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Cholecystokinin and substance P immunoreactive projections to the paraventricular thalamic nucleus in the rat
Neuroscience Research 51 (2005) 383–394 www.elsevier.com/locate/neures
Cholecystokinin and substance P immunoreactive projections to the paraventricular thalamic nucleus in the rat Kazuyoshi Otake* Section of Neuroanatomy, Department of Systems Neuroscience, Division of Cognitive and Behavioral Medicine, Tokyo Medical and Dental University Graduate School, Tokyo 113-8519, Japan Received 28 August 2004; accepted 9 December 2004 Available online 15 January 2005
Abstract Cholecystokinin (CCK) and substance P (SP) are thought to play an important role in a variety of stress responses. Both CCK- and SPpositive fibers innervating the thalamus are found principally in the midline nuclei, including the paraventricular thalamic nucleus (PVT), which has strong reciprocal connections with the medial prefrontal cortex. In the present study, we determined the source of the CCK- and SPimmunoreactive fibers to the PVT, employing combination of retrograde neuronal tracing and immunohistochemistry in the rat. The PVTprojecting neurons showing CCK immunoreactivity were detected in the dorsomedial nucleus of the hypothalamus, and ventral mesencephalic periaqueductal gray, including the Edinger-Westphal nucleus and the dorsal raphe nucleus. Sources of SP afferents to the PVT were detected in the Edinger-Westphal nucleus, the mesopontine tegmentum and the medullary raphe nucleus. CCK- and SP-immunoreactive fibers may exert modulatory influence on the prefrontal cortical activity via the PVT and regulate behavioral components of stress-adaptation responses. # 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Midline thalamus; Cholecystokinin; Substance P; Retrograde tracing; Immunohistochemistry; Dorsomedial hypothalamic nucleus; EdingerWestphal nucleus; Stress
1. Introduction The densities of neuropeptide-immunoreactive cells and fibers in the thalamus have been reported as being relatively low with a few notable exceptions. Among these exceptions, the midline region of the thalamus, whose functions are not well understood, is heavily innervated by various peptidergic fibers, e.g. substance P (SP), cholecystokinin (CCK), a corticotrophin-releasing factor, neuropeptide Y, and somatostatin (Bentivoglio et al., 1991). The paraventricular thalamic nucleus (PVT), the most dorsal component of the thalamic midline, is known to be strongly activated following a variety of stressors and thus might be suggested to play a role in stress-related information targeted for viscerolimbic areas in the brain.
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Among neuropeptides innervating the PVT, SP and CCK are of particular interest, due to the roles ascribed to these peptides as neuromodulators in the integrated hypothalamic stress response, mediating stress–neuroendocrine interaction (Siegel et al., 1987). SP controls vomiting and various behavioral, neurochemical and cardiovascular responses to stress, besides its best-known role as a pain neurotransmitter. For example, recent clinical trials have confirmed the efficacy of NK1 receptor (i.e., SP receptor) antagonists to alleviate depression and emesis (Rupniak and Kramer, 1999). CCK has been shown to be involved in numerous physiological functions such as feeding behavior, central respiratory control and cardiovascular tonus, vigilance states, memory processes, nociception, and emotional and motivational responses (Fink et al., 1998; Noble and Roques, 1999) and has been associated with several neuropsychiatric diseases such as schizophrenia as well as anxiety and panic attacks (Bourin et al., 1998; Bradwejn et al., 1995).
0168-0102/$ – see front matter # 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2004.12.009
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Although it seems important to identify the origins of the SP-ergic and CCK-ergic innervation to the PVT in the contexts mentioned above, only limited researchers have investigated this issue so far. For example, Bhatnager et al. (2000) identified the origins of CCK innervations mainly in brainstem areas, including the lateral parabrachial, periaqueductal gray and dorsal raphe. However, previous findings of altered CCK levels in various hypothalamic regions during acute stress exposure suggested the importance of hypothalamic CCK, related to the negative feedback actions of glucocorticoids (Siegel et al., 1987). Intracerebroventricular administration of CCK produced a wide spectrum of endocrine effects, likely via a hypothalamic site of action (Vijayan et al., 1979). The origin of SP innervation of the PVT has not been so far documented. The aim of the present study was therefore to determine the detailed sites of origin of CCK- and SP-projection fibers to the PVT by immunohistochemistry for CCK and SP combined with retrograde tract tracing. A preliminary report of this study was presented in abstract form (Otake and Nakamura, 2003).
