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Parafascicular nucleus-raphe projections and termination patterns in the rat
BRAIN RESEARCH Brain Research 690 (1995) 177-184
ELSEVIER
Research report
Parafascicular nucleus-raphe projections and termination patterns in the rat Gabriella Marini b, Giovanni Tredici a,, a lstituto di Anatomia Umana, Universitd degli Studi, via Mangiagalli 31, Milano, ltalv b lstituto di Neuroscienze e Bioimmagini-C.N.R., via Mario Bianco 9. Milano, Italy
Accepted 2 May 1995
Abstract
Thalamic projections from the parafascicularis nucleus to the raphe system were studied by means of anterograde techniques, utilizing both biocytin and dextran amine in rats. Both tracers injections in the parafascicularis nucleus resulted in the labeling of descending bundles of fibers running along the brainstem. Labeled terminal fields were found in all the raphe nuclei except the nucleus raphe pallidus. Three different types of labeled terminals (numerous small boutons, less numerous large claw-like terminals, varicosities in close apposition to blood vessel walls) originating from the parafascicular nucleus were present in the raphe system. Ultrastructural data suggest an inhibitory nature for the parafascicular-raphe projections. Our results confirm and extend previous retrograde data by indicating the trajectories, the terminal fields, the fine structure of terminal axonal arborizations and boutons. Based on previous retrograde data and our observations, we conclude that the relationship between the parafascicular nucleus and raphe system is reciprocal and concerns most of the raphe nuclei, suggesting that parafascicularis cell population may be involved in many of the functions ascribed to the raphe system. Keywords: Parafascicular nucleus; Thalamus; Raphe system; Biocytin; Dextran amine; Motor control; Rat
I. Introduction
The intralaminar thalamic nuclei (both rostral and caudal group) have long been considered as part of the nonspecific ascending activating system and, like the entire thalamus, mainly viewed as a gateway (open during waking, closed during sleep) through which signals must pass to reach the cortex. Therefore, anatomical investigations on the efferents of the intralaminar complex have particularly focused on their ascending projections [15,29]. The notion of the non-specificity of intralaminar projections has become less clear than it had previously been thought to be in view of subsequent anatomical retrograde studies showing that the intralaminar system and the caudal intralaminar neurons (centre median-parafascicularis, CMPF), in particular, project to restricted areas of the cerebral cortex revealing specificity of connections [for reviews see [8] and [18]]. The parafascicularis (PF) cells also give rise to dense
* Corresponding author. Fax: (39) (2) 2364082. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 q 3 ( 9 5 ) 0 0 6 1 7 - 6
projection innervation to the striatum [14,18,36]. The preferential targeting of parafascicular (PF) neurons to the striatum [2,36,37] and to structures known to be involved in the control of movements (e.g. subthalamic nucleus [32]), suggests their involvement in movement-related activities. In this context, it is noteworthy that recent experiments [20] have shown that stimulation of the lateral part of PF participates in the regulation of motor inhibition and that this effect is blocked by raphe lesion. The connections between the raphe system and the PF have been the subject of intense investigation by means of retrograde tracing methods. Most retrograde studies have addressed the issue of the ascending projections from the raphe nuclei to the PF [3,4,15,22,23,26,30]. The descending projection from the PF to the raphe nuclei have, however, been investigated less [25,31] and information has been presented in rather general terms, since the retrograde technique only provides information regarding the origin of the projection. The introduction of the recent techniques of labeling axonal ramifications down to the terminal boutons by anterograde transport has given an opportunity to investigate the possible descending projections from PF to raphe system, their spatial distribution and patterns of termina-
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tion. In this study we present anatomical evidence of the PF-raphe projections and report the detailed morphology of the axonal terminals visualized with anterograde transported biocytin [16] and biotinylated amine dextran [38]. In rodents, the PF is a unique element of the caudal group of the intralaminar complex of the thalamus and consists of cells located among the fibers of the internal medullary lamina posteriorly. The fasciculus retroflexus divides the PF into a thin rim of grey matter located medially, which does not send fibers to the raphe [31] and into a much larger lateral part, formed by small and medium-sized neurons [13], where the present injections were aimed. Preliminary results were reported at the 25th annual meeting of the European Neuroscience Association [191.
