pergamon

PII:

Neuroscience Vol. 82, No. 2, pp. 443–468, 1998 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00268-6

FOREBRAIN AFFERENTS TO THE RAT DORSAL RAPHE NUCLEUS DEMONSTRATED BY RETROGRADE AND ANTEROGRADE TRACING METHODS C. PEYRON,* J.-M. PETIT, C. RAMPON, M. JOUVET and P.-H. LUPPI INSERM U52, CNRS ERS 5645, Universite´ Claude Bernard, 8 Avenue Rockefeller, 69373 Lyon, cedex 8, France Abstract––The dorsal raphe nucleus through its extensive efferents has been implicated in a great variety of physiological and behavioural functions. However, little is know about its afferents. Therefore, to identify the systems likely to influence the activity of serotonergic neurons of the dorsal raphe nucleus, we re-examined the forebrain afferents to the dorsal raphe nucleus using cholera toxin b subunit and Phaseolus vulgaris-leucoagglutinin as retrograde or anterograde tracers. With small cholera toxin b subunit injection sites, we further determined the specific afferents to the ventral and dorsal parts of the central dorsal raphe nucleus, the rostral dorsal raphe nucleus and the lateral wings. In agreement with previous studies, we observed a large number of retrogradely-labelled cells in the lateral habenula following injections in all subdivisions of the dorsal raphe nucleus. In addition, depending on the subdivision of the dorsal raphe nucleus injected, we observed a small to large number of retrogradelylabelled cells in the orbital, cingulate, infralimbic, dorsal peduncular, and insular cortice, a moderate or substantial number in the ventral pallidum and a small to substantial number in the claustrum. In addition, we observed a substantial to large number of cells in the medial and lateral preoptic areas and the medial preoptic nucleus after cholera toxin b subunit injections in the dorsal raphe nucleus excepting for those located in the ventral part of the central dorsal raphe nucleus, after which we found a moderate number of retrogradely-labelled cells. Following cholera toxin b subunit injections in the dorsal part of the central dorsal raphe nucleus, a large number of retrogradely-labelled cells was seen in the lateral, ventral and medial parts of the bed nucleus of the stria terminalis whereas only a small to moderate number was visualized after injections in the other dorsal raphe nucleus subdivisions. In addition, respectively, a substantial and a moderate number of retrogradely-labelled cells was distributed in the zona incerta and the subincertal nucleus following all tracer injections in the dorsal raphe nucleus. A large number of retrogradely-labelled cells was also visualized in the lateral, dorsal and posterior hypothalamic areas and the perifornical nucleus after cholera toxin b subunit injections in the dorsal part of the central raphe nucleus and to a lesser extent following injections in the other subdivisions. We further observed a substantial to large number of retrogradely-labelled cells in the tuber cinereum and the medial tuberal nucleus following cholera toxin b subunit injections in the dorsal part of the central dorsal raphe nucleus or the lateral wings and a small to moderate number after injections in the two other dorsal raphe nucleus subdivisions. A moderate or substantial number of labelled cells was also seen in the ventromedial hypothalamic area and the arcuate nucleus following cholera toxin injections in the dorsal part of the central dorsal raphe nucleus and the lateral wings and an occasional or small number with injection sites located in the other subdivisions. Finally, we observed, respectively, a moderate and a substantial number of retrogradely-labelled cells in the central nucleus of the amygdala following tracer injections in the ventral or dorsal parts of the central dorsal raphe nucleus and a small number after injections in the other subnuclei. In agreement with these retrograde data, we visualized anterogradely-labelled fibres heterogeneously distributed in the dorsal raphe nucleus following Phaseolus vulgaris-leucoagglutinin injections in the lateral orbital or infralimbic cortice, the lateral preoptic area, the perifornical nucleus, the lateral or posterior hypothalamic areas, the zona incerta, the subincertal nucleus or the medial tuberal nucleus. Altogether our retrograde and anterograde results clearly indicate, in contrast to previous studies, that each dorsal raphe nucleus subdivision receives a differential distribution of afferents from numerous forebrain structures. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: cortex, basal forebrain, hypothalamus, serotonin, sleep, mood disorder.

*To whom correspondence should be addressed. Abbreviations: CLiP, corticotropin-like intermediate lobe peptide; CTb, cholera toxin b subunit; CTb+, retrogradely-labelled cells; DAB, 3,3*-diaminobenzidine; DRN, dorsal raphe nucleus; 5-HT, serotonin; HRP, horseradish peroxidase; PAP, peroxidase–antiperoxidase; PB, phosphate buffer; PBS, phosphatebuffered saline; PBST, PBS containing 0.3% Triton X-100; PBST-Az, PBST with 0.1% sodium azide; PHA-L, Phaseolus vulgaris-leucoagglutinin; WGA–HRP, wheat germ agglutinin–horseradish peroxidase

The dorsal raphe nucleus (DRN) is located on the midline in the brainstem at the joining level of the mesencephalon and pons. The DRN is a large, distinct cell group which can be divided into rostral, central and caudal subdivisions based on the distribution of the serotonin (5-HT) cells. In addition to these subdivisions, a lateral extension called the ‘‘lateral wings’’ of the DRN has been described. It is well developed at the level of the central DRN.46 All these components can be further divided into dorsal and ventral portions.

443

444

C. Peyron et al.

The DRN through its extensive efferents53 has been implicated in a great variety of physiological and behavioural functions in particular sleep, pain and mood disorders.6,17,18,57 However, little is known about the afferent connections of the DRN. Indeed, only two retrograde tracing studies using horseradish peroxidase (HRP) or wheat germ agglutinin– horseradish peroxidase (WGA–HRP) have been realized in rats. These studies reported that the most striking afferent to the DRN both in terms of selectivity and density is the lateral habenula.1,19 Using tritiated -aspartate, Kale´n et al.21 further showed that this input used an excitatory amino acid as a transmitter. In both reports, minor descending inputs to the DRN were also identified from the horizontal band of Broca and the bed nucleus of the stria terminalis. Aghajanian and Wang,1 unlike Kale´n et al.21 found other minor descending afferents from the prefrontal cortex and the medial, lateral and magnocellular preoptic areas. Kale´n et al.21 in contrast to Aghajanian and Wang1 described afferent projections from the posterior hypothalamic areas and the submammillothalamic nucleus. Furthermore, in these reports, the HRP or WGA–HRP injection sites involved the entire DRN and therefore did not reveal whether the afferent projections to the DRN are topographically organized. Such detailed examination of the topography of the afferents to the different subdivisions of the DRN seems necessary in view of the chemical heterogeneity of each DRN subdivision. Indeed, the ventral part of the central DRN contains nearly exclusively serotonergic neurons while the other subdivisions also contain peptidergic or dopaminergic neurons. For example, the rostral DRN contains dopaminergic neurons, the dorsal parts of the central and rostral DRN neuropeptide Y neurons and the most rostral and the most caudal parts of the DRN cholecystokinin neurons.27,43–46,54 Therefore, to completely determine the afferents to the different subdivisions of the DRN, we reexamined the cortical, telencephalic and diencephalic afferents to the DRN with a sensitive retrograde tracer, the cholera toxin b subunit (CTb) injected by microiontophoresis in the ventral or dorsal parts of the central DRN, the rostral DRN or the lateral wings. We confirmed the specificity of the afferents to the different subdivisions of the DRN using two anterograde tracers, CTb and Phaseolus vulgarisleucoagglutinin (PHA-L) injected in some of the areas containing retrogradely-labelled cells following CTb injections in the DRN. In a companion study, we will report the mesencephalic, pontine and medullary afferents to the DRN.33 EXPERIMENTAL PROCEDURES

Preparation of the tracer (Cholera toxin b subunit and Phaseolus vulgaris-leucoagglutinin) Lyophilized CTb (List Biological Lab.) was first reconstituted in distilled water, then concentrated to 1% and buffer

exchanged (with 0.1 M phosphate buffer [PB], pH 6.0) by two repeated 1 h 30 min ultrafiltration (7000 r.p.m., at 4)C) with a centricon-10 microconcentrator (Amicon). The lyophilized PHA-L (Vector Lab.) was reconstituted at 2.5% in 0.01 M phosphate-buffered saline (PBS), pH 8.0. Tracer injection protocol Glass capillary tubes (1 mm o.d., Clark Electromedical Instruments) were heated, pulled and the tips broken to 3–5 µm diameter under microscopic control. The micropipettes were then filled with CTb or PHA-L solutions. Prior to CTb injections, male OFA rats (IFFA Credo, France) weighing 260–310 g were anaesthetized with sodium pentobarbital (40 mg/kg, i.p.) and placed in a stereotaxic apparatus. A scalp incision was made, a hole was drilled in the skull overlying the dorsal raphe nucleus, and the dura was reflected. The stereotaxic coordinates of the DRN were defined with the atlas of Paxinos and Watson30 (1 mm rostral to the interaural (lambda point), 0–600 µm lateral to the midline and approximately 5.8 mm ventral from the skull). Furthermore, an extracellular recording of the 5-HT neurons was made to localize the DRN. In agreement with previous electrophysiological studies in anaesthetized rats,9 recorded 5-HT neurons were characterized by a slow, regular pattern of discharge (1–2 spikes/s) and biphasic action potential of long duration (>2 ms). After placing the tip of the pipette in the DRN, the CTb was ejected iontophoretically by a pulsed positive current 7 s ‘on’, 7 s ‘off’ generated by a Midgard CS-4. The size of the CTb injection depended on the current intensity applied (0.5– 1 µA) as well as the duration of the ejection (10–15 min). Then, the micropipette was left inside the brain for 10 min before removal in order to prevent leakage along the pipette track. The ventral (n = 10) or dorsal (n = 4) parts of the central DRN, the rostral DRN (n = 9) or the lateral wings (n = 3) received CTb injections. In a second series of experiments, PHA-L or CTb were injected in structures containing retrogradely-labelled cells after CTb injections in the DRN. PHA-L- or CTbcontaining micropipettes (10–15 µm tip micropipette) were stereotaxically lowered in these structures. Then, PHA-L was iontophoretically ejected with 5 µA and CTb with 2 µA pulsed current 7 s ‘on/off’ for 15–30 min. Targets structures were the lateral orbital (PHA-L, n = 1), and infralimbic cortice (PHA-L, n = 1), the lateral preoptic area (CTb, n = 4; PHA-L, n = 2), the perifornical area (CTb, n = 2; PHA-L, n = 2), the lateral hypothalamic area laterodorsal to the fornix (CTb, n = 2; PHA-L, n = 3), the lateral hypothalamic area dorsomedial to the subthalamic nucleus (CTb, n = 1; PHA-L, n = 1), the posterior hypothalamic area (PHA-L, n = 2), the medial tuberal nucleus (PHA-L, n = 1), the zona incerta (PHA-L, n = 1) and the subincertal nucleus ventral to the zona incerta (PHA-L, n = 1). Histology One to two weeks after the injections, the animals were deeply anaesthetized and perfused through the ascending aorta with a Ringer lactate solution containing 0.1% heparin followed by 500 ml of an ice-cold fixative composed of 4% paraformaldehyde, 0.25% glutaraldehyde and 0.2% picric acid in 0.1 M PB (pH 7.4). The brains were postfixed overnight at 4)C in the same fixative without glutaraldehyde. They were cryoprotected by immersion in 0.1 M PB containing 30% sucrose, for 48–72 h at 4)C. Then, the brains were rapidly frozen with CO2 gas and cut on a cryostat in 20 µm-thick coronal sections. The free-floating sections were stocked in 0.1 M PB, pH 7.4 containing 0.9% NaCl, 0.3% Triton X-100 (PBST) and 0.1% sodium azide (PBST–Az).

