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Brain Research 1019 (2004) 64–67

Research report

Serotonergic collateralized projections from Barrington’s nucleus to the

medial preoptic area and lumbo-sacral spinal cord

Antonella Russoa,*, Sebastiana Monacoa, Rosa Romeob, Rosalia Pellitteric, Stefania Stanzania

aDepartment of Physiological Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, ItalybDepartment of Anatomy, Diagnostic Pathology, Phorens Medicine, Hygiene and Publich Health, University of Catania, Via Santa Sofia, Catania, Italy

c Institute of Neurological Sciences, National Research Counsil, Viale R. Margherita 6, 95123, Catania, Italy

Accepted 10 March 2004

Available online 8 July 2004

Abstract

In this study, we employed triple fluorescent labelling to reveal the distribution of the direct serotonergic neurons within Barrington’s

nucleus (BN) that supply branching collateral input to the medial preoptic area (MPA) and to the lumbo-sacral spinal cord (LSC).

Immunocytochemical detection of the monoclonal antibody raised against serotonin was used for identification of the neurons. The

projections were defined by injections of two retrograde tracers: fluoro gold and rhodamine in the MPA and LSC, respectively. The aim of

this study is to identify the direct projections to BN and MPA and/or LSC. The present study confirms findings of others describing BN–LSC

projections and extends previous findings by demonstrating an single or collateralized fibers with MPA, and serotonergic immunoreactive

fibers.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Barrington’s nucleus; Medial preoptic area; Lumbo-sacral spinal cord; Serotonergic neuron; Fluoro gold; Rhodamine

1. Introduction strated that the neurons of Barrington’s nucleus (BN)

Micturition is a spino-bulbo-spinal reflex: a coordinated

action between the detrusor muscle of the bladder and the

external striated urethral sphincter. In adult mammals, the

area responsible for the synergistic action of both muscles

(detrusor–sphincter) is identifiable in the brainstem. The

region involved is located in the dorsolateral pons and is

known as Barrington’s area, M-region or pontine micturi-

tion center in different species [1–3,8,9]. This area is

indicated as a small group of neurons lying just ventrome-

dial to the rostral pole of the nucleus locus coeruleus (LC),

and receives afferent fibers from brainstem nuclei and

forebrain limbic structures. Electrical or chemical stimula-

tion of this region in rats elicits bladder contraction and

increases discharge of bladder postganglionic nerve

[11,15]. Moreover, lesions to this nucleus or applications

of opiates inhibit micturition [19]. Anatomical studies

utilizing anterograde and retrograde tracers have demon-

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2004.03.080

* Corresponding author. Tel.: +39-95-7384041; fax: +39-95-330645.

E-mail addresses: antrusso@mbox.unict.it (A. Russo),

stanzani@mbox.unict.it (S. Stanzani).

project to the intermediolateral column of the sacral spinal

cord in the region of preganglionic neurons that innervate

the bladder [5,6,16]. It has been suggested that the spinal

neurons convey bladder-filling information directly to BN;

there have been some studies on this pathway with trans-

neuronal virus labelling [10], c-fos expression [4], and

physiological techniques [11]. In addition, BN receives

widespread afferents from the brain including hypothala-

mus [2] and, in particular, the medial preoptic area (MPA)

that can be involved in the modulation of the micturition

reflex, in the rat [7]; in addition, BN contains not only

projecting neurons to the spinal cord, but also some

projecting neurons to the paraventricular thalamic nucleus,

periacqueductal gray and other regions, through axon

collaterals [12,17]. In contrast to the numerous studies of

the BN and spinal cord, little information exists regarding

the connections of BN with supraspinal structures. Some

studies have involved the BN in the simultaneous trans-

mission of signals [17] with corticotropin-releasing hor-

mone (CRH) release. Nonetheless, there have been no

systematic investigations focusing on various neurotrans-

mitters in BN [14,18]. Here, we raise the question of

whether single neurons in BN sending projection fibers to

A. Russo et al. / Brain Research 1019 (2004) 64–67 65

supraspinal region also project directly to the spinal cord,

and what kind of neurotransmitter these fibers contain.