2. Materials and methods The present observations are based on the analysis of materials from 14 adult Wistar rats. All surgical procedures were carried out under anesthesia with chloral hydrate (300 mg/kg, i.p.). Animals received unilateral stereotaxic injections of biocytin (Sigma, 1.0-1.5 /xl of 0.05 M Tris-HCL buffer at pH = 7.6) (10 rats) or biotinylated amine dextran (Molecular Probes Inc.) 5% in double distilled water (4 rats) in the PF nucleus. The last mentioned animals have been used for electron microscopy
observations that will be the subject of a detailed report elsewhere. The tracers were freshly dissolved and pressure injected. Target coordinates, according to Paxinos and Watson's atlas [24] were: A - 4 . 2 ; L 1.3; H 5.2 with respect to bregma. Biocytin was used for light microscopy. The animals were allowed to survive 2 days after biocytin injections. They were then deeply reanesthetized, perfused transcardially with 200 ml saline solution (0.9%) followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer at pH = 7.4. The brain was dissected, cryoprotected for 2-3 days in 30% sucrose solution in phosphate buffer at 4°C. Frozen sections (50 /z thick) were cut in the frontal plane or in the horizontal plane with a cryostat and treated immunohistochemically (see [1,39]) using 3,3'-diaminobenzidine (DAB; Sigma) as chromogen, with nickel intensification. Sections were incubated overnight at room temperature with the avidinbiotin-peroxidase complex (standard Vectastain ABC Kit PK-4000 from Vector Laboratories, dilution l:500 in phosphate buffer with 1% Triton X-100), then incubated for 30 min in a similar solution to which 0.006% of hydrogen peroxide has been added and finally rinsed three times for 5 min in Tris-HCL buffer. After completion of all reactions, the sections were mounted onto slides, air-dried, dehydrated and coverslipped. To avoid misinterpretations in recognising labeled terminal varicosities, sections were examined unstained with
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Fig. 1. A: photomicrograph illustrating a typical biocytin injection site in the parafascicular nucleus (PF) unilaterally. Transverse section at approximately rostrocaudal level AP - 4 . 1 6 , illustrated in B (modified from Paxinos and Watson's atlas [22]). The tracer injection was centered in the nucleus and confined to the portion lateral to the fasciculus retroflexus (fr). Note that there is no spread of the tracer to the fr. Bar: 100 /xm.
G. Marini, G. Tredici / Brain Research 690 (1995) 177-184
both bright- and dark-field illumination. In selected slides, the cloverslip was removed in xylene, and the sections were rehydratated and counterstained with Cresyl violet or
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Neutral red, in order to relate the distributions of fibers and terminals to the cytoarchitecture of the corresponding brain regions.
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Fig. 2. In A: X - Y plotter reconstruction of three representative horizontal sections through the brainstem showing the distribution of anterogradely labeled fibers after injection of biocytin in PF. The sections are arranged in a dorsal to ventral direction. In B: bright-field photomicrograph illustrating the trajectory of densely labeled axons crossing at mid-pontine level. Vessels mark the position of the midline. Bar: 50 Arm.
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3. Results 3.1. Injection site
in the lateral part of the brainstem of both sides (Fig. 2A). These lateral components generally terminated in the trigeminal complex and pontine reticular formation.
Fig. 1 illustrates a typical biocytin injection site in the PF. The tracer injection was centered in the nucleus and confined to the portion lateral to the fasciculus retroflexus (fr), which was not contaminated. In some cases, a few neurons of the small medial part of the PF had also taken up the tracer. In two experiments, the injections were made slightly posteriorly to the borders of the PF and the pattern of anterograde labeling was quite different. Biotinylated amine dextran injections were similarly confined to the PF nucleus.
3.3. Distribution of labeled terminals in the raphe system
3.2. Location and trajectories o f PF-raphe labeled descending fibers
3.4. Features o f labeled terminals
After injection of biocytin or biotinylated amine dextran into the lateral part of the parafascicular nucleus, descending bundles of labeled fibers were found running along the rostral brainstem. In the raphe nuclei axons which ramified to give rise to the labeled terminals derived from the largest of these bundles. This was made up of 2 . 5 - 3 . 5 / x m fibers descending near the midline. The majority of the fibers crossed the midline at mid-pontine level (Fig. 2A, B) and formed a crossed bundles, which descended further as far as the spinal cord. Other minor, smaller bundles, consisting of a few thinner fibers (1.5-2 /zm), descended
Labeled terminals were found in all the raphe nuclei except for the nucleus raphe pallidus in the medulla oblungata. Labeled terminals were bilaterally distributed in all the raphe nuclei that receive a PF projection, but in the nucleus raphe dorsalis the majority of them were ipsilateral to the injection site. In the small raphe nuclei of the midline, labeled terminals were often seen arising as collaterals of the descending fibers which cross the midline.
