Serotonergic collateralized projections from Barrington's nucleus to the medial preoptic area and lumbo-sacral spinal cord

Brain Research 1019 (2004) 64 – 67 www.elsevier.com/locate/brainres

Research report

Serotonergic collateralized projections from Barrington’s nucleus to the medial preoptic area and lumbo-sacral spinal cord Antonella Russo a,*, Sebastiana Monaco a, Rosa Romeo b, Rosalia Pellitteri c, Stefania Stanzani a b

a Department of Physiological Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy Department 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 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 micturition center in different species [1 – 3,8,9]. This area is indicated as a small group of neurons lying just ventromedial to the rostral pole of the nucleus locus coeruleus (LC), and receives afferent fibers from brainstem nuclei and forebrain limbic structures. Electrical or chemical stimulation 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-

* Corresponding author. Tel.: +39-95-7384041; fax: +39-95-330645. E-mail addresses: [email protected] (A. Russo), [email protected] (S. Stanzani). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.03.080

strated that the neurons of Barrington’s nucleus (BN) 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 transneuronal virus labelling [10], c-fos expression [4], and physiological techniques [11]. In addition, BN receives widespread afferents from the brain including hypothalamus [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 transmission of signals [17] with corticotropin-releasing hormone (CRH) release. Nonetheless, there have been no systematic investigations focusing on various neurotransmitters in BN [14,18]. Here, we raise the question of whether single neurons in BN sending projection fibers to

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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 elucidate the circuitry of BN regulation to MPA and lumbosacral 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 demonstrate 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 (23 F 1 jC) and lighting (lights on 07:00 –19:00 h); laboratory chow diet and water were available ad libitum; the in

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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 rhodamine-labelled bead (RLB) were injected into MPA (stereotaxic 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 reanaesthetized and perfused through ascending aorta with 60 ml saline solution, followed by 300 ml ice-cold 4% paraformaldeyde 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% sucrose 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 fluorescence 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 antiserotonin antibody (Chemicon). The primary antibody was

Fig. 1. Injection zones in the CNS. Microphotographs showing injection zones: (A) RLB injection site and drawing in MPA (black area), scale bar = 400 Am; (B) FG injection site and drawing in LSC (black area), scale bar = 90 Am.

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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

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.

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) fluorescence. To determine the number of neurons containing both retrograde tracers, cell count was performed on the series

All injections of the retrograde tracers remained relatively localized as has previously reported. Fig. 1 shows microphotographs and schematic drawings example of the injection 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, demonstrating 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 summarized 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-HTpositive neurons are about 88.33% ipsilateral, and 69.31% Table 1 Ipsilateral and contralateral labelled cells in the whole Barrington’s nucleus

FG RLB FG + RLB FG + 5-HT RLB + 5-HT FG + RLB + 5-HT

Ipsilateral

Contralateral

17.5 F 0.1 8.8 F 1.7 5.4 F 1.0 10.3 F 1.0 6.1 F 0.2 1.8 F 0.2

12.8 F 0.3 6.0 F 0.8 2.1 F 0.3 8.4 F 0.4 5.3 F 0.2 No neurons

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

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

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 projection exists [7]; in addition, the presence of collateralized fibers of the BN neurons includes the possibility of the ascending and descending simultaneous control. The discovery that BN laterodorsal tegmental nucleus modulates micturition has been defined [1– 3,8,9]. BN receives bladder-filling information through ascending projection pathways, 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-serotonergic axon collaterals. Future researches will include investigating 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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