2. Materials and methods 2.1. Animals and tissue preparation Sprague–Dawley rats (male; 200–250 g; Nippon BioSupp. Center, Tokyo, Japan) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). All efforts were made to minimize the number of animals used and their suffering. Injections of a 1% solution of cholera toxin B-subunit (CT-b, Research Biochemicals International, Natick, MA, USA) diluted with 0.2 M sodium phosphate buffer (PB, pH 7.5) were delivered from glass micropipettes in the dorsal midline thalamus using coordinates from the atlas of Paxinos and Watson (Paxinos and Watson, 1998). Iontophoretic ejections were made using positive pulses (7 mA, 7 s on/off) from a Midgard precision current source (Stoelting, Wood Dale, IL, USA) for 15– 20 min. Three to four days later, these rats were reanesthetized and colchicine (150 mg/10 ml saline) was slowly injected unilaterally into the lateral ventricle to build up transmitter concentration in cell bodies, and then, after 1 more day, the animals were re-anesthetized and perfused transcardially with 200 ml heparinized saline followed by 500 ml of Zamboni’s fixative. The surgical procedures and pre- and post-operative care of the animals were approved by the Tokyo Medical and Dental University Animal Care Committee and were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication 80-23, revised 1996). The brains were removed and immersed overnight at 4 8C in 0.1 M PB containing 10% sucrose. They were sectioned in the transverse plane at 40-mm on a freezing microtome and
each 1-in-4 series was immunostained by a double or triple labeling method as follows. All histological procedures were carried out at room temperature. 2.2. Double immunohistochemistry for CT-b and CCK/CT-b and SP Two 1-in-4 series were processed for CT-b and SP or CCK reactivity. The sections were immersed for 1 h in PB containing 1% bovine serum albumin (Wako, Osaka, Japan) and incubated overnight in a mixture of goat anti-CT-b antiserum (1:20,000; List Biological, Inc., Campbell, CA, USA) and rabbit anti-SP (1:200; Peninsula Labs, Belmont, CA, USA) or rabbit anti-CCK antibody (1:200; Chemicon, Temecula, CA, USA). The sections were rinsed in PB and then incubated for 2 h in biotinylated horse anti-goat IgG (1:1000; Vector, Burlingame, CA, USA). After additional rinsing, sections were immersed for 2 h in a mixture of avidin-TRITC (1:250; Vector) and FITC-conjugated swine anti-rabbit IgG (1:20; DakoCytomation, Glostrup, Denmark). Sections were mounted in a glycerol-mounting medium containing 0.1% p-phenylenediamine (Sigma, Saint-Louis, MO, USA), examined in a Nikon fluorescent microscope (Eclipse E600) equipped with appropriate filter combinations, and photographed by using a cooled CCD camera (Keyence VB 6000/6010, Osaka, Japan) attached to a microscope. The distribution of single- and multi-labeled neurons was mapped onto drawings prepared from digital photomontage of the total area of the representative sections. 2.3. Triple immunohistochemistry for CT-b, SP and serotonin Since many SP-containing neurons also contained serotonin in the raphe nuclei in cat (Arvidsson et al., 1994; Lovick and Hunt, 1983) as well as in human (Baker et al., 1991), coexistence of serotonin with SP and CT-b immunoreactivity was analyzed by use of the triple-labeling technique. A 1-in-4 series through the medulla was first processed for SP and serotonin using the same procedure. In brief, the sections were incubated overnight in a mixture of rat anti-serotonin (1:100; Chemicon) and rabbit anti-SP antiserum (1:200; Peninsula). The sections were placed for 2 h in biotinylated goat anti-rat antibody (1:1000; Vector), and then for 2 h in a mixture of avidin-TRITC (1:250; Vector) and FITC-conjugated swine anti-rabbit IgG (1:20; DakoCytomation). Sections were mounted in a glycerolmounting medium. After examining and photographing the relevant areas, the sections were then immunostained for CT-b. To eliminate potential cross-reaction between avidin/ biotin system reagents, the sections first went through a blocking procedure (Ferri et al., 1999); the sections were incubated in the free avidin solution (Avidin/Biotin Blocking Kit, #SP-2001, Vector) for 40 min, in 10% formaldehyde for 40 min, and then in a free biotin solution
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(Avidin/Biotin Blocking Kit, #SP-2001, Vector) for 40 min. The sections were then incubated overnight in goat anti-CTb antiserum (1:40,000; List), for 45 min in biotinylated horse anti-goat IgG (1:2000) and for 30 min in avidin/biotin peroxidase complex (ABC Elite kit; Vector). Enzymatic development was done in a solution of 0.02% diaminobenzidine (Sigma) in 0.1 M Tris–buffer containing 0.003 and 0.6% nickel ammonium sulfate. The control experiment was conducted to verify the absence of cross-reactivity by processing sections from a rat without CT-b injection in the same triple-labeling protocol. 2.4. Double immunohistochemistry for SP and ChAT Almost all of the SP-containing neurons in the rat mesopontine tegmentum have been shown to be immunoreactive for choline acetyltransferase (ChAT) (Standaert et al., 1986; Sutin and Jacobowitz, 1988; Vincent et al., 1983, 1986). We conducted double immunohistochemistry for SP and ChAT to confirm the co-localization of these two substances. The sections were incubated overnight in a mixture of mouse anti-ChAT (1:100; Chemicon) and rabbit anti-SP antiserum (1:200; Peninsula). The sections were placed for 2 h in biotinylated horse anti-mouse antibody (1:1000; Vector), and then for 2 h in a mixture of avidinTRITC (1:250; Vector) and FITC-conjugated swine antirabbit IgG (1:20; DakoCytomation). Sections were mounted in a glycerol-mounting medium.