Light microscope examination showed that the projections from the PF nucleus to the raphe nuclei exhibited three different types of terminals. Some were small boutons (both terminaux or en passant) (Fig. 3A) arising from the labeled fibers. Less frequently, but not rarely, thicker terminals were also observed originating from larger labeled fibers (Fig. 3B). These large terminals had a claw-like aspect with many short fingerlike processes that could clearly be seen at different planes of focusing (Fig. 4). A third type of labeled terminal was also observed: labeled axonal ramifications were seen coursing towards and around blood vessels exhibiting en passage varicosi-
Fig. 3. Light micrographsillustrating a few examplesof the three types of labeled terminals of the thalamo-rapheprojections. In A: arrows point to small en passant or terminaux bouton-likeendings. Bar: 50 /xm. In B: the open arrows indicate claw-like terminals; arrows indicate small terminaux boutons; arrowheads indicate axonal varicositiesin close appositionwith vessel wall. Bar: 20 /xm.
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t 81
Fig. 4. A: photomicrographs showing an example of a claw-like termination. Bar: 10/xm. B and C: different focal planes. Bar: 5/xm.
Fig. 5. Bright-field photomicrographs illustrating labeled varicosities in apposition to vessel wall (arrowheads). Bar: 10 p.m. In B: arrow points to a labeled collateral emerging from a long-running labeled fiber. Bar: 20 /.Lm.
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ties or terminal swellings in close apposition to the vessel wall (Fig. 3B and Fig. 5). Preliminary electron microscopy analysis showed that labeled terminal boutons contained pleomorphic vescicles and made symmetrical synaptic junctions suggesting an inhibitory nature for these synaptic terminals.
pallidus, receive the PF innervation is consistent with the data of Petrovicky [25]. The absence of raphe pallidus terminals originating from the PF has been confirmed in a recent retrograde study on the afferents to the nucleus raphe pallidus examined by iontophoretic application of cholera-toxin B [9]. 4.3. Features of labeled terminals
4. Discussion
Efferent connections from the intralaminar PF to the raphe system, in particular to the dorsal raphe nucleus [25] and the nucleus raphe magnus [31], in rats, have been alluded to in previous studies using retrograde tracing techniques, but these procedures are not suitable for visualizing the details of the course of axons and their terminations. The present study provides further anatomical evidence for a descending projection from the PF to the raphe system. Using the highly sensitive anterograde tracers biocytin, the trajectories, the pattern of termination, and the synaptic characteristics of this projection were elucidated. 4.1. Methodological considerations
The modern anterograde tracer substances biocytin and biotinylated amine dextran provide excellent sensitivity and resolution [16,27]. Another advantage of these tracers is that they are not taken up by fibers of passage [16,17,27], as is also demonstrated by the absence of contamination of the fibers of the fr in all our cases. Furthermore, biotinylated amine dextran permits superior labeling of terminals due to the fact that ultrastructural details are better preserved [27,41]. The anterograde techniques allow a global view of the efferents of a given cell population to be obtained and, specifically in the case of the PF, of the distribution of labeled terminals to the raphe system, including the small nuclei. Using retrograde axonal tracing, the difficulty in injecting the entire complex of the nuclei in the same animal may have resulted in the cells in the thalamus failing to be labeled [6]. In the present experiments, the injection site was confined almost exclusively to the lateral part of the PF, from where it had previously been shown, with retrograde markers, that the projection to the nucleus raphe magnus originates [31]. 4.2. Trajectories and distribution of labeled fibers
In all rats, descending bundles of labeled fibers were found running along the brainstem. Neither the main bundle, which ran medially and crossed the midline at midpontine level, nor the lateral bundles followed the course of known long-ascending and descending systems of the brainstem [33]. That all the raphe nuclei, except the nucleus raphe