Forebrain afferents to the rat dorsal raphe Immunohistochemistry of cholera toxin b subunit The free-floating sections were successively incubated in (i) a goat antiserum to CTb (List Biological Lab., 1:40,000 in PBST–Az) for three to four days at 4)C; (ii) a biotinylated rabbit anti-goat immunoglobulin (Vector Lab., 1:2000 in PBST) for 90 min at room temperature; and (iii) streptavidin–HRP (Jackson ImmunoResearch Lab., 1:40,000 in PBST) for 90 min at room temperature. Each incubation was carried out under gentle stirring and stopped by 2#15 min washes in PBST. Afterwards, the sections were immersed in 0.025% 3,3*-diaminobenzidine–4HCl solution (DAB; Sigma) containing 0.6% nickel ammonium sulphate (DAB–Ni) in 0.05 M Tris–HCl buffer (pH 7.6) and 0.003% H2O2 for 15–30 min at room temperature. The reaction was stopped by two rinses in PBST–Az. The CTb staining appeared as black punctate granules in the soma and proximal dendrites of retrogradely-labelled neurons (CTb+). Finally, the sections were mounted on gelatin-coated glass slides, dried, dehydrated and coverslipped with Depex. Phaseolus vulgaris-leucoagglutinin immunohistochemical procedures For PHA-L immunohistochemistry, the sections were successively incubated in (i) a rabbit antiserum to PHA-L (DAKO, 1:5000 in PBST–Az) for three to four days at 4)C; (ii) a biotinylated goat anti-rabbit immunoglobulin (Vector Lab., 1:2000 in PBST) for 90 min at room temperature; (iii) streptavidin–HRP (Jackson ImmunoResearch Lab., 1:40,000 in PBST) for 90 min at room temperature. Finally, the sections were immersed for 10–15 min in 0.025% DAB–Ni in 0.05 M Tris–HCl buffer with 0.003% H2O2 at room temperature. The reaction was terminated by extensive washes in PBST–Az. Immunohistochemistry of serotonin To determine precisely the location of the injection sites with regard to the 5-HT serotonergic neurons, serotonin immunohistochemistry was performed on CTb-stained sections at the level of the DRN (see Fig. 1). These sections were incubated in (i) a rabbit antiserum to serotonin (Incstar Corporation; 1:20,000 in PBST–Az) for three to four days at 4)C; (ii) a donkey anti-rabbit immunoglobulin (Jackson ImmunoResearch Lab., 1:1000 in PBST) for 90 min at room temperature and (iii) a rabbit peroxidase– antiperoxidase complex (PAP; Dakopatts, 1:1000 in PBST) for 90 min at room temperature. Then, the sections were immersed for 10–15 min in 0.025% DAB in 0.05 M Tris–HCl buffer with 0.003% H2O2 at room temperature. The cytoplasm of the serotonergic neurons were coloured homogeneously brown (Fig. 1A). Data analysis of the retrograde labelling Sections immunoreacted for CTb were viewed with a Leitz Orthoplan microscope equipped with an X-Y-sensitive stage and a video camera connected to a computerized image analysis system (Biocom, France). Outlines of sections and major structures were drawn at low power magnification (with a 2.5# objective) while retrogradelylabelled cells were plotted at higher magnification (with a 16# objective). To determine the respective contribution of each structure projecting to the DRN, counts of the retrogradelylabelled cells were made in each structure on one hemisphere for five injection sites in the ventral part of the central DRN, three in the dorsal part of the central DRN, two in the lateral wings and three in the rostral DRN. The coronal 20 µm-thick sections analysed were taken every 160 µm. The number of sections counted for each structure depended on its rostrocaudal extension. The maximum number of cells found per structure on one section for two large sites and one small site in the ventral, two in the dorsal

445

parts of the central DRN, two in the rostral DRN and two in the lateral wing is shown in Table 1. In addition, drawings were made of rostrocaudal sections from representative large injections (700–800 µm diameters) in the ventral (RD27), dorsal (S5) parts of the central DRN, lateral wing (K1) and rostral DRN (N4). The photomicrographs were taken with a Kodak Ektar film (25 ASA) using a Leitz Ortoplan microscope connected to a camera (Vario-orthomat). The negatives were then scanned and the digital microphotographs were stored on a Kodak Photo CD disk. To get an optimal reproduction of the CTb and PHA-L staining, we modified the contrast, luminosity and colour dominance of the scrude scans with Photoshop 3.0 (Adobe). The illustration plates were then printed with a colour dye-sublimation printer (Sony UPD7000E). RESULTS

The DRN can be divided into rostral, central and caudal parts. In addition to these subdivisions, a lateral extension called the ‘‘lateral wings’’ has been described. It is well developed only at the level of the central DRN.46 All these subdivisions can be further subdivided into ventral and dorsal parts. The ventral part of the central DRN is located just dorsal to the medial longitudinal fasciculi at the level of the anterior tegmental nucleus. The dorsal part of the central DRN is situated on the midline, just ventral to the aqueduct. The rostral DRN is located at the level of the trochlear nucleus and the caudal DRN at the level of the ventral tegmental nucleus of Gudden. Retrograde experiments Cholera toxin subunit b injection sites. Application of CTb by iontophoresis through micropipettes allowed us to obtain injection sites limited to one subdivision of the DRN (Fig. 1). The CTb injection sites were characterized by a dense central area surrounded by a peripheral zone containing diffuse tracer and stained fibres. Usually, a crack was visible at the centre of the site. This crack was certainly due to the shrinkage of the tissue induced by the dense deposit. Indeed, the morphology of the cells in the injection site appeared normal in adjacent untreated, counterstained sections. Following small, medium or large CTb injections in the same part of the DRN, the distribution of retrogradely-labelled cells was similar while their number decreased in parallel with the size of the injection site. To precisely determine the respective contribution of each input to the different parts of the DRN, we compared the number of retrogradelylabelled cells (Table 1, Figs 2–6) between injection sites in the different parts of the DRN. Small (200–270 µm in diameter, n = 4, Fig. 1B), medium (450–600 µm, n = 3) and large (700–800 µm, n = 3, Fig. 1C) injection sites were obtained in the ventral part of the central DRN. The large sites had a small extension in the dorsal part of the central DRN. The centre of all these sites was located at the level of the anterior tegmental nucleus where the

446

C. Peyron et al.

ventral part of the DRN contains nearly exclusively 5-HT cells. The large sites extended from the trochlear nucleus (IV) to the rostral part of the ventral tegmental nucleus. Among the three CTb injection sites located in the dorsal part of the central DRN, one was small-sized (380 µm in large diameter) and restricted to this subdivision. The other two were larger (750 µm) with a small involvement of the rostral DRN (Fig. 1D). They extended from the trochlear nucleus to the rostral part of the ventral tegmental nucleus. In the rostral DRN, we obtained small (300 µm, n = 1), medium (450–550 µm, n = 4) and large (800 µm, n = 2, Fig. 1F) injection sites. They were centred in the ventral part between the medial longitudinal fasciculi excepting for one which involved the dorsal part of the rostral DRN just ventral to the aqueduct. All sites extended from the caudal part of the oculomotor nucleus to the caudal part of the trochlear nucleus.

In the lateral wings, medium (450–500 µm, n = 2) and large (700–950 µm, n = 3, Fig. 1E) injection sites extended rostrocaudally from the caudal part of the trochlear nucleus to the laterodorsal tegmental nucleus. All these sites were localized in the dorsal part of the lateral wing excepting a large one centred in the ventral part of this subdivision. Cortex (Fig. 2). A large number of retrogradelylabelled cells (22–35 cells) was observed in the lateral orbital cortex after CTb injections in the ventral parts of the central DRN or lateral wing (Fig. 7A). In contrast, a small to moderate number (5–12 cells) was seen with injection sites located in the dorsal part of these subdivisions and the rostral DRN. A large number of CTb+ cells (20–27 cells) was observed in the medial orbital cortex following tracer injections in the lateral wing (Fig. 7B). In contrast, a moderate to substantial number (2–9 cells) was

Abbreviations used in the figures 3 3V ac aca Acb AHC AI AO AOP Aq Arc BLA BSTL BSTM BSTLP BSTMP BSTV cc CeA Cg CG Cl CPu DA DP DMH End EP f fmi fr Fr Fstr GI GP HDB Hi HLA ic IL LHb lo LO

oculomotor nucleus third ventricle anterior commissure anterior commissure, anterior accumbens nucleus anterior hypothalamic area, central agranular insular cortex anterior olfactory nucleus anterior olfactory nucleus, posterior aqueduct arcuate nucleus basolateral amygdaloid nucleus bed nucleus of the stria terminalis, lateral bed nucleus of the stria terminalis, medial bed nucleus of the stria terminalis, lateroposterior bed nucleus of the stria terminalis, medioposterior bed nucleus of the stria terminalis, ventral corpus callosum central amygdaloid nucleus cingulate cortex central gray claustrum caudate putamen dorsal hypothalamic area dorsal peduncular cortex dorsomedial hypothalamic nucleus endopiriform nucleus entopeduncular nucleus fornix forceps minor corpus callosum fasciculus retroflexus frontal cortex fundus striati granular insular cortex globus pallidus horizontal limb diagonal band hippocampus lateral hypothalamic area internal capsule infralimbic cortex lateral habenular nucleus lateral olfactory tract lateral orbital cortex

LPO LS LV MCPO MeA MHb ml mlf MO MnPO MPA MPO MS mt MTu opt ox Pa Par1 PeF PF PH Pir PVP Rt SCh SFO SHi SHy sm SOR sox st SI STh Subl TT VDB VMH VLO VP VO ZI ZID ZIV

lateral preoptic nucleus lateral septal nucleus lateral ventricle magnocellular preoptic nucleus medial amygdaloid nucleus medial habenular nucleus medial lemniscus medial longitudinal fasciculus medial orbital cortex median preoptic nucleus medial preoptic area medial preoptic nucleus medial septal nucleus mammillothalamic tract medial tuberal nucleus optic tract optic chiasm paraventricular hypothalamic nucleus parietal cortex area 1 perifornical nucleus parafascicular thalamic nucleus posterior hypothalamic area piriform cortex paraventricular thalamic nucleus reticular thalamic nucleus suprachiasmatic nucleus subfornical organ septohippocampal nucleus septohypothalamic nucleus stria medullaris thalamic supraoptic nucleus retrochiasmatic supraoptic decussation stria terminalis substantia innominata subthalamic nucleus subincertal nucleus tenia tecta vertical limb diagonal band ventromedial hypothalamic nucleus ventrolateral orbital cortex ventral pallidum ventral orbital cortex zona incerta zona incerta, dorsal zona incerta, ventral

Forebrain afferents to the rat dorsal raphe

Fig. 1. Photomicrographs of frontal sections at the level of the DRN. (A) Illustration of the central DRN after serotonin immunohistochemistry. The dorsal (just ventral to the aqueduct), ventral (interfascicular area) parts and the lateral wings can be easy distinguished based on the distribution of the serotonergic cells. (B) Illustration of a small CTb injection site (E2) restricted to the ventral part of the central DRN (150 µm in diameter). (C) Illustration of a large representative CTb injection site (RD27) centred on the ventral part of the central DRN (700 µm large diameter). The distribution of the retrograde labelling obtained following this injection is illustrated in Figs 2–5 and Fig. 6 (A–C). (D) Photomicrograph of a large injection site (S5) limited to the dorsal part of the central DRN (750 µm large diameter). The distribution of the neurons retrogradely labelled following this injection is shown in Figs 2–5 and Fig. 6 (A*–C*). (E) Photomicrograph of a large injection site (K1) located in the lateral wing with a small involvement of the ventrolateral part of the periaqueductal gray (850 µm large diameter). The distribution of the cells retrogradely labelled after this injection is shown in Figs 2–5 and Fig. 6 (A+–C+). (F) Illustration of a large injection site (N4) located in the rostral DRN (800 µm length) at the level of the trochlear nucleus (4). The distribution of the retrograde labelling obtained following this injection is illustrated in Figs 2–5 and Fig. 6 (A***–C***). Scale bars = 150 µm.