3. Results 3.1. Injection sites and patterns of retrograde transport In the dorsal part of the diencephalon, CCK- (Fig. 1) and SP-immunoreactive varicose fibers and punctate processes (Fig. 2a) were heavily concentrated in the PVT. Five cases were selected on the basis that the injection sites of CT-b were limited to the PVT and overlapped the area displaying the heavy CCK or SP innervation (Fig. 2b). The pattern of retrograde labeling was similar to that reported in our previous studies (Otake et al., 1994, 2002; Otake and Ruggiero, 1995). 3.2. CCK-immunoreactive afferents Principal sources of CCK-immunoreactive PVT afferents were detected in the hypothalamus and mesencephalon. The caudal part of the hypothalamus was the main forebrain region containing double-labeled neurons. CCKcontaining neurons heavily concentrated in the lateral region of the compact part of the dorsomedial hypothalamus at its most caudal level (Paxinos and Watson, 1998). Circumscribed subsets of CT-b-labeled neurons were found in this hypothalamic nucleus which overlaps the distribution of the CCK-immunoreactive neurons. Almost
Fig. 1. CCK innervation of the PVT: (a) the region of the PVT is indicated by the box in a section of rat forebrain and (b) photomicrograph showing axonal fibers and terminal-like varicosities immunoreactive for CCK in the PVT, corresponding to the boxed area in (a).
half of the CT-b-labeled neurons in this region (47.5 6.3%; n = 5) were also immunoreactive for CCK (Fig. 3). In the forebrain, the paraventricular hypothalamic nucleus and supramammillary nucleus occasionally contained double-labeled neurons. In the midbrain, double-labeled neurons were distributed in the midline of the ventral part of the mesencephalic periaqueductal gray (Fig. 4), corresponding to the EdingerWestphal nucleus, and the rostral linear raphe nucleus as well as the dorsal raphe nucleus. Small numbers of doublelabeled neurons were consistently found in the rostral part of the Edinger-Westphal nucleus. Although CCK immunoreactivity was present in a large proportion of the mesencephalic dopamine neurons, i.e., ventral tegmental area, no double-labeled cells were found in this area (not illustrated). In the pons, the lateral parabrachial nucleus occasionally contained double-labeled neurons (not illustrated).
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Fig. 2. The photomicrographs of the PVT and its vicinity showing axonal fibers and terminal-like varicosities immunoreactive for SP (a) and a CT-b deposit (b) observed on the same section under different illumination. Some neurons retrogradely labeled from CT-b (d) are immunoreactive for SP (c) on the same section under different illumination in the mesopontine tegmentum. Double-labeled neurons are indicated with arrows in (c) and (d).