This study provides new information about the morphology of the PF-raphe terminals. The three types of labeled terminals (numerous small boutons, less numerous giant claw-like terminals, and varicosities close to the vessels) in the raphe system originating from the thalamic PF nucleus support the presence of heterogeneous subpopulations in the PF [31], although their functional significance has yet to be elucidated. Conversely, the ultrastructural features of labeled synaptic boutons are similar. All labeled boutons contain pleomorphic vescicles and symmetrical junctions suggesting that the terminals originating from the PF are of an inhibitory nature ([7,35], see for review: [34]). Regarding the first two types of labeled endings, they are reminiscent of the two morphologically distinct terminals (small and giant boutons) observed in the cortico-thalamic sensory [10,11] and motor systems [28]. The claw-like terminals (as suggested for the giant endings in the corticothalamic terminals) may provide strong synaptic transmissions. With respect to the giant endings, the fingerlike processes of the PF-raphe claw-like terminals have an even larger area of apposition with the contacted postsynaptic elements and, therefore, may enhance the synaptic strength even further. 4.4. Functional significance of the PF-raphe projection
The serotoninergic raphe system facilitates motor output and inhibits sensory information processing [12]. The long-held belief that the raphe system is involved in nociception could be explained by the mechanism of sensory depression, since serotoninergic raphe neurons are activated neither by a variety of painful stimuli [12] nor by an analgesic dose of morphine [12]. We present anatomical evidence that the intralaminar PF neurons have dense projections to the raphe system. Such close anatomical connections suggest an involvement of the PF in motor control and sensory modulation. In this context, it is noteworthy that recent findings in rats showed that the PF receives corticothalamic input (collaterals of long-range corticofugal axons) from layer V cells supporting a role in movement execution [5]. Additional support to the concept of a state-dependent tonic facilitation of spinal motor mechanisms by the serotoninergic system is the known suppression of spontaneous activity of raphe neurons during rapid eye movement sleep (see [12]), which is accompanied by a marked reflex depression and atonia. Our electron microscopic observations suggest that the PF-raphe
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projection m a y be o f an inhibitory nature. It is t e m p t i n g to speculate that the PF inhibitory input to raphe nuclei m a y participate in the c o m p l e x intrinsic drive of R E M sleep-related processes. Further lesion and single-unit activity e x p e r i m e n t s in Pf nuclei are n e e d e d to c o n f i r m this. A s regards the labeled axonal ramifications originating f r o m PF-neurons, c o u r s i n g towards and around b l o o d vessels and exhibiting b o u t o n swellings, these m a y represent the possible anatomical substrate for m e d i a t i n g the cerebrovascular responses, recently found after stimulation o f the caudal intralaminar c o m p l e x in rats [21]. G i v e n the high density o f b l o o d vessels in the raphe c o m p l e x , a possible function o f raphe neurons as c h e m o s e n s o r s for circulating substances has been s u g g e s t e d [40]. H o w e v e r , studies e x a m i n i n g unit activity of raphe neurons and b l o o d pressure in rats have failed to find any relationship [12]. The present light m i c r o s c o p i c observations suggest a possible thalamic rather than a rapheal influence on cardiovascular function.
Acknowledgements T h e help p r o v i d e d by Dr. Sara Tazzari and Dr. Laura Torri Tarelli in the preliminary e l e c t r o n m i c r o s c o p y study is greatly appreciated. The authors w i s h to a c k n o w l e d g e to excellent technical w o r k o f O s v a l d o De N e g r i and Roberto G i g l i o and to thank Dr. L. Jenton for reading the manuscript.
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[31] Senba, E., Takagi, H., Shiosaka, S. et al., On the afferent projections from some dieneephalic nuclei to n. raph6 magnus of the rat, Brain Res., 211 (1981) 387-392. [32] Sugimoto, T., Hattori, T., Mizuno, N., Itoh, K. and Sato, M., Direct projections from the centre median-parafascicular complex to the subthalamic nucleus in the cat and rat, J. Comp. Neurol., 241 (1983) 209-216. [33] Tork, I., Raphe nuclei and serotonin containing systems. In G. Paxinos (Ed.), The Rat Nervous System, Academic Press, Sydney, 1985, pp. 43-78. [34] Tredici, G., Torri Tarelli, L., Cavaletti, G. and Marmiroli, P., Ultrastruetural organization of lamina VI of the spinal cord of the rat, Prog. Neurobiol., 24 (1985) 293-331. [35] Uchizono, K., Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat, Nature, 207 (1965) 642-643. [36] Van der Kooy, D., The organization of the thalamic, nigral and raph6 cells projecting to the medial vs. lateral caudate-putamen in rat. A fluorescent retrograde double labeling study, Brain Res., 169 (1979) 381-387.