447

448

C. Peyron et al.

Table 1. Distribution of the retrogradely labeled cells in cortical, telencephalic and diencephalic areas Ventral small E2

Large RD27

RD11

S5

RD7

A6

N4

Lateral ipsi contro K1 L3 K1 L3

Cortex Cingulate Frontal Insular Orbital, lateral Orbital, medial Orbital, ventral Infralimbic Dorsal peduncular Tenia tecta

3 2 4 9 4 0 2 0 0

19 7 9 31 9 5 6 4 2

21 6 8 25 9 4 7 7 2

14 2 13 9 0 18 22 15 4

25 2 12 6 9 12 20 22 4

37 3 12 7 2 10 5 14 5

34 5 12 12 7 24 6 22 11

56 11 3 5 27 9 19 13 5

35 6 5 35 7 0 4 13 0

38 2 2 1 20 2 17 4 1

5 2 5 13 6 2 2 0 0

Amygdala Amygdaloid nucleus, anterior Medial amygdaloid nucleus Central amygdaloid n. Anterior cortical amygdaloid n. Basomedial amygdaloid n.

0 0 1 0 0

4 5 10 3 2

3 2 5 0 1

0 9 16 1 2

0 6 18 3 1

0 0 5 0 0

0 0 5 0 0

2 5 5 0 0

2 4 4 0 0

0 0 0 0 0

0 0 0 0 0

Basal forebrain Diagonal band, horizontal Diagonal band, vertical Septal nucleus, medial Septal nucleus, lateral Septohypothalamic n. Accumbens n. Accumbens shell Bed nucleus, medial Bed nucleus, lateral Bed nucleus, ventral Bed nucleus, medial posterior Bed nucleus, lateral posterior Substantia innominata Ventral pallidum Fundus striati Claustrum Endopiriform n.

1 0 0 0 0 0 0 1 0 1 1 0 4 3 2 1 0

2 1 1 3 0 0 1 2 2 7 0 0 6 7 9 5 1

3 2 2 6 2 0 0 5 2 7 2 3 10 11 7 3 1

5 2 1 5 4 0 2 10 11 20 26 17 8 24 8 17 1

2 2 2 7 3 0 2 6 8 32 25 15 10 16 6 12 2

1 0 1 3 1 0 3 5 1 6 2 5 9 6 5 10 1

2 2 2 5 1 0 5 4 2 8 4 2 14 10 3 12 2

2 4 3 8 6 8 9 5 4 4 9 8 14 9 5 15 0

2 6 1 5 4 7 5 6 3 4 6 7 13 9 7 8 0

0 1 0 2 1 1 1 2 1 0 3 3 2 3 3 4 0

0 1 0 1 1 0 0 1 1 0 3 4 1 1 1 3 0

Preoptic areas Median preoptic n. Medial preoptic n. Medial preoptic area Lateral preoptic area Magnocellular preoptic n.

0 2 5 7 2

3 9 6 12 3

2 11 10 18 6

9 38 53 36 8

7 27 44 22 5

2 20 19 41 1

4 24 20 55 2

7 20 17 52 4

6 23 15 48 4

4 4 6 9 1

4 5 5 6 1

Anterior hypothalamic areas Lateroanterior hypothalamic n. Anterior hypothalamic area ant. Anterior hypothalamic area cont. Paraventricular hypothalamic n. Dorsal hypothalamic area Dorsomedial hypothalamic n. Ventromedial hypothalamic n. Arcuate n. Tuber cinereum Tuberomammillary n. Medial tuberal n. Perifornical n. Lateral hypothalamic area Posterior hypothalamic area

0 0 0 1 2 2 0 0 1 0 1 4 5 2

2 0 1 3 18 15 2 0 3 1 5 21 28 10

2 1 1 5 12 12 1 1 4 1 7 15 34 13

4 0 10 11 21 12 10 5 12 1 21 26 50 38

3 0 7 13 29 21 17 10 16 3 26 32 39 44

0 0 1 8 6 10 3 0 6 1 4 18 20 30

0 0 2 7 10 19 7 4 7 2 7 19 26 34

3 2 4 9 32 16 18 3 12 3 11 25 52 50

3 2 3 9 31 18 14 6 12 1 10 24 46 45

1 1 1 4 11 6 5 2 5 0 6 17 20 14

1 1 1 2 7 8 4 1 2 0 5 12 18 15

Epithalamus Lateral habenula Paraventricular thalamus n.

5 0

26 4

33 2

30 8

28 11

22 3

27 5

32 5

28 5

28 2

27 1

Subthalamus Zona incerta Subincertal n.

3 1

8 10

6 7

10 7

11 6

6 7

4 8

18 23

17 21

10 13

7 11

Location of injection sites

Dorsal

Rostral

Forebrain afferents to the rat dorsal raphe

A

. .... Fr . . ventral . . (RD27) .. . . . .... .. .. ..... . .. ..... ......... AI MO .. . LO

B

C

. .

. .

Cg

.

IL

... . . ... ........ LO

. .. .. . . . . .. . .

DP

Fr

fmi Par1

x

AO

449

..

.

.. . Cl .

. TT

aca

VO

. IL

. ........ AI

End

Acb

lo

A'

B'

Cg

. ... .. .... .... ..... .

dorsal (S5)

MO/VO ..

.

AI

.. .. .

VLO

... .. . .

TT

A"

.

lateral (K1) MO

Acb

Fr

LO AO

.

Cg fmi Par1

. ...... . DP ........ IL

............... AI

CPu

. .... . . ... . . . ... AI .

. .

..

AOP

. .. . . .. ........ . .... . . fmi ......... . IL ..... .. . ..... .. .. .. . . . . LO . ... . DP .. ... TT

B"

Cg . . .... .

. .. . . ........ ... .... . . .. ..... .. ..... .. ... VLO

. . . .. . .... .. . .. . IL ...... .. ........ .. ..... Cl ........... .......... VO ..... ... TT

lo

C'

Fr

C"

Cg

Fr

.

. .... Par1

TT

..

IL ..... SHi

Pir

. . . ... .

GI .... .... . . . ... AI .

Pir lo

A"' rostral (N4)

. .. ... . .. fmi ........ ..... . ... VLO .

B"'

. . ... AI ....

MO/VO

.

.

C"'

Fr

... . . ... IL ... .. . .... . . . Cl .. . ... . . .. TT

Cg Par1

. . ..... AI

.. . .....

CPu Acb

. .

GI

... . .

aca Pir Fig. 2. Schematic drawings of 20 µm rostrocaudal frontal sections at the prefrontal cortex level illustrating the distribution of retrogradely-labelled cells after CTb injections in the ventral (A–C), dorsal (A*–C*) parts of the central DRN, the lateral wings (A+–C+) or the rostral DRN (A***–C***). Every dot represents one retrogradely-labelled neuron.

450

C. Peyron et al.

A

B

cc

CPu

.

ventral . (RD27)

SHy

.

BSTM CPu

.

BSTV

. . VDB .. aca. . .. VP . Fstr .. . . HDB ... ......... . .. . . . . .

. ..

aca

LS

MS

BSTL

Cl Acb

.

.

VP

.

lo

A' LV

.

dorsal (S5)

B'

. .

CPu

. . . . . ... Acb . . .. . . . ..

.. End

. LV CPu

. ... .. . .

LS . . ... BSTM . aca . . .. ... . .......... .. . . .... Acb .. .... ..... . .. ... . .. .. .... . . HDB .. VP .

.. .. .

VP

A"

LV

lateral (K1) ..

B"

cc

CPu

.

.

aca

LS

SHi

LS

.

...

Acb

aca

.

.

.

. LV

. .

SHi

CPu

MS . .

.. .. HDB

.. . .

. Acb

.

A"'

B"'

cc LV LS

rostral (N4)

. .

. CPu

. ..

LV

CPu

. . aca . . .. .. .. VP . .

. .. VP

VP

.

. .. . . . . VDB .

.. . .

BSTM

.. .. .. . . .. . . .. .

.

Fig. 3. Schematic drawings of 20 µm rostrocaudal frontal sections at the basal forebrain level illustrating the distribution of retrogradely-labelled cells after CTb injections in the ventral (A–B), dorsal (A*–B*) parts of the central DRN, the lateral wings (A+–C+) or the rostral DRN (A***–C***). Every dot represents one retrogradely-labelled neuron.

visualized with injection sites located in the other subdivisions of the DRN. With injection sites located in the rostral DRN or the dorsal part of the central DRN, a substantial number of cells (10–24 cells) was observed in the ventral orbital cortex defined by Krettek and

Price,22 located dorsally to the olfactory ventricle and the rostral part of the accumbens nucleus. A moderate number of retrogradely-labelled cells (4–9 cells) was observed in this cortex after CTb injections in the other subnuclei of the DRN (Fig. 8C,D).

Forebrain afferents to the rat dorsal raphe

A ventral (RD27)

BSTMP

BSTM

f

.

BSTL

B

LS

LV

ic

ic

.

sm

GP

.. . CPu ac ... . . . . . . . . . . . . . . . . . . .. . VP . ... . .. LPO . ..... . . .. . . .. . . . . . . . . . . HDB . .... .. .

st

.

.

aca

451

. .

SI . . . . .. . StHy

.HDB . .

f

. . 3V . . . . . . ..

.. .. . . ... .. ... . . .. .. . . LPO. . . MPA

MCPO

A' dorsal (S5)

. MS . . SHy . .... . . ... . . . . .. CPu . .. .. BSTV .. . . . . . . . ... .... . . .. . . .. . .......... ..... . .. . . .... .. ...... .. ... . ...... . 3V ... .. . .. . ......... . Fstr . .. ..... . .. . .. ........... ... . . . .... .. ...... . . MPA . . .. . .. . . .

A"

BSTL

f

lateral (K1)

A"' rostral (N4)

.. PS . MnPO . ...... . .. . .. . . ... . . . . .. . .. .. ... . ...... .. ........... ..... . . .. VP . . . . . . . .. . . . . .Fstr . . . ox . SI

GP BSTV

LV

. .