In this study, we used two retrograde tracers to eluci-

date the circuitry of BN regulation to MPA and lumbo-

sacral spinal cord (LSC). Furthermore, to determine which

neurotransmitter is present in BN neurons, we initiated a

systematic investigation using immunocytochemical 5-HT

(serotonin) detection, and later on, we could assay other

specific antibodies. The aim of this study was to demon-

strate the presence and distribution of BN neurons that

project via axon collaterals to the medial preoptic area

(MPA) and lumbo-sacral spinal cord (LSC), and the

participation of serotonin in these pathways, presumed to

controlling micturition.

2. Materials and methods

All animal experiments were carried out in accordance

with current institutional guidelines for the care and use of

experimental animals. Experiments were performed on 12

adult male Wistar rats weighing 250–300 g (Morini, Italy),

maintained under controlled conditions of room temperature

(23F 1 jC) and lighting (lights on 07:00–19:00 h); labo-

ratory chow diet and water were available ad libitum; the in

Fig. 1. Injection zones in the CNS. Microphotographs showing injection zones: (A

(B) FG injection site and drawing in LSC (black area), scale bar = 90 Am.

vivo experimental procedure was performed during the

daytime (10:00–13:00 h). Animals were anaesthetized with

chloral hydrate (400 mg/kg i.p.). Briefly, 0.08 Al of rhoda-mine-labelled bead (RLB) were injected into MPA (stereo-

taxic planes: AP=� 0.30; L= 0.5; V=� 8.5) [13], whereas

0.04 Al of FG solution (6%in saline) were injected into the

ventral portion of LSC monolaterally. Both tracers were

pressure-injected at a rate of 50 nl/min using 1 Al Hamilton

micro-syringe.

Seven days after injections, the animals were reanaes-

thetized and perfused through ascending aorta with 60 ml

saline solution, followed by 300 ml ice-cold 4% parafor-

maldeyde phosphate buffer (pH 7.4). The brains were

removed, immersed in the same fixative for 3–4 h, and

cryo-protected overnight in phosphate-buffered 20% su-

crose solution. Two series of coronal sections (40 Am) were

cut on a cryostat and collected in phosphate buffer (PBS, pH

7.4, 0.1 M). One series was serially mounted on microscope

slides and immediately observed with a Reichert fluores-

cence microscope. After the first observation of BN region,

we employed the second series for immunocytochemical

processing. The series designated for immunocytochemical

visualization of serotonin was incubated as free-floating

sections for 16–18 h with anti-mouse monoclonal anti-

serotonin antibody (Chemicon). The primary antibody was

) RLB injection site and drawing in MPA (black area), scale bar = 400 Am;

Fig. 2. Microphotographs of triple-labelled neurons FG-RLB-FITC in

Barrington’s nucleus (BN). (A) Cell stained positively for FG (excitation

wavelength 330 nm); (B) the same cell stained positively for RLB

(excitation wavelength 560 nm) indicating the existence of a collateral

axon; (C) the same cell is positive to the serotonin (FITC, excitation

wavelength 450 nm). Scale bar = 20 Am. (D) Schematic drawing of frontal

brainstem section including BN region: the black dots show the labelled

neurons within the BN.

Table 1

Ipsilateral and contralateral labelled cells in the whole Barrington’s nucleus

Ipsilateral Contralateral

FG 17.5F 0.1 12.8F 0.3

RLB 8.8F 1.7 6.0F 0.8

FG+RLB 5.4F 1.0 2.1F 0.3

FG+ 5-HT 10.3F 1.0 8.4F 0.4

RLB+ 5-HT 6.1F 0.2 5.3F 0.2

FG+RLB+5-HT 1.8F 0.2 No neurons

The results (cells number per section) are summarized in this table.

A. Russo et al. / Brain Research 1019 (2004) 64–6766

diluted (1:200) in a solution of 0.3% Triton X-100 in PBS.