[37] Veening, J.G., Cornelissen. F.M. and Lieven, P.A.J.M., The topical organization of the afferents to the caudoputamen of the rat: a horseradish peroxidase study, Neuroscience, 5 (1980) 1253-1268. [38] Veenman, C.L., Reiner, A. and Honig, M.G., Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies, J. Neurosci. Methods, 41 (1992) 239-254. [39] Wan, X.S.T., Liang, F., Moret, V., Wiesendanger, M. and Rouiller, E.M., Mapping of the motor pathways in rats: c-los induction by intraconical microstimulation of the motor cortex correlated with afferent connectivity of the site of cortical stimulation. Neuroscience, 49 (1992) 749-761. [40] Wolf, W.A., Kuhn, D.M. and Lovenberg, W., In P.M. Vanhoutte (Ed.), Serotonin and Central Regulation Of Arterial Blood Pressure, Raven, New York, 1985, pp. 63-73. [41] Wouterlood, F.G. and Jorritsma, B., The anterograde neuroanatomical tracer biotinylated dextran-amine: comparison with the tracer Phaseolus t,ulgaris-leucoagglutinin in preparations for electron microscopy, J. Neurosci. Methods, 48 (1993) 75-87.
ELSEVIER
Research report
Parafascicular nucleus-raphe projections and termination patterns in the rat Gabriella Marini b, Giovanni Tredici a,, a lstituto di Anatomia Umana, Universitd degli Studi, via Mangiagalli 31, Milano, ltalv b lstituto di Neuroscienze e Bioimmagini-C.N.R., via Mario Bianco 9. Milano, Italy
Accepted 2 May 1995
Abstract
Thalamic projections from the parafascicularis nucleus to the raphe system were studied by means of anterograde techniques, utilizing both biocytin and dextran amine in rats. Both tracers injections in the parafascicularis nucleus resulted in the labeling of descending bundles of fibers running along the brainstem. Labeled terminal fields were found in all the raphe nuclei except the nucleus raphe pallidus. Three different types of labeled terminals (numerous small boutons, less numerous large claw-like terminals, varicosities in close apposition to blood vessel walls) originating from the parafascicular nucleus were present in the raphe system. Ultrastructural data suggest an inhibitory nature for the parafascicular-raphe projections. Our results confirm and extend previous retrograde data by indicating the trajectories, the terminal fields, the fine structure of terminal axonal arborizations and boutons. Based on previous retrograde data and our observations, we conclude that the relationship between the parafascicular nucleus and raphe system is reciprocal and concerns most of the raphe nuclei, suggesting that parafascicularis cell population may be involved in many of the functions ascribed to the raphe system. Keywords: Parafascicular nucleus; Thalamus; Raphe system; Biocytin; Dextran amine; Motor control; Rat
I. Introduction
The intralaminar thalamic nuclei (both rostral and caudal group) have long been considered as part of the nonspecific ascending activating system and, like the entire thalamus, mainly viewed as a gateway (open during waking, closed during sleep) through which signals must pass to reach the cortex. Therefore, anatomical investigations on the efferents of the intralaminar complex have particularly focused on their ascending projections [15,29]. The notion of the non-specificity of intralaminar projections has become less clear than it had previously been thought to be in view of subsequent anatomical retrograde studies showing that the intralaminar system and the caudal intralaminar neurons (centre median-parafascicularis, CMPF), in particular, project to restricted areas of the cerebral cortex revealing specificity of connections [for reviews see [8] and [18]]. The parafascicularis (PF) cells also give rise to dense
* Corresponding author. Fax: (39) (2) 2364082. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 q 3 ( 9 5 ) 0 0 6 1 7 - 6
projection innervation to the striatum [14,18,36]. The preferential targeting of parafascicular (PF) neurons to the striatum [2,36,37] and to structures known to be involved in the control of movements (e.g. subthalamic nucleus [32]), suggests their involvement in movement-related activities. In this context, it is noteworthy that recent experiments [20] have shown that stimulation of the lateral part of PF participates in the regulation of motor inhibition and that this effect is blocked by raphe lesion. The connections between the raphe system and the PF have been the subject of intense investigation by means of retrograde tracing methods. Most retrograde studies have addressed the issue of the ascending projections from the raphe nuclei to the PF [3,4,15,22,23,26,30]. The descending projection from the PF to the raphe nuclei have, however, been investigated less [25,31] and information has been presented in rather general terms, since the retrograde technique only provides information regarding the origin of the projection. The introduction of the recent techniques of labeling axonal ramifications down to the terminal boutons by anterograde transport has given an opportunity to investigate the possible descending projections from PF to raphe system, their spatial distribution and patterns of termina-
178
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tion. In this study we present anatomical evidence of the PF-raphe projections and report the detailed morphology of the axonal terminals visualized with anterograde transported biocytin [16] and biotinylated amine dextran [38]. In rodents, the PF is a unique element of the caudal group of the intralaminar complex of the thalamus and consists of cells located among the fibers of the internal medullary lamina posteriorly. The fasciculus retroflexus divides the PF into a thin rim of grey matter located medially, which does not send fibers to the raphe [31] and into a much larger lateral part, formed by small and medium-sized neurons [13], where the present injections were aimed. Preliminary results were reported at the 25th annual meeting of the European Neuroscience Association [191.