3.3. SP-immunoreactive afferents Principal sources of SP-immunoreactive PVT afferents were detected in regions in the brainstem. Besides, doublelabeled cells were occasionally detected in the medial preoptic area, ventrolateral and dorsomedial hypothalamic nuclei and medial prefrontal cortex. In the midbrain, SP-immunoreactive neurons were widely distributed in the periaqueductal gray, and doublelabeled neurons were observed especially in the midline of its ventral part (Fig. 5). This median area corresponds to the Edinger-Westphal nucleus. Double-labeled neurons in the Edinger-Westphal nucleus were detected from the level of the Bragma 5.20 to 6.04, corresponding to the whole extent of the red nucleus. In each section, more than 50% (up to 100%) of retrogradely labeled neurons were also stained with SP, although many the EdingerWestphal neurons were stained only with SP. The SP-positive cells in the area of Edinger-Westphal nucleus seem to extend further rostrally than CCK-containing neurons. In the mesopontine tegmentum, double-labeled neurons were consistently observed in the laterodorsal tegmental
nucleus and lateral parabrachial nucleus (Fig. 6). Doublelabeling experiments of ChAT and SP confirmed that all of the SP-containing neurons in this area are also immunoreactive for ChAT (Fig. 7), indicating that PVT-projecting SP-immunoreactive neurons are cholinergic. The pedunculopontine tegmental nucleus also contained double-labeled neurons. In the medulla, double-labeled neurons were distributed in the midline region corresponding to the raphe nuclei (Figs. 8 and 9). The degree of coexistence (percentage of double-labeled cells) varied with the medullary level. The nucleus raphe magnus, at more rostral locations, showed a lower proportion of double labeling. Most of the doublelabeled neurons in the raphe were found in the nucleus raphe obscurus. Few double-labeled neurons were detected in the raphe pallidus (Fig. 9). From triple-labeled sections it was concluded that many PVT projecting neurons containing SP also contained serotonin. As shown in Fig. 9, a subpopulation of serotonin-immunoreactive neurons lacked SP-immunoreactivity and a subpopulation of SPimmunoreactive neurons lacked serotonin-immunoreactivity. Quantitatively, 63.4 22.2% of PVT-projecting neurons were immunoreactive for SP, 51.0 33.8% of which
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Fig. 3. (a) Line drawing of the forebrain section containing the dorsomedial hypothalamic nucleus. CCK-immunoreactive neurons (c) and retrogradely labeled neurons (d) on the same section in the area corresponding to the box in (a) are shown under different illumination. In (b), the distribution of retrogradely labeled and/or CCK-immunoreactive neurons is illustrated based on the photomicrographs (c) and (d). Retrogradely labeled neurons are indicated by crosses, CCKimmunoreactive neurons by open circles and double-labeled neurons by filled circles.
were immunoreactive for serotonin. The presence of all three compounds (CT-b, SP and serotonin) in one and the same cell body could be demonstrated principally in the caudal raphe obscurus nucleus.
critical role of these neurochemicals in regulating a neural network subserving complex spontaneous and reactive behaviors via the PVT (Brown et al., 1992; Robinson and Mishkin, 1968). 4.1. CCK
4. Discussion It has been previously reported that certain extrahypothalamic limbic CCK and SP neuronal systems (mesolimbic, mesocortical, septal and hippocampal) are responsive to stress (Siegel et al., 1985). Thus, both CCK and SP may play roles as neuromodulators in the integrated function of the limbic system and may be key elements in the biology of several psychiatric disorders, including depression and schizophrenia. The present study focused on the neuroanatomical substrates involved in regulation of the limbic thalamostriatal system by SP- and CCK-containing neurons. The importance of our study is underscored by the
CCK has been associated with several neuropsychiatric diseases, including panic attack and schizophrenia. The tetrapeptide form of CCK (CCK-4) is reported to be panicogenic in man and is considered to provide a pharmacological model of panic attack (Bradwejn et al., 1990), although the efficacy of CCK antagonist in panic disorder or anxiety has not yet been reported. In the present study, principal sources of CCKimmunoreactive neurons projecting to the PVT were detected in the dorsomedial hypothalamic nucleus and Edinger-Westphal nucleus as well as in the mesencephalic dorsal raphe nucleus.
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importance for the motor behavior in several psychiatric diseases. The mesolimbic CCK-containing neurons, including those in the ventral tegmental area, do not project to the PVT (Bhatnager et al., 2000). This is consistent with the previous study showing that the mesolimbic dopaminergic neurons do not project to the PVT, in spite of rich dopaminergic innervation of the PVT (Otake and Ruggiero, 1995). Mesolimbic dopamine neurons instead supply heavy innervation to the nucleus accumbens (Wang, 1981), which constitutes the limbic thalamostriatal system together with the PVT. Thus mesolimbic neurons that co-express dopamine and CCK may influence thalamostriatal systems implicated in behavioral responses via the nucleus accumbens.