After a 15-min rinse in PBS, the sections were incubated for

30 min with fluorescein isothiocyanate (FITC) conjugated to

sheep anti-mouse Ig G (1:100; Boehringer). The sections

were air-dried, mounted and observed with a Reichert

fluorescence microscope equipped with filter combinations

revealing red (RLB), yellow (FG) or green (FITC) fluores-

cence. To determine the number of neurons containing both

retrograde tracers, cell count was performed on the series

that was not stained immunocytochemically. The incidence

of triple-labelled cells was estimated directly from the series

processed immunocytochemically by sequentially viewing

tissue with the three different filters. For every animal, three

non-adjacent sections were evaluated and the labelled cells

were plotted onto schematic drawings of the BN region

level. Thus, cell numbers were expressed as the average

number/section calculated from these three sections.

3. Results

All injections of the retrograde tracers remained relatively

localized as has previously reported. Fig. 1 shows micro-

photographs and schematic drawings example of the injec-

tion site of the retrograde tracers in the MPA and LSC; only

those cases where microscopic analysis of the injection site

revealed that the tracer deposits were correctly positioned

were included in this study. Injection of RBL into MPA and

of FG into LSC resulted in a large number of retrogradely

labelled neurons in the whole ipsi- and contralateral BN

region (stereotaxic planes: � 9.16/� 9.80 [13]; Fig. 2D).

More BN neurons from the ipsilateral LSC and MPA

were labelled than contralateral (Table 1).

The fluorescence microscopy revealed a substantial

number of double-labelled neurons, thus providing evidence

of collateralization to the MPA and LSC. These neurons

were generally of small size (25–30 Am).

The multiple staining protocol followed in the present

study evidences patterns of neurons in the brainstem, dem-

onstrating serotonin-like immunoreactivity (5-HT) together

with retrograde labelling by each of the tracers employed.

Only a limited number of labelled cells (FG +RLB) were

found to bifurcate to the contralateral MPA and LSC (10%),

whereas a consistent population of cells were found to

branch to MPA and LSC ipsilaterally (20%).

A relatively low number of FG/RLB/FITC triple labelled

(Fig. 2A–C) were scattered mainly at the ipsilateral BN

level (33.33% of the total immunoreactive population).

The results (BN cells number per section) are summa-

rized in Table 1.

Our results show that: (1) BN single neurons directly

project to MPA, and confirm that BN single neurons directly

project to LSC [7]; (2) by separate counting, the RLB-5-HT-

positive neurons are about 88.33% ipsilateral, and 69.31%

A. Russo et al. / Brain Research 1019 (2004) 64–67 67

contralateral; whereas the FG-5-HT neurons are 65.62%

ipsilateral and 58.85% contralateral; (3) BN neurons supply,

via collaterals, branching inputs to the MPA and LSC; (4)

about one third of these double-labelled neurons in BN are

serotonergic.

4. Discussion

These results implicate the distribution of neuronal single

projection in the BN; in particular, they confirm other

findings [5–7,16] regarding the projections to LSC and

visualize new projections to MPA. This result is very

interesting because the demonstration of direct MPA pro-

jection exists [7]; in addition, the presence of collateralized

fibers of the BN neurons includes the possibility of the

ascending and descending simultaneous control. The dis-

covery that BN laterodorsal tegmental nucleus modulates

micturition has been defined [1–3,8,9]. BN receives blad-

der-filling information through ascending projection path-

ways, directly or indirectly via periacqueductal gray.

Besides, the direct projections from MPA to the BN neurons

directly projecting to the LSC [7] were found. This result

involves the MPA function in control of BN that regulates

the micturition.

5. Conclusion

In conclusion, this study reveals the presence of a

projection from the Barrington’s nucleus to MPA and this

ascending serotonergic pathway may close a control loop.

Furthermore, the BN neurons send projection fibers to both

the MPA and LSC by way of serotonergic and non-seroto-

nergic axon collaterals. Future researches will include in-

vestigating the other possible neurochemical nature of these

BN projections.

Acknowledgements

This study was supported by MIUR. We thank Mr. Silvio

Bentivegna for help in adjustment of figure.

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