2. Materials and methods The present observations are based on the analysis of materials from 14 adult Wistar rats. All surgical procedures were carried out under anesthesia with chloral hydrate (300 mg/kg, i.p.). Animals received unilateral stereotaxic injections of biocytin (Sigma, 1.0-1.5 /xl of 0.05 M Tris-HCL buffer at pH = 7.6) (10 rats) or biotinylated amine dextran (Molecular Probes Inc.) 5% in double distilled water (4 rats) in the PF nucleus. The last mentioned animals have been used for electron microscopy
observations that will be the subject of a detailed report elsewhere. The tracers were freshly dissolved and pressure injected. Target coordinates, according to Paxinos and Watson's atlas [24] were: A - 4 . 2 ; L 1.3; H 5.2 with respect to bregma. Biocytin was used for light microscopy. The animals were allowed to survive 2 days after biocytin injections. They were then deeply reanesthetized, perfused transcardially with 200 ml saline solution (0.9%) followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer at pH = 7.4. The brain was dissected, cryoprotected for 2-3 days in 30% sucrose solution in phosphate buffer at 4°C. Frozen sections (50 /z thick) were cut in the frontal plane or in the horizontal plane with a cryostat and treated immunohistochemically (see [1,39]) using 3,3'-diaminobenzidine (DAB; Sigma) as chromogen, with nickel intensification. Sections were incubated overnight at room temperature with the avidinbiotin-peroxidase complex (standard Vectastain ABC Kit PK-4000 from Vector Laboratories, dilution l:500 in phosphate buffer with 1% Triton X-100), then incubated for 30 min in a similar solution to which 0.006% of hydrogen peroxide has been added and finally rinsed three times for 5 min in Tris-HCL buffer. After completion of all reactions, the sections were mounted onto slides, air-dried, dehydrated and coverslipped. To avoid misinterpretations in recognising labeled terminal varicosities, sections were examined unstained with
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Fig. 1. A: photomicrograph illustrating a typical biocytin injection site in the parafascicular nucleus (PF) unilaterally. Transverse section at approximately rostrocaudal level AP - 4 . 1 6 , illustrated in B (modified from Paxinos and Watson's atlas [22]). The tracer injection was centered in the nucleus and confined to the portion lateral to the fasciculus retroflexus (fr). Note that there is no spread of the tracer to the fr. Bar: 100 /xm.
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both bright- and dark-field illumination. In selected slides, the cloverslip was removed in xylene, and the sections were rehydratated and counterstained with Cresyl violet or
179
Neutral red, in order to relate the distributions of fibers and terminals to the cytoarchitecture of the corresponding brain regions.
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Fig. 2. In A: X - Y plotter reconstruction of three representative horizontal sections through the brainstem showing the distribution of anterogradely labeled fibers after injection of biocytin in PF. The sections are arranged in a dorsal to ventral direction. In B: bright-field photomicrograph illustrating the trajectory of densely labeled axons crossing at mid-pontine level. Vessels mark the position of the midline. Bar: 50 Arm.
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3. Results 3.1. Injection site
in the lateral part of the brainstem of both sides (Fig. 2A). These lateral components generally terminated in the trigeminal complex and pontine reticular formation.