.

3V

.

B"

LV

LS

B'

.

SFO sm

ic BSTLP GP

.. VP ..

f

. ... . . . . . ..... ..... ... . ........ . ...... SI .. . .. . . .. .... .

st

B"' MPO

. f

MPA

.

BSTMP .. . .. .. .. . ... ....... .. ............ ... ...... . ..... .... . . . .. .............................. . . . . . .... ....... ......... .. . . . .. .. .. . . . . .. . . ... . MCPO .

LS

3V .. . ... . ... . ac . . ....... ... . . . .. . .. ... .... . .... .. . . . . . .. . . . ....... . .. ......... ........ .. . . .. . ... VP . . . . . .. . . ... . . . ..... . . . ox . . .. ..MCPO

BSTMP

LV

BSTLP . . ..... . . ... . f . . . . . . .... .. . .. . . .. . . . . ................................. .. ............................ . . . ............. .. ... ...................... .. . . ..... .. . .. . ................ . .. ... . .. . . .. . .. .... . .............. .. ........................... . .. . .. ... .. ..... . . . . . . . . .... .. . .. . .. .. . . .. ..... .. .. HDB . . ... . SCh

f . . . .. .... ........ ... .... . .. . 3V ... . ................. VP .. .. . . . . ox .. . LPO

ic

. ... . ... GP . .. ... ............... . . . . . .. ... ... ... .. .......... . .. . . . . .. .. . .... HDB

Fig. 4. Schematic drawings of 20 µm rostrocaudal frontal sections at the preoptic areas level illustrating the distribution of retrogradely-labelled cells after CTb injections in the ventral (A–B), dorsal (A*–B*) parts of the central DRN, the lateral wings (A+–C+) or the rostral DRN (A***–C***). Every dot represents one retrogradely-labelled neuron.

After tracer injections in the lateral wing or the rostral DRN, a large number of retrogradely-labelled cells (34–56 cells) was seen in the rostral cingulate cortex. A substantial number was observed (14–25 cells) in this cortex following tracer injections in the central DRN (Fig. 8A,B). After CTb injections localized in the dorsal part of the lateral wing or the dorsal part of central DRN, a substantial number of retrogradely-labelled cells (16–20 cells) was observed in the infralimbic cortex.

In contrast, a small to moderate number of labelled cells (2–7 cells) was seen after injections in the ventral parts of the same subdivisions or the rostral DRN (Fig. 9A). A substantial number of retrogradely-labelled cells (8–15 cells) was observed in the insular cortex after injections in the rostral or central DRN. A small number of labelled cells (3–5 cells) was seen in this cortex after tracer injections in the lateral wing.

452

C. Peyron et al.

A

B

sm

ZI

. .. f .

.

ic

.

. .. . . HLA .. .. .. . ... ..

MeA

ACo

A'

CPu

Rt

DMH

. .. . .... ....... ... ... . . . TC . . .

.. . . . . .. .

AHC

MCPO

B'

..

...

..

ZI

GP

. . . ........... . . . . .. . . . .. .. . .......... . . EP ..... . . . . . . . . . . . . . . .. . . . . . .. .. ..... ... . . . .. . .... . . . . . . ....... .. . . ... .. . . . ... . ... . . . . . ..... . . .. . . . . .. CeA. .... .. . . .. . . . . ..

B"

.. .

........... ....

MHb

lateral (K1)

Arc

mt

GP . . .. ... .. .. . ....... .. . ... . ... . . . . .. ....... . .. 3V . .... . . . .. . . . ........ . .. . .. . .. . . . . . ox .. . ... ... . . . . . ... .. CeA

.....

A"

. .. . . .. ... . ...... . . ... ..... .... opt . . .

.

3V

VMH

PVN

. . .. . ... . . .. . .. . .. . . sox

.......... ..

LHb

ZI

. ... .. ... . .. . ........ .. . . EP . . ..... .. .. . .. . . .. ... ....... .... . . .. . . AHA . . . . . . . ... . ... . .. . . .. . . CeA . . . . . . . . . .. .. . BLA MeA

. . . . . .. ... . . . . . . . . ... .... .... .... . .. TC ... ..

ic

mt

.................. . ... .... ic . ..... ..... . ....... . . . ........ .............. .... .. . ... .. opt . . . .. . . SOR

Arc

B"'

A"'

. .......

sm LHb

rostral (N4)

LHb Rt

. ...

dorsal (S5)

..

.

MHb

Rt

ventral (RD27)

sm

. .. .. . .

.......

Rt mt

PVN

. .. . . . . .... . . .. . .... . . . . ... . . 3V . . . . . .. . . ox ..

.. ic ... . . . .. .. HLA . . . . . . CeA

ZI

. . . . . . ... . .. . . EP ... . .. ... .. .. . . . . . . . .... ....... . .. ...... 3V . . ... . . .... . ... . . . ... CeA . . .. . SOR

VMH

Fig. 5. Schematic drawings of 20 µm rostrocaudal frontal sections at the anterior hypothalamus level illustrating the distribution of retrogradely-labelled cells after CTb injections in the ventral (A–B), dorsal (A*–B*) parts of the central DRN, the lateral wings (A+–C+) or the rostral DRN (A***–C***). Every dot represents one retrogradely-labelled neuron.

A moderate number of labelled cells (6–11 cells) was observed in the frontal cortex following tracer injections in the lateral wing or the ventral part of the central DRN. An occasional to small number of CTb+ cells was seen following injections in the other subnuclei of the DRN (1–5 cells).

A substantial number of cells (14–22 cells) was located in the dorsal peduncular cortex (localized dorsal to the tenia tecta and ventral to the infralimbic cortex) after injections in the rostral DRN or the dorsal part of the central DRN. A moderate number of CTb+ cells (4–13 cells) was seen after

Forebrain afferents to the rat dorsal raphe

A

453

B LHb

ventral (RD27)

.. .

.

. . . .. .. . fr ... . PVP

ml mt

. ... . PeF .....DA ...... . . ..... .. .. . .... ... . .. .. .. . ......... .. .. ..DMH .... ...... .. . ......... . . . . ... .. . . .. . MTu

. mt ... . . .. . . . . .PH. . .. ......... . . . ic . . .... .... . ... .. ..... . ... ... .. ... ... ... .......... . . . . . . .. 3V . HLA .

ic opt

. ... . .

..

B'

..........

LHb

. . .. .. . . . . ..... . . . . . . fr ZID . . . . Subl ZIV . . . . 3V .. . . . . . . . mt.. . .. . .. . . . .. . ......... .............. PH ic . .. .. .... .f. . ... . . .... . . .. .... . .. .. . .... . . HLA 3V . .. ... Arc VMH

Rt

dorsal (S5)

Subl ic ZI .. .... .... . .... DA.. .... . ...... . .. ... ... ........... ....... ...... . .. .. . . ........ ... ... . . ......... . . ...... .... .. ...... ......... ......... . . . .. .... .... . . .. . . . ........... .. . 3V .... ................... .. . ... ...... .. HLA .. ... . ... MTu

A" lateral (K1)

. ........ . .. BLA

B"

MHb

. ... . . . .. . . .. . . . .. . PVP . . . . .. . .. ..... ... ..... .. . ... .. .. .. . . . ..... . . . . . . . .

ml

.

....... . .. .. . . ................. ... ..... . ...................................... ..... . ... . ..... ... ...... .. ........... ..

A"'

.

STh

.

B"'

.

. ... . .. . .. ... . . .. ... ..... . .. ... ... ................... . . . .............. ....... ......... .................... ......... .. .. .. ... .. ..... .......... . ..... . . . .. .... . ... . .

ZIV

. .. . ..... .... .... ... .... ... . ........ .... . . ... .... .. ..... ic ........... ...... . ..... .... ... .. . 3V ... .. . .. . .. ...... . . .

Arc

. . .........

. .......... .... PF

MTu

. .. .

PVP ZID

ml

(N4)

.

. .

DMH

MHb

. . ..... .... .....

mt

STh .. . . . ... .. .. .. . .. . . ...... ... ................. HLA . . .. f. ... . . .

PeF

Arc

rostral

. . ...... .. . . .. ............ ... . . .. . fr .. ml

Subl mt . .

STh

VMH

VMH

A'

ml

ZI

opt

. . . . .. PH . . . .. . ............. .. ..... . ...... .... .. .. . STh . . ... ...... . ... .. ... .. . . . . ... . .. . .. . . ..... VMH DMH

Fig. 6. Schematic drawings of 20 µm rostrocaudal frontal sections at the posterior hypothalamus level illustrating the distribution of retrogradely-labelled cells after CTb injections in the ventral (A–B), dorsal (A*–B*) parts of the central DRN, the lateral wings (A+–C+) or the rostral DRN (A***–C***). Every dot represents one retrogradely-labelled neuron.

454

C. Peyron et al.

Fig. 7. Photomicrographs showing the medial and lateral orbital cortice projections to the different subdivisions of the DRN. (A–B) Illustrations of retrogradely-labelled cells located in the lateral orbital cortex following a CTb injection in the ventral part of the central DRN (A) (RD27) and in the medial orbital cortex after a CTb injection in the lateral wing (B) (K1). Scale bars = 500 µm. (C) Illustration of a PHA-L injection site in the lateral orbital cortex (X1). Scale bar = 700 µm. (D) Dark-field photomicrograph showing numerous anterogradely-labelled fibres in the ventral part of the central DRN obtained with the PHA-L injection site illustrated in (C). Note that numerous varicose fibres course in the ventral part of the central DRN while no or occasional fibres are present in its dorsal part and the lateral wings. Scale bar = 200 µm.

tracer injections in the other subdivisions of the DRN. A moderate number of labelled cells (5–11 cells) was visualized in the tenia tecta with injection sites located in the rostral DRN and an occasional to small number was seen following injections in the other subdivisions (1–5 cells). Amygdala (Fig. 5). In the central nucleus of the amygdala, a substantial number of retrogradelylabelled cells (16–18 cells) was seen with injection sites located in the dorsal part of the central DRN (Fig. 10D). A small to moderate number of labelled cells (4–10 cells) was visualized after CTb injections in the other subnuclei of the DRN. In the medial nucleus of the amygdala, a small to moderate number of labelled cells (2–9 cells) was seen

following CTb injections in all subnuclei of the DRN excepting for those located in the rostral DRN after which no labelled cell was observed. In the anterior nucleus of the amygdala, a small number of CTb+ cells (2–4 cells) was observed following tracer injections in the ventral part of the central DRN. No or occasional labelled cells (0–2 cells) were visualized with injection sites in the other subdivisions of the DRN. Finally, occasional cells (1–3 cells) were observed in the anterior cortical and the basomedial amygdaloid nuclei only after injections in the ventral or dorsal parts of the central DRN. Basal forebrain (Fig. 3). In the anterior portion of the medial and lateral bed nucleus of the stria terminalis (BNST), a moderate number of labelled cells

Fig. 8. (A) Photomicrographs of coronal sections showing retrogradely-labelled cells in the cingulate cortex following a CTb injection in the lateral wing of the DRN (K1). Note that the labelled cells are predominantly located in the layer V excepting a few smaller cells in layer VI. Scale bar = 250 µm. (B) Enlargement of (A) showing that the retrogradely-labelled cells in layer V are pyramidal cells. Scale bar = 100 µm. (C–D) Illustrations showing the retrogradely-labelled cells located in the ventral orbital cortex with an injection site located in the rostral (C) (N4) or the ventral part of the central DRN (D) (RD27). The difference in cell number between the two cases clearly show the topographical organization of this projection to the DRN. Scale bars = 200 µm.

456

C. Peyron et al.

Fig. 9. A series of photomicrographs of frontal sections illustrating the projection of the infralimbic cortex to the DRN. (A) Photomicrograph showing retrogradely-labelled cells in the caudal part of the infralimbic cortex after a CTb injection in the rostral DRN (N4). The labelled cells are located in layers V and VI. Scale bar = 150 µm. (B) Photomicrograph of a PHA-L injection site in the infralimbic (IL1) cortex. Scale bar = 300 µm. (C) Dark-field photomicrograph illustrating the distribution of the anterogradely-labelled cells in the DRN following the PHA-L injection in the infralimbic cortex shown in C. Scale bar = 200 µm.

was seen with injection sites located in the dorsal part of the central DRN (6–12 cells, Fig. 10A) while an occasional to small number (1–6 cells) was visualized after tracer injections in the other subdivisions of the DRN (Fig. 10B). A substantial to large number of retrogradely-labelled cells was observed in the posterior portion of the medial (25–26 cells), lateral (15–17 cells) and ventral (20–32 cells) subdivisions of the BNST after CTb injections in the dorsal part of the central DRN (Fig. 10C). A small to moderate number of CTb+ cells was seen in these areas after tracer injections in the other subdivisions of the DRN (2–10 cells). A substantial number of labelled cells (14–24 cells) was observed in the ventral pallidum after tracer injections in the dorsal part of the central DRN. A moderate number (6–13 cells) was seen with injection sites located in the other regions of the DRN.

In the horizontal band of Broca, an occasional to small number of retrogradely-labelled cells (1–4 cells) was observed following CTb injections in all regions of the DRN. In the vertical band, a small number of retrogradely-labelled cells (4–6 cells) was seen following CTb injections in the lateral wing and occasional cells (1–2 cells) after injections in the other subdivisions of the DRN. In the lateral septal nucleus, a small to moderate number of retrogradely-labelled cells was observed after tracer injections in all subdivisions of the DRN (2–9 cells). In the medial septal and septohypothalamic nuclei (Fig. 4), an occasional to small number of retrogradely-labelled cells (1–5 cells) was observed for all CTb injections in the DRN. A moderate to substantial number of retrogradelylabelled cells (6–17 cells) was observed in the substantia innominata after CTb injections in the different subdivisions of the DRN.

Forebrain afferents to the rat dorsal raphe

457

Fig. 10. (A–B) Illustrations showing the distribution of labelled neurons in the rostral part of the bed nucleus of the stria terminalis following CTb injections in the dorsal (A) (S5) or ventral (B) (M4) parts of the central DRN. Note the large difference in number of labelled cells between the two cases. Scale bars = 250 µm. (C) Photomicrograph showing labelled cells in the posterior region of the lateral and medial parts of the bed nucleus of the stria terminalis after a CTb injection in the dorsal part of the central DRN (S5). Scale bar = 100 µm. (D) Illustration of CTb+ cells in the central amygdaloid nucleus following a tracer injection in the dorsal part of the central DRN (S5). Scale bar = 150 µm.

A small to moderate number of labelled cells (3–10 cells) was seen in the fundus striati after injections in all subdivisions of the DRN. A moderate number of CTb+ cells (5–9 cells) was observed in the nucleus accumbens and its shell part after injections in the lateral wings. A small to

occasional number was seen following tracer injections in the other subdivisions (1–5 cells). A substantial number of labelled cells (6–17 cells) was located in the claustrum after injections in all subdivisions of the DRN excepting for injection sites located in the ventral part of the central DRN after

458

C. Peyron et al.

which a small number of labelled cells (3–5 cells) was observed. No or occasional cells (1–2 cells) were seen in the endopiriform nucleus for all injections in the DRN. Preoptic areas (Fig. 4). A very large number of retrogradely-labelled cells (44–58 cells) was visualized in the medial preoptic area (MPA) with CTb injection sites located in the dorsal part of the central DRN (Fig. 11D). A substantial number of labelled cells (10–21 cells) was observed following tracer injections in the rostral DRN or the lateral wings. A moderate number (5–10 cells) was seen following CTb injections in the ventral part of the central DRN (Fig. 11C). A large number of labelled cells was observed in the medial preoptic nucleus (MPO) following CTb injections in the rostral DRN, the lateral wings (20–24 cells) or the dorsal part of the central DRN (27–44 cells). A moderate number (9–11 cells) was seen after CTb injections in the ventral part of the central DRN. For all injections, no labeled cells were seen in the nuclear core of the MPO. A large number of labelled cells was visualized in the lateral preoptic area after tracer injections in the lateral wing, the rostral DRN (40–55 cells) or the dorsal part of the central DRN (22–36 cells) (Fig. 11F). A substantial number (7–18 cells) were seen following CTb injections in the ventral part of the central DRN (Fig. 11E). A small to moderate number of labelled cells were seen in the magnocellular preoptic nucleus with all injection sites localized in the DRN (3–8 cells) excepting for those localized in the rostral DRN after which only occasional cells (1–2 cells) were observed. In the median preoptic nucleus (MnPO), a small to moderate number of CTb+ cells (2–9 cells) was observed following all tracer injections in DRN. Anterior areas of the posterior hypothalamus (Fig. 5). A substantial number of retrogradelylabelled cells (10–13 cells) were seen in the parvocellular hypothalamic nucleus following tracer injections in the dorsal part of the central DRN. A small to moderate number of CTb+ cells (3–9 cells) were seen with injection sites located in the other subnuclei of the DRN. No or a small number of CTb+ cells (1–4 cells) were seen in the lateroanterior hypothalamic nucleus following all CTb injections in all subnuclei of the DRN. A moderate number of labelled cells (7–12 cells) were visualized in the central portion of the anterior hypothalamic area following injections in the dorsal part of the central DRN. An occasional to small number of cells (1–4 cells) were observed with injection sites located in the other portions of the DRN. Posterior areas of the posterior hypothalamus (Figs 5, 6). A large number of retrogradely-labeled cells (20–50 cells) were observed over the entire

rostrocaudal extent of the lateral hypothalamic area for all CTb injections in the DRN (Fig. 12C,D). Nevertheless, a larger number of labelled cells were seen in this area after injections in the lateral wing and the dorsal part of the central DRN than the ventral part of the central DRN and the rostral DRN (Table 1). A substantial to large number of retrogradelylabelled cells (15–32 cells) were seen in the perifornical nucleus after all tracer injections in the DRN. A large number of retrogradely-labelled cells (30–52 cells) were seen in the posterior hypothalamic area after all tracer injections in the DRN excepting those in the ventral part of the central DRN for which a substantial number of cells (10–15 cells) were observed. A large number of CTb+ cells (21–32 cells) were seen in the dorsal hypothalamic area with injection sites localized in the lateral wings or the dorsal part of the central DRN. A moderate to substantial number of labelled cells (6–18 cells) were observed following CTb injections in the other subdivisions of the DRN. A substantial number of labelled cells (10–21 cells) were observed in the dorsomedial hypothalamic nucleus for all tracer injections in the DRN. A substantial number of CTb+ (10–20 cells) were visualized in the ventromedial hypothalamic nucleus following tracer injections in the lateral wing or the dorsal part of the central DRN. An occasional to small number of labelled cells (1–7 cells) were seen after CTb injections in the other subdivisions. A substantial number of retrogradely-labelled cells (12–16 cells) were visualized in the tuber cinereum after tracer injections in the lateral wing or the dorsal part of the central DRN. A small to moderate number (2–7 cells) were observed after injections in the other subdivisions of the DRN. A large number of labelled cells (21–26 cells) were seen in the medial tuberal nucleus with injection sites located in the dorsal part of the central DRN. A moderate number (4–7 cells) were observed after CTb injections in the other subdivisions of the DRN. A moderate number of retrogradely-labelled cells (4–10 cells) were observed in the arcuate nucleus with CTb injections located in the dorsal parts of the lateral wing, rostral DRN or central DRN. No or occasional labelled cells (0–3 cells) were seen with tracer injections located in the ventral part of the lateral wing, rostral DRN or central DRN. For all tracer injections, occasional cells (1–3 cells) were visualized in the tuberomammillary nucleus. Epithalamus (Fig. 5, 6). A large number of retrogradely-labelled cells (19–35 labeled cells) were observed in the lateral habenula nucleus, mainly its medial part, whatever the location of the injection sites in the DRN (Fig. 12A,B).

Fig. 11. Series of photomicrographs showing the distribution of retrogradely-labelled cells in the medial and lateral preoptic areas. (A–B) Overviews of the preoptic areas showing the location of retrogradelylabelled cells after a CTb injection in the ventral part of the central DRN (A) (M4) or the dorsal part of the central DRN (B) (S5). (C–D) Enlargements of (A) and (B) showing the morphology and density of CTb+ cells in the medial preoptic nucleus and area. (E–F) Enlargements of (A) and (B) showing the morphology and density of retrogradely-labelled cells in the lateral preoptic area. Note that a large number of retrogradely-labelled cells are located in the medial preoptic nucleus and area with the injection site in the dorsal part of the central DRN (D) while only a moderate number are visible for the CTb injection in the ventral part of the central DRN (C). Note also that with the injection site located in the dorsal part of the central DRN, a larger number of CTb+ cells is situated in the medial preoptic area (D) than in the lateral preoptic area (F). In contrast, with the injection site located in the ventral part of the central DRN, a larger number of labelled cells is visible in the lateral preoptic area (E) than in the medial preoptic area (C). Scale bars for C, D, E, F = 200 µm.

Fig. 12. A series of photomicrographs of frontal sections illustrating the projections from the lateral hypothalamic area and the lateral habenula to the DRN. (A) Photomicrograph showing retrogradelylabelled cells in the lateral habenula following a CTb injection in the ventral part of the central DRN (RD27). Note that the labelled cells are mainly situated in the medial portion of the lateral habenula. Scale bar = 100 µm. (B) Enlargement of (A) showing at higher magnification the morphology of the retrogradely-labelled cells in the medial portion of the lateral habenula. Scale bar = 50 µm. (C) Low-power photomicrograph showing retrogradely-labelled cells in the lateral hypothalamic area of the posterior hypothalamus following a CTb injection in the ventral part of the central DRN (M4). Scale bar = 300 µm. (D) Enlargement of (A) showing the morphology of the retrogradely-labelled cells. Scale bar = 50 µm. (E) Illustration of a PHA-L injection site in the lateral hypothalamic area (PeF1). Scale bar = 300 µm. (F) Photomicrograph showing PHA-L anterogradely-labelled fibres in the DRN after the injection illustrated in (C). Note that numerous labelled fibres are distributed in the ventral part of the central DRN and slightly more in the dorsal part of the central DRN, the lateral wing and moreover the ventrolateral part of the periaqueductal gray. Scale bar = 300 µm.