Fig. 1 illustrates a typical biocytin injection site in the PF. The tracer injection was centered in the nucleus and confined to the portion lateral to the fasciculus retroflexus (fr), which was not contaminated. In some cases, a few neurons of the small medial part of the PF had also taken up the tracer. In two experiments, the injections were made slightly posteriorly to the borders of the PF and the pattern of anterograde labeling was quite different. Biotinylated amine dextran injections were similarly confined to the PF nucleus.
3.3. Distribution of labeled terminals in the raphe system
3.2. Location and trajectories o f PF-raphe labeled descending fibers
3.4. Features o f labeled terminals
After injection of biocytin or biotinylated amine dextran into the lateral part of the parafascicular nucleus, descending bundles of labeled fibers were found running along the rostral brainstem. In the raphe nuclei axons which ramified to give rise to the labeled terminals derived from the largest of these bundles. This was made up of 2 . 5 - 3 . 5 / x m fibers descending near the midline. The majority of the fibers crossed the midline at mid-pontine level (Fig. 2A, B) and formed a crossed bundles, which descended further as far as the spinal cord. Other minor, smaller bundles, consisting of a few thinner fibers (1.5-2 /zm), descended
Labeled terminals were found in all the raphe nuclei except for the nucleus raphe pallidus in the medulla oblungata. Labeled terminals were bilaterally distributed in all the raphe nuclei that receive a PF projection, but in the nucleus raphe dorsalis the majority of them were ipsilateral to the injection site. In the small raphe nuclei of the midline, labeled terminals were often seen arising as collaterals of the descending fibers which cross the midline.
Light microscope examination showed that the projections from the PF nucleus to the raphe nuclei exhibited three different types of terminals. Some were small boutons (both terminaux or en passant) (Fig. 3A) arising from the labeled fibers. Less frequently, but not rarely, thicker terminals were also observed originating from larger labeled fibers (Fig. 3B). These large terminals had a claw-like aspect with many short fingerlike processes that could clearly be seen at different planes of focusing (Fig. 4). A third type of labeled terminal was also observed: labeled axonal ramifications were seen coursing towards and around blood vessels exhibiting en passage varicosi-
Fig. 3. Light micrographsillustrating a few examplesof the three types of labeled terminals of the thalamo-rapheprojections. In A: arrows point to small en passant or terminaux bouton-likeendings. Bar: 50 /xm. In B: the open arrows indicate claw-like terminals; arrows indicate small terminaux boutons; arrowheads indicate axonal varicositiesin close appositionwith vessel wall. Bar: 20 /xm.
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t 81
Fig. 4. A: photomicrographs showing an example of a claw-like termination. Bar: 10/xm. B and C: different focal planes. Bar: 5/xm.
Fig. 5. Bright-field photomicrographs illustrating labeled varicosities in apposition to vessel wall (arrowheads). Bar: 10 p.m. In B: arrow points to a labeled collateral emerging from a long-running labeled fiber. Bar: 20 /.Lm.
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ties or terminal swellings in close apposition to the vessel wall (Fig. 3B and Fig. 5). Preliminary electron microscopy analysis showed that labeled terminal boutons contained pleomorphic vescicles and made symmetrical synaptic junctions suggesting an inhibitory nature for these synaptic terminals.
pallidus, receive the PF innervation is consistent with the data of Petrovicky [25]. The absence of raphe pallidus terminals originating from the PF has been confirmed in a recent retrograde study on the afferents to the nucleus raphe pallidus examined by iontophoretic application of cholera-toxin B [9]. 4.3. Features of labeled terminals
4. Discussion
Efferent connections from the intralaminar PF to the raphe system, in particular to the dorsal raphe nucleus [25] and the nucleus raphe magnus [31], in rats, have been alluded to in previous studies using retrograde tracing techniques, but these procedures are not suitable for visualizing the details of the course of axons and their terminations. The present study provides further anatomical evidence for a descending projection from the PF to the raphe system. Using the highly sensitive anterograde tracers biocytin, the trajectories, the pattern of termination, and the synaptic characteristics of this projection were elucidated. 4.1. Methodological considerations
The modern anterograde tracer substances biocytin and biotinylated amine dextran provide excellent sensitivity and resolution [16,27]. Another advantage of these tracers is that they are not taken up by fibers of passage [16,17,27], as is also demonstrated by the absence of contamination of the fibers of the fr in all our cases. Furthermore, biotinylated amine dextran permits superior labeling of terminals due to the fact that ultrastructural details are better preserved [27,41]. The anterograde techniques allow a global view of the efferents of a given cell population to be obtained and, specifically in the case of the PF, of the distribution of labeled terminals to the raphe system, including the small nuclei. Using retrograde axonal tracing, the difficulty in injecting the entire complex of the nuclei in the same animal may have resulted in the cells in the thalamus failing to be labeled [6]. In the present experiments, the injection site was confined almost exclusively to the lateral part of the PF, from where it had previously been shown, with retrograde markers, that the projection to the nucleus raphe magnus originates [31]. 4.2. Trajectories and distribution of labeled fibers
In all rats, descending bundles of labeled fibers were found running along the brainstem. Neither the main bundle, which ran medially and crossed the midline at midpontine level, nor the lateral bundles followed the course of known long-ascending and descending systems of the brainstem [33]. That all the raphe nuclei, except the nucleus raphe
This study provides new information about the morphology of the PF-raphe terminals. The three types of labeled terminals (numerous small boutons, less numerous giant claw-like terminals, and varicosities close to the vessels) in the raphe system originating from the thalamic PF nucleus support the presence of heterogeneous subpopulations in the PF [31], although their functional significance has yet to be elucidated. Conversely, the ultrastructural features of labeled synaptic boutons are similar. All labeled boutons contain pleomorphic vescicles and symmetrical junctions suggesting that the terminals originating from the PF are of an inhibitory nature ([7,35], see for review: [34]). Regarding the first two types of labeled endings, they are reminiscent of the two morphologically distinct terminals (small and giant boutons) observed in the cortico-thalamic sensory [10,11] and motor systems [28]. The claw-like terminals (as suggested for the giant endings in the corticothalamic terminals) may provide strong synaptic transmissions. With respect to the giant endings, the fingerlike processes of the PF-raphe claw-like terminals have an even larger area of apposition with the contacted postsynaptic elements and, therefore, may enhance the synaptic strength even further. 4.4. Functional significance of the PF-raphe projection
The serotoninergic raphe system facilitates motor output and inhibits sensory information processing [12]. The long-held belief that the raphe system is involved in nociception could be explained by the mechanism of sensory depression, since serotoninergic raphe neurons are activated neither by a variety of painful stimuli [12] nor by an analgesic dose of morphine [12]. We present anatomical evidence that the intralaminar PF neurons have dense projections to the raphe system. Such close anatomical connections suggest an involvement of the PF in motor control and sensory modulation. In this context, it is noteworthy that recent findings in rats showed that the PF receives corticothalamic input (collaterals of long-range corticofugal axons) from layer V cells supporting a role in movement execution [5]. Additional support to the concept of a state-dependent tonic facilitation of spinal motor mechanisms by the serotoninergic system is the known suppression of spontaneous activity of raphe neurons during rapid eye movement sleep (see [12]), which is accompanied by a marked reflex depression and atonia. Our electron microscopic observations suggest that the PF-raphe
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projection m a y be o f an inhibitory nature. It is t e m p t i n g to speculate that the PF inhibitory input to raphe nuclei m a y participate in the c o m p l e x intrinsic drive of R E M sleep-related processes. Further lesion and single-unit activity e x p e r i m e n t s in Pf nuclei are n e e d e d to c o n f i r m this. A s regards the labeled axonal ramifications originating f r o m PF-neurons, c o u r s i n g towards and around b l o o d vessels and exhibiting b o u t o n swellings, these m a y represent the possible anatomical substrate for m e d i a t i n g the cerebrovascular responses, recently found after stimulation o f the caudal intralaminar c o m p l e x in rats [21]. G i v e n the high density o f b l o o d vessels in the raphe c o m p l e x , a possible function o f raphe neurons as c h e m o s e n s o r s for circulating substances has been s u g g e s t e d [40]. H o w e v e r , studies e x a m i n i n g unit activity of raphe neurons and b l o o d pressure in rats have failed to find any relationship [12]. The present light m i c r o s c o p i c observations suggest a possible thalamic rather than a rapheal influence on cardiovascular function.
Acknowledgements T h e help p r o v i d e d by Dr. Sara Tazzari and Dr. Laura Torri Tarelli in the preliminary e l e c t r o n m i c r o s c o p y study is greatly appreciated. The authors w i s h to a c k n o w l e d g e to excellent technical w o r k o f O s v a l d o De N e g r i and Roberto G i g l i o and to thank Dr. L. Jenton for reading the manuscript.
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