Forebrain afferents to the rat dorsal raphe

A moderate number of retrogradely-labelled cells (8–11 cells) were seen in the posterior part of the paraventricular thalamic nucleus after CTb injections in the dorsal part of the central DRN and a small number (2–5 cells) after injections in the other parts of the DRN. Subthalamus (Figs 5, 6). A substantial number of retrogradely-labelled cells (10–18 cells) were observed in the zona incerta after tracer injections in the lateral wing or the dorsal part of the central DRN. A moderate number of CTb+ (4–8 cells) were seen following injections in the other regions of the DRN. A large number of labelled cells (21–23 cells) were observed in the subincertal nucleus following CTb injections in the lateral wings. A moderate number of labelled cells (6–12 cells) were seen with injection sites located in the other subdivisions of the DRN. Anterograde tract tracing experiments General remarks. In the present anterograde study, we used two tracers, CTb (which is a retrograde and anterograde tracer) and PHA-L. The CTb injection sites were darkly stained and sharply delineated (Fig. 1) but the labelling of the CTb anterogradelylabelled fibres was in general stronger for their varicosities than for the thinner non-varicose parts of the axons. In contrast, the PHA-L fibres were more strongly and completely labelled (Fig. 12F) but the extent of the injection sites was more difficult to evaluate. Indeed, diffuse immunoreactivity was visualized in the neuropil surrounding the central core of the injection sites (Fig. 12E). Cortex. With a small PHA-L injection site (n = 1) restricted to the rostral portion of the lateral orbital cortex (Fig. 7C), many long varicose fibres coursed in the ventral part of the central DRN (Figs 7D, 14B). A few fibres were distributed in the other subdivisions (Figs 7D, 14 A,B). The highest number of fibres was visualized in the ventrolateral part of the periaqueductal gray just dorsolateral to the medial longitudinal fasciculus and lateral to the DRN (Fig. 7D). After a small PHA-L injection (n = 1) in the caudal part of the infralimbic cortex (Fig. 9B), long varicose fibres were sparsely distributed in the ventral part of the central DRN and the rostral DRN. Slightly more short varicose fibres were localized in the dorsal part of the central DRN (mainly in its periaqueductal region) and moreover the lateral wings (Fig. 9C). Preoptic area. After CTb (n = 2) or PHA-L (n = 2) injections limited to the lateral preoptic area, many varicose fibres were distributed in the ventral and dorsal parts of the central DRN (Fig. 14D). A higher density of fibres was observed in the rostral DRN and the lateral wings (Fig. 14C). The greatest number of labelled fibres at the level of the DRN was visualized in the ventrolateral periaqueductal gray ipsilateral to the injection site (Figs 13A, 14C,D).

461

Hypothalamic areas. Following two injections in the perifornical nucleus, the first dorsolateral (CTb) and the second dorsomedial to the fornix (PHA-L), descending varicose fibres covered the entire DRN. Slightly more fibres were distributed in the lateral wing and moreover the ventrolateral and lateral parts of the periaqueductal gray. With injection sites in the lateral hypothalamic area (CTb, n = 3; PHA-L, n = 3), a high density of long varicose fibres ran throughout the entire DRN. Slightly more filled fibres were localized in the dorsal part of the central DRN, the lateral wing and moreover the ventrolateral part of the periaqueductal gray (Fig. 12F). With injection sites in the posterior hypothalamic area (CTb, n = 2; PHA-L, n = 2), many long varicose fibres were seen throughout the entire DRN. Slightly more filled fibres were localized in the lateral wing and moreover the ventrolateral part of the periaqueductal gray (Fig. 14E,F). After a very large CTb injection site in the medial tuberal nucleus, lateral to the ventromedial hypothalamic nucleus and ventral to the fornix, labelled fibres were sparsely distributed in the ventral part of the central DRN and the rostral DRN. A higher density of labelling was seen in the dorsal part of the central DRN and the lateral wing ipsilateral to the injection site. Subthalamus. Following a very small PHA-L injection site localized in the medial portion of the zona incerta just ventral to the mammillothalamic tract, sparse varicose fibres were seen in the ventral and dorsal parts of the central DRN and the rostral DRN. More terminal-like boutons were distributed in the lateral wing (Fig. 13B). The same distribution was observed in the DRN with a PHA-L injection site situated in the subincertal nucleus ventrally to the zona incerta. DISCUSSION

Our results indicate in accordance with previous retrograde studies1,21 that the DRN receives a strong projection from the lateral habenula nucleus. In addition, our retrograde and anterograde results also showed that the DRN receives extensive projections from different areas of the prefrontal cortex, the medial and lateral preoptic areas, the lateral, dorsal and posterior hypothalamic areas and the perifornical nucleus. Furthermore, we described a differential distribution of the afferents to the different subdivisions of the DRN. Technical considerations In this study, using CTb as a retrograde tracer, we observed afferents to the DRN either not reported or underestimated in the two previous studies studying the afferents to the DRN with HRP or

462

C. Peyron et al.

Fig. 13. (A) Dark-field photomicrograph of a frontal section illustrating PHA-L immunolabelled fibres differentially distributed in the DRN after a PHA-L injection in the lateral preoptic area. Note that anterogradely-labelled fibres are distributed with a higher density in the lateral wing and moreover the ventrolateral part of the periaqueductal gray. (B) Illustration of anterogradely-labelled fibres in the DRN following a PHA-L injection in the zona incerta (HLA4) showing more anterogradely-labelled fibres in the lateral wing than in the ventral and dorsal parts of the central DRN. Scale bars = 200 µm.

WGA–HRP.1,21 For example, we saw extensive projections from many cortical and hypothalamic areas either not reported or considered as minor inputs in these studies. Such discrepancies can be attributed to the superior sensitivity of CTb over WGA–HRP and moreover HRP. Indeed, using CTb, we demonstrated a large number of pathways not previously observed in cats with HRP or WGA–HRP.26 Moreover, recently in rats, we demonstrated a large number of afferents to the locus coeruleus25 not seen with WGA–HRP or FluoroGold as retrograde tracers.2 The more complete retrograde staining obtained

with CTb cannot be attributed to an uptake of CTb by fibres of passage as suggested recently by Chen and Aston-Jones.7 These authors realized in the olivocerebellar pathway (in the ventrolateral medulla), iontophoretic ejections of CTb with 2 µA current during 15 min and observed numerous retrogradely-labelled cells in the inferior olivary complex indicating that CTb has been taken up and transported by passing fibres. In agreement with their results, we observed that iontophoretic injections of CTb in fibre bundles such as the fornix with currents of 2 µA or above in rats and 5 µA or above in cats26

Forebrain afferents to the rat dorsal raphe

Fig. 14. (A–B) Schematic drawings of anterogradely-labelled fibres in the anterior (A) and central DRN (B) following an injection in the lateral orbital cortex (X1). Note that the fibres are essentially distributed in the ventral part of the DRN. (C–D) Schematic drawings of anterogradely-labelled fibres located in the rostral (C) and central DRN (D) after a PHA-L injection in the lateral preoptic area (LPO4). More fibres are covering the rostral DRN and the lateral wing than the ventral and dorsal parts of the central DRN. (E–F) Schematic drawings showing anterogradely-labelled fibres in the rostral (E) and central DRN (F) following a PHA-L injection in the posterior hypothalamic area (HLA6). More fibres are located in the lateral wing than in the rostral DRN and the ventral and dorsal parts of the central DRN.

463

464

C. Peyron et al.

induced artefactual retrograde labelling. However, we also determined that when CTb was ejected in the medial longitudinal fasciculus close to the DRN, with 0.5–1 µA currents during 10–15 min, no artefactual retrograde labelling could be noted. Moreover, further supporting that CTb is not taken up by fibres of passage in our experimental conditions, afferents to the DRN found in this report or to the locus coeruleus by Luppi et al.25 were confirmed by anterograde tract-tracing using PHA-L. In the next chapter, we discuss in detail the projections observed and their differential distribution in the DRN.

parts of the central DRN. In contrast, a small number of CTb+ cells was observed after tracer injections in the lateral wing or the rostral DRN. Previous retrograde experiments1,21 found no retrogradely-labelled cells in the amygdala after HRP or WGA–HRP injections covering the entire DRN. However, supporting our results, anterograde studies13,38 revealed a heavy anterograde labelling from the central nucleus of the rat amygdala to the ventral half of the periaqueductal gray including the DRN using tritiated leucine or WGA–HRP anterograde tracers.

Cortical areas

Basal forebrain

In the present study, depending on the subdivision of the DRN injected, a small to large number of retrogradely-labelled cells was observed in the orbital, cingulate, infralimbic, dorsal peduncular, and insular cortice. Using anterograde tracing with PHA-L, we confirmed that the lateral orbital cortex preferentially project to the ventral part of the central DRN and the infralimbic cortex to the dorsal part of the central DRN and the lateral wings. Aghajanian and Wang1 using HRP as a retrograde tracer consistently observed retrogradely-labelled cells in the prefrontal cortex with no further description. Further supporting our data, using autoradiographic or PHA-L tract-tracing techniques, efferent projections to the DRN have been shown from the cingulate, insular, medial orbital and infralimbic cortice, with no information concerning their topographical organization.35,36,51 Besides, in agreement with the topographical organization we observed, it has been shown using PHA-L that the medial orbital cortex mainly project to the lateral wing,40 and the infralimbic cortex to the dorsal part of the central DRN and the lateral wing.15 In addition, Wyss and Sripanidkulchai58 using autoradiographic tracttracing technique showed that the cingulate cortex give rise to a stronger projection to the lateral wing and the rostral DRN than to the other subdivisions. Altogether, these and our results support the existence of topographical projections from the lateral orbital, medial orbital, infralimbic and cingulate cortice to the different subdivisions of the DRN. Although our retrograde results also indicate the existence of a topographical organization of the projections from the insular, ventral orbital and dorsal peduncular cortice to the DRN, anterograde tracing experiments are necessary to confirm these results.

After CTb injections in the different subdivisions of the DRN, we observed a moderate to substantial number of retrogradely-labelled cells in the ventral pallidum and a small to substantial number in the claustrum. In addition, a small to moderate number of cells was observed in the fundus striati and the lateral septal nucleus and an occasional to small number in the horizontal and vertical bands of Broca. In contrast to us, previous tracing studies after WGA–HRP or True Blue injections in the DRN reported the presence of retrogradely-labelled cells in the horizontal band of Broca rather than in the adjacent ventral pallidum.20,39 Such discrepancy seems to be due to a misplacement of the retrogradely-labelled cells in the previous studies. Indeed, in contrast to these studies, we localized the retrogradely-labelled cells on counterstained sections. By this mean, we observed that the retrogradelylabelled cells were localized in an area composed of dispersed cells in fibre bundles corresponding to the ventral pallidum and not in the horizontal band of Broca which contains a dense group of small oriented cells as defined by Paxinos and Watson.30 Kale´n and Wiklund20 have further shown combining True Blue retrograde tracing with acetylcholinesterase histochemistry that retrogradely-labelled cells in this region are non-cholinergic. Previous studies reported the presence of retrogradely-labelled cells in the medial septum1,20,39 after injections covering the DRN. After CTb injections in the different subdivisions of the DRN, we observed only occasional retrogradely-labelled cells in the medial septum. Following tritiated leucine injections in the medial septal nucleus, Kale´n and Wiklund20 observed a substantial number of anterogradely-labelled fibres only in the most caudal part of the DRN. Altogether these and our results suggest that the medial septum specifically project to the most caudal part of the DRN. In agreement with previous studies, after injections of CTb in the DRN, we saw a few retrogradelylabelled cells in the lateral septum.1,20,39 The exact terminal field in the DRN of this small projection and those we showed from the fundus striati and the

Amygdala In the present study, we observed, respectively, a moderate and a substantial number of retrogradelylabelled cells in the central nucleus of the amygdala following CTb injections in the ventral or dorsal

Forebrain afferents to the rat dorsal raphe

claustrum, remain to be determined with anterograde tracing methods. We observed a large number of retrogradelylabelled cells in the lateral, ventral and medial parts of the bed nucleus of the stria terminalis following CTb injections in the dorsal part of the central DRN. Only a small to moderate number was observed in this nucleus after CTb injections in the other DRN subdivisions. In earlier retrograde tracing reports, Aghajanian and Wang,1 Kale´n et al.21 and Semba et al.39 consistently observed retrogradely-labelled neurons in the bed nucleus of the stria terminalis following HRP or WGA–HRP injections covering the entire DRN. Besides, using [3H]leucine, a strong projection to the DRN has been described from the bed nucleus of the stria terminalis.8,12,49 Altogether these and our data indicate that the bed nucleus of the stria terminalis project to the DRN. Additional anterograde studies are necessary to confirm that this projection is mainly directed to the dorsal part of the central DRN. We also saw a small to moderate number of retrogradely-labelled cells in the substantia innominata and the magnocellular preoptic nucleus following CTb injections in the DRN. Aghajanian and Wang1 saw retrogradely-labelled cells only in the magnocellular preoptic nucleus. Subsequent anterograde tracing studies with autoradiographic and PHA-L tracers confirmed that the substantia innominata provide a moderate projection to the DRN.10,50 Preoptic areas Following CTb injections in the ventral part of the central DRN, we found a moderate number of retrogradely-labelled cells in the medial and lateral preoptic areas and the medial preoptic nucleus. In contrast, a substantial to large number of cells were seen in these nuclei following tracer injections in the other subdivisions of the DRN. In earlier retrograde tracing reports, Aghajanian and Wang1 and Semba et al.39 consistently observed retrogradely-labelled neurons in the medial, lateral and magnocellular preoptic areas with HRP or WGA–HRP injections covering the entire DRN. Subsequent anterograde tracing studies with [3H]leucine or PHA-L confirmed that the medial preoptic nucleus and area,8,42 and the lateral preoptic area,42,48,50 strongly project to the DRN. However, these anterograde tracing studies gave no information concerning the topographical organization of these inputs in the DRN. Nevertheless, supporting the existence of a topographical organization, we confirmed, using PHA-L as an anterograde tracer, that the lateral preoptic area gives rise to a smaller projection to the ventral part of the central DRN than to the other subdivisions. We also saw a small to moderate number of retrogradely-labelled cells in the median preoptic nucleus following CTb injections in the DRN. Subsequent anterograde tracing studies with

465

autoradiographic and PHA-L tracers confirmed that the median preoptic nucleus60 provides a moderate projection to the DRN. Posterior hypothalamic level We observed a large number of retrogradelylabelled cells in the lateral, dorsal and posterior hypothalamic areas and the perifornical nucleus after CTb injections in the dorsal part of the central DRN and the lateral wings and to a lesser extent following injections in the ventral parts of the central DRN and the rostral DRN. Peschanski and Besson31 and Kale´n et al.21 but not Aghajanian and Wang1 described a large number of retrogradely-labelled cells in the lateral, dorsal and posterior hypothalamic areas and the perifornical nucleus following injection of retrograde tracers in the DRN. Further supporting our results, a large number of anterogradely-labeled fibres was observed in the DRN following [3H]leucine or PHA-L injections in the dorsal or lateral hypothalamic areas3,8,14,55,56 and the perifornical nucleus.3 These studies did not reveal a topographical organization of these inputs in the DRN. However, confirming the topography revealed by our retrograde tracing, after CTb or PHA-L injections in the posterior hypothalamic area, we observed slightly more anterogradely-labelled fibres in the lateral wing than in the other subnuclei of the DRN. We, Peschanski and Besson31 and Kale´n et al.21 but not Aghajanian and Wang1 described a large number of retrogradely-labelled cells in the dorsomedial hypothalamic nucleus and a moderate number in the parvocellular hypothalamic nucleus following injection of retrograde tracer in the DRN. Supporting these results, Ter Horst and Luiten.52 after PHA-L injections in the dorsomedial hypothalamic nucleus observed a substantial number of anterogradely-labelled fibres homogeneously in all subdivisions of the DRN. Moreover, a moderate number of anterogradely-labelled fibres was observed in the DRN following PHA-L injections in the paraventricular hypothalamic nucleus.24 We further observed a substantial to large number of retrogradely-labelled cells in the tuber cinereum and the medial tuberal nucleus following CTb injections in the dorsal part of the central DRN and the lateral wings, and a small to moderate number following injections in the other DRN subdivisions. Supporting these results, after CTb injections in the medial tuberal nucleus, we observed numerous anterogradely-labelled fibres in the dorsal part of the central DRN and the lateral wing and less fibres in the other subdivisions of the DRN. The topographically organized projection from the tuber cinereum to the DRN remains to be confirmed. We also observed a moderate to substantial number of CTb+ cells in the ventromedial and arcuate hypothalamic nuclei following CTb injections in

466

C. Peyron et al.

the dorsal part of the central DRN and the lateral wings, and an occasional to small number following injections in the other DRN subdivisions. Supporting our results, after PHA-L injections in the ventromedial hypothalamic nucleus, Veening et al.55 found a substantial number of anterogradely-labelled fibres in the lateral wing and a small number in the other subnuclei of the DRN. A moderate number of anterogradely-labelled fibres with no further precision was also observed in the DRN following PHA-L injections in the arcuate hypothalamic nucleus.41 However, supporting the existence of a topographical organization of the projection from the arcuate nucleus to the DRN, Zheng et al.61 showed that numerous CLIP/adrenocorticotropic hormone-immunoreactive fibres are present in the dorsal part of the central DRN, and the rostral DRN and only a few in the other subdivisions of the DRN. Indeed, it is likely that these fibres originate from the CLIP/adrenocorticotropic hormone neurons nearly exclusively located in the arcuate nucleus.59 We observed occasional retrogradely-labelled cells in the tuberomammillary nucleus after CTb injections in the DRN. Steinbush47 found histaminergic fibres in the DRN by immunohistochemistry. Lin et al.23 further observed terminal boutons making contacts with 5-HT neurons of the DRN. Besides, it has been shown that the tuberomammillary nucleus contains nearly exclusively histaminergic neurons.28,47 Altogether these data suggest the existence of a small histaminergic projection from the tuberomammillary nucleus to the DRN. Epithalamus Our retrograde data confirms that the lateral habenula (predominantly its medial portion) strongly projects to all subdivisions of the DRN. Indeed, this projection has been reported by numerous retrograde and anterograde tracing studies using HRP, WGA– HRP, tritiated amino acids and PHA-L.1,4,11,21,29 After tritiated []-aspartate injection in the DRN, Kale´n et al.21 further showed a large number of retrogradely-labelled cells in the lateral habenula indicating that this input operates with an excitatory amino acid transmitter. Subthalamus We observed, respectively, a substantial and a moderate number of retrogradely-labelled cells in the zona incerta and the subincertal nucleus following CTb injections in the lateral wings and the other subdivisions of the DRN. Using HRP or WGA– HRP retrograde tracing, only Peschanski and Besson31 saw retrogradely-labelled cells in the zona incerta. Ricardo37 using [3H]leucine as an anterograde tracer also found a small projection from the zona incerta to the DRN but gave no description on its topographic organization. However, confirming

our retrograde tracing results, after PHA-L injections in the medial part of the zona incerta or the subincertal nucleus, we observed a stronger projection to the lateral wing than to the other DRN subdivisions. Besides, combining CTb retrograde tracing with tyrosine hydroxylase immunohistochemistry, we previously showed that 19% of the neurons of the medial portion of the zona incerta projecting to the lateral wing contain dopamine.32 CONCLUSION AND PHYSIOLOGICAL IMPLICATIONS

Our retrograde and anterograde results indicate in accordance with previous retrograde studies1,21 that the DRN receives a strong projection from the lateral habenula nucleus. In addition, we found that it receives extensive inputs from orbital, cingulate, infralimbic and insular cortices, medial and lateral preoptic areas, lateral, dorsal and posterior hypothalamic areas and the perifornical nucleus. Furthermore, we described a differential distribution of these afferents to the different subdivisions of the DRN. These results indicate that a large number of forebrain structures are in a position to modulate the activity of serotonergic neurons of the DRN. Such results are particularly puzzling since electrophysiological recordings have shown that only very few physiological stimuli induce a modification of the regular and pacemaker-like activity of serotonergic neurons during wakefulness.15,16 The only major change of activity of serotonergic neurons occurred during sleep when they progressively decrease their activity to stop firing at the entrance of and during paradoxical sleep.15 A possibility might be that some of these pathways are activated by untested physiological conditions. Otherwise, these afferents could also terminate on the numerous non-serotonergic cells present in the DRN. Concerning the modulation of activity of serotonergic neurons during sleep, it would be important to determine the role of the afferents from the preoptic and the posterior hypothalamic areas respectively known to play a crucial role in the genesis of sleep and wakefulness.19 Beside sleep, the projections from the prefontal cortex, in particular the medial and lateral orbital cortices to the DRN found in this study, might play a role in obsessive compulsive disorders or depression. Indeed, these two pathologies are characterized by an increase in regional blood flow in the orbital cortex and are alleviated by serotonin uptake inhibitor treatment.5,16,34 Moreover, the increase of metabolic activity seen in obsessive compulsive disorder normalize with serotonin uptake inhibitor treatment.16 Serotonergic pathways from the dorsal raphe nucleus to the orbital cortice have previously been demonstrated.53 In the present study, we revealed strong projections from the lateral and medial orbital cortices to the DRN. Therefore, in view of all these data, reciprocal connections between serotonergic neurons of the DRN and the orbital

Forebrain afferents to the rat dorsal raphe

cortices might play a crucial role in mood regulation and dysfunction of this loop might lead to pathologies such as obsessive compulsive disorder and depression.

467

Acknowledgements—This work has been supported by ‘‘Ministe`re de la Recherche et de l’Enseignement Supe´rieur’’, INSERM and CNRS.

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

Aghajanian G. K. and Wang R. Y. (1977) Habenular and other midbrain raphe afferents demonstrated by a modified retrograde tracing technique. Brain Res. 122, 229–242. Aston-Jones G., Ennis M., Pieribone V. A., Nickell W. T. and Shipley M. T. (1986) The brain nucleus locus coeruleus: restricted afferent control of a broad efferent network. Science 234, 734–737. Allen G. V. and Cechetto D. F. (1992) Functional and anatomical organisation of cardiovascular pressor and depressor sites in the lateral hypothalamic area: I. Descending projections. J. comp. Neurol. 315, 313–332. Araki M., McGeer P. L. and Kimura H. (1988) The efferent projections of the rat lateral habenular nucleus revealed by the PHA-L anterograde tracing method. Brain Res. 441, 319–330. Baldwin D. and Rudge S. (1995) The role of serotonin in depression and anxiety. Int. clin. Psychopharmac. 9, 41–45. Behbehani M. M., Park M. R. and Clement M. E. (1988) Interactions between the lateral hypothalamus and the periaqueductal gray. J. Neurosci. 8, 2780–2787. Chen S. and Aston-Jones G. (1995) Evidence that cholera toxin subunit (CTb) can be avidly taken up and transported by fibers of passage. Brain Res. 674, 107–111. Conrad L. C. A. and Pfaff D. W. (1976) Efferents from medial basal forebrain and hypothalamus in the rat. An autoradiographic study of the medial preoptic area. J. comp. Neurol. 169, 185–211. Gallager D. W. (1978) Benzodiazepines: potentiation of a GABA inhibitory response in the dorsal raphe nucleus. Eur. J. Pharmac. 49, 133–143. Grove E. A. (1988) Neural associations of the substantia innominata in the rat: afferent connections. J. comp. Neurol. 277, 315–346. Herkenham M. and Nauta W. J. (1979) Efferent connections of the habenular nuclei in the rat. J. comp. Neurol. 187, 19–47. Holstege G., Meiners L. and Tan K. (1985) Projections of the bed nucleus of the stria terminalis to the mesencephalon, pons and medulla oblongata in the cat. Expl Brain Res. 58, 379–391. Hopkins D. A. and Holstege G. (1978) Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat. Expl Brain Res. 32, 529–547. Hosoya Y. and Matsushita M. (1981) Brainstem projections from the lateral hypothalamic area in the rat, as studied with autoradiography. Neurosci. Lett. 24, 111–116. Hurley K. M., Herbert H., Moga M. M. and Saper C. B. (1991) Efferent projections of the infralimbic cortex of the rat. J. comp. Neurol. 308, 249–276. Insel T. R. and Winslow J. T. (1992) Neurobiology of obsessive compulsive disorder. Psychiatr. Clin. North Am. 15, 813–824. Jacobs B. L., Wilkinson L. O. and Fornal C. A. (1990) The role of brain serotonin. A neurophysiologic perspective. Neuropsychopharmacology 3, 473–479. Jacobs B. L. and Azmitia E. C. (1992) Structure and function of the brain serotonin system. Physiol. Rev. 72, 165–229. Jouvet M. (1988) The regulation of paradoxical sleep by the hypothalamo-hypophysis. Archs ital. Biol. 126, 259–274. Kale´n P. and Wiklund L. (1989) Projections from the medial septum and diagonal band of Broca to the dorsal and central superior raphe nuclei: a non-cholinergic pathway. Expl Brain Res. 75, 401–416. Kale´n P., Karlson M. and Wiklund L. (1985) Possible excitatory amino acid afferents to nucleus raphe dorsalis of the rat investigated with retrograde wheat germ agglutinin and -(3H)aspartate tracing. Brain Res. 360, 285–297. Krettek J. E. and Price J. L. (1977) The cortical projections of the mediodorsal nucleus and adjacent thalamic nuclei in the rat. J. comp. Neurol. 171, 157–192. Lin J. S., Hou Y., Sakai K. and Jouvet M. (1996) Histaminergic descending inputs to the mesopontine tegmentum and their role in the control of cortical activation and wakefulness in the cat. J. Neurosci. 16, 1523–1537. Luiten P. G. M., Ter Horst G. J., Karst H. and Steffens A. B. (1985) The course of paraventricular hypothalamic efferents to autonomic structures in medulla and spinal cord. Brain Res. 329, 374–378. Luppi P. H., Aston-Jones G., Akaoka H., Chouvet G. and Jouvet M. (1995) Afferent projections to the rat locus coeruleus demonstrated by retrograde and anterograde tracing with cholera-toxin B subunit and Phaseolus vulgaris-leucoagglutinin. Neuroscience 65, 119–160. Luppi P. H., Fort P. and Jouvet M. (1990) Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons. Brain Res. 534, 209–224. Ochi J. and Shimizu K. (1978) Occurrence of dopamine-containing neurons in the midbrain raphe nuclei of the rat. Neurosci. Lett. 8, 317–320. Panula P., Pirvola U., Auvinen S. and Airaksinen M. S. (1989) Histamine-immunoreactive nerve fibers in the rat brain. Neuroscience 28, 585–610. Pasquier D. A., Anderson C., Forbes W. B. and Morgane P. J. (1976) Horseradish peroxidase tracing of the lateral habenular-midbrain raphe nuclei connections in the rat. Brain Res. Bull. 1, 443–451. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. Academic Press, North Ryde, Australia. Peschanski M. and Besson J. M. (1984) Diencephalic connections of the raphe nuclei of the rat brainstem: an anatomical study with reference to the somatosensory system. J. comp. Neurol. 224, 509–534.

468 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61.

C. Peyron et al. Peyron C., Luppi P. H., Fort P., Rampon C. and Jouvet M. (1995) Lower brainstem catecholamine afferents to the dorsal raphe nucleus. J. comp. Neurol. 364, 402–413. Peyron C., Rampon C., Petit J. M., Herman D. M., Jouvet M. and Luppi P. H. (1997) Mesencephalic, pontine and medullar afferent projections to the rat dorsal raphe nucleus demonstrated by retrograde and anterograde tracing methods. (In preparation). Price J. L., Carmichael S. T. and Drevets W. C. (1996) Networks related to the orbital and medial prefontal cortex; a substrate for emotional behavior? Prog. Brain Res. 107, 523–536. Reep R. L. and Winans S. S. (1982) Afferent connections of dorsal and ventral agranular insular cortex in the hamster Mesocricetus auratus. Neuroscience 7, 1265–1288. Reep R. L., Corwin J. V., Hashimoto A. and Watson R. T. (1987) Efferent connections of the rostral portion of medial agranular cortex in rats. Brain Res. Bull. 19, 203–221. Ricardo J. A. (1981) Efferent connections of the subthalamic region in the rat. II. The zona incerta. Brain Res. 214, 43–60. Rizvi T. A., Ennis M., Behbehani M. M. and Shipley M. T. (1991) Connections between the central nucleus of the amygdala and the midbrain periaqueductal gray: topography and reciprocity. J. comp. Neurol. 303, 121–131. Semba K., Reiner P. B., McGeer E. G. and Fibiger H. C. (1989) Brainstem projecting neurons in the rat basal forebrain: neurochemical, topographical, and physiological distinctions from cortically projecting cholinergic neurons. Brain Res. Bull. 22, 501–509. Sesack S. R., Deutch A. Y., Roth R. H. and Bunney B. S. (1989) Topographical organisation of the efferent projections of the medial prefrontal cortex in the rat: an anterograde tract-tracing study with Phaseolus vulgaris leucoagglutinin. J. comp. Neurol. 290, 213–242. Sim L. J. and Joseph S. A. (1991) Arcuate nucleus projections to brainstem regions which modulate nociception. J. chem. Neuroanat. 4, 97–109. Simerly R. B. and Swanson L. W. (1988) Projections of the medial preoptic nucleus: a Phaseolus vulgaris leucoagglutinin anterograde tract-tracing study in the rat. J. comp. Neurol. 270, 209–242. Smith G. S. T., Savery D., Marden C., Lopez Costa J. J., Averill S., Priestley J. V. and Rattray M. (1994) Distribution of messenger RNAs encoding enkephalin, substance P, somatostatin, galanin, vasoactive intestinal polypeptide, neuropeptide Y, and calcitonin gene-related peptide in the midbrain periaqueductal grey in the rat. J. comp. Neurol. 350, 23–40. Smits R. P., Steinbusch H. W. and Mulder A. H. (1990) Distribution of dopamine-immunoreactive cell bodies in the guinea-pig brain. J. chem. Neuroanat. 3, 101–123. Stamp J. A. and Semba K. (1995) Extent of colocalization of serotonin and GABA in the neurons of the raphe nuclei. Brain Res. 677, 39–49. Steinbusch H. W. M. (1981) Distribution of serotonin-immunoreactivity in the central nervous system of the rat – cell bodies and terminals. Neuroscience 6, 557–618. Steinbusch H. W. M. (1991) Distribution of histaminergic neurons and fibers in the rat brain. Acta otolar. Suppl. 479, 12–23. Swanson L. W. (1976) An autoradiographic study of the efferent connections of the preoptic region in the rat. J. comp. Neurol. 167, 227–256. Swanson L. W. and Cowan W. M. (1979) The connections of the septal region in the rat. J. comp. Neurol. 186, 621–656. Swanson L. W., Mogenson G. J., Gerfen C. R. and Robinson P. (1984) Evidence for a projection from the lateral preoptic area and substantia innominata to the mesencephalic motor region in the rat. Brain Res. 295, 161–178. Takagishi M. and Chiba T. (1991) Efferent projections of the infralimbic (area 25) region of the medial prefrontal cortex in the rat: an anterograde tracer PHA-L study. Brain Res. 566, 26–39. Ter Horst G. J. and Luiten P. G. M. (1986) The projections of the dorsomedial hypothalamic nucleus in the rat. Brain Res. Bull. 16, 231–248. To¨rk I. (1985) Raphe nuclei and serotonin containing systems. In The Rat Nervous System (ed. Paxinos G.), pp. 43–78. Academic, Australia. Van der Kooy D., Hunt S. P., Steinbusch H. W. M. and Verhofstad A. A. J. (1981) Separate population of cholecystokinin and 5-hydroxytryptamine-containing neuronal cells in the rat dorsal raphe, and their contribution to the ascending raphe projections. Neurosci. Lett. 26, 25–30. Veening J., Buma P., Ter Horst G. J., Roeling T. A. P., Luiten P. G. M. and Nieuwenhuys R. (1991) Hypothalamic projections to the PAG in the rat: topographical, immuno-electromicroscopical and functional aspects. In The Midbrain Periaqueductal Gray Matter (eds Deapulis A. and Bandler R.), pp. 387–415. Plenum, New York. Villalobos J. and Ferssiwi A. (1987) The differential descending projections from the anterior, central and posterior regions of the lateral hypothalamic area: an autoradiographic study. Neurosci. Lett. 81, 95–99. Wang Q. P. and Nakai Y. (1994) The dorsal raphe: an important nucleus in pain modulation. Brain Res. Bull. 34, 575–585. Wyss J. M. and Sripanidkulchai K. (1984) The topography of the mesencephalic and pontine projections from the cingulate cortex of the rat. Brain Res. 293, 1–15. Yoshida M. and Taniguchi Y. (1988) Projection of pro-opiomelanocortin neurons from the rat arcuate nucleus to the midbrain central gray as demonstrated by double staining with retrograde labeling and immunohistochemistry. Archs Histol. Cytol. 51, 175–183. Zardetto-Smith A. M. and Johnson A. K. (1995) Chemical topography of efferent projections from the median preoptic nucleus to pontin monoaminergic cell groups in the rat. Neurosci. Lett. 199, 215–219. Zheng Z., Le´ger L., Cespuglio R. and Jouvet M. (1991) Distribution of the pro-opiomelanocortin-immunoreactive axons in relation to the serotoninergic neurons in the dorsal raphe nucleus of the rat. Neurosci. Lett. 130, 17–21. (Accepted 12 May 1997)