Fig. 4. Line drawings showing the location of CT-b-labeled and/or CCKimmunoreactive neurons in the mesencephalic periaqueductal gray. Boxed areas in (a) and (b), which frame the vicinity of the Edinger-Westphal nucleus, are enlarged and shown in (a0 ) and (b0 ). Retrogradely labeled neurons are indicated by crosses, CCK-immunoreactive neurons by open circles and double-labeled neurons by filled circles.
4.1.1. Dorsomedial hypothalamic nucleus The dorsomedial hypothalamic nucleus contained mutually exclusive populations of GABA and glutamate neurons (Ziegler et al., 2002), both of which supply projections to the paraventricular hypothalamic nucleus (Tasker et al., 1998). The location of CCK-containing neurons in the dorsolateral part of this nucleus overlaps that of glutamatergic neurons (Ziegler et al., 2002), whereas GABA-expressing neurons almost avoid the location of the CCK-immunoreactive neurons in the ventrolateral part. The role of the dorsomedial nucleus in the HPA integration may be subregionally differentiated; GABAergic neurons in the dorsomedial nucleus show c-fos activation following swim stress (Cullinan et al., 1996), whereas the dorsolateral part, presumably containing glutamate and/or CCK, does not show c-fos activation following immobilization stress (K. Otake, unpublished observation). Thus, many CCK-containing neurons in the dorsolateral part, presumably coexpressing glutamate, may project to the PVT as well as to the hypothalamic paraventricular nucleus, promoting ACTH secretion (Bailey and Dimicco, 2001) and activating limbic thalamo-striatal systems implicated in behavioral responses. Under stressful conditions, these neurons are inhibited, presumably by local GABAergic neurons, and thus disfacilitate neuroendocrine and behavioral responses. Large populations of mesolimbic dopaminergic neurons co-express CCK and are supposed to be of crucial
4.1.2. Edinger-Westphal nucleus Neurons in a well-defined cell group in the midline of the rostral, ventral periaqueductal gray, including the Edinger-Westphal nucleus, contained both SP- and CCKlike peptides (Skirboll et al., 1982). Recently it has been shown that virtually all of the neurons in the EdingerWestphal nucleus contain a newly discovered neuropeptide urocortin (Skelton et al., 2000), suggesting that SP and CCK might co-localize with urocortin within this nucleus. Urocortin is a corticotropin-releasing hormone-related neuropeptide and, when injected into the brain, it has been demonstrated to cause an increase in anxiety-like behavior (Moreau et al., 1997). The multiple peptidergic systems in this nucleus might be implicated in the delicate balance critical for maintenance of mental and physical homeostasis under stressful conditions. Following immobilization stress, the Edinger-Westphal neurons projecting to the PVT, parts of which presumably contain SP and/or CCK, express Fos protein (Otake et al., 2002) (see also Section 4.2.1). 4.2. SP Although SP has been best known as a pain neurotransmitter, it also plays an important role in the regulation of emotional states and the development of anxiety disorders and depression. Recent clinical trials have confirmed the efficacy of NK1 receptor antagonists to diminish anxious behavior and stress-related responses (Stout et al., 2001). It has also been shown that selective deletion of the Tac1 gene, which encodes SP and neurokinin A, yielded an anxiolytic profile in animal models of anxiety and depression (BilkeiGorzo et al., 2002). SP and its preferring NK1 receptor are widely expressed throughout the CNS implicated in fear processing, including the PVT (Mantyh et al., 1984; Ribeiro-da-Silva and Hokfelt, 2000). Our present study demonstrated that principal sources of SP-immunoreactive neurons projecting to the PVT were located in the three brainstem regions, i.e., the Edinger-Westphal nucleus, the mesopontine tegmentum and the medullary raphe nuclei.
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Fig. 5. Line drawings showing the location of CT-b-labeled and/or SP-immunoreactive neurons in the mesencephalic periaqueductal gray. Boxed areas in (a), (b) and (c), frame the vicinity of the Edinger-Westphal nucleus, are enlarged and shown in (a0 ), (b0 ) and (c0 ). Retrogradely labeled neurons are indicated by crosses, SP-immunoreactive neurons by open circles and double-labeled neurons by filled circles.
4.2.1. Edinger-Westphal nucleus In this study, several SP- and/or CCK-immunoreactive neurons were demonstrated to send ascending projection fibers from the Edinger-Westphal nucleus to the PVT. It has long been believed that the Edinger-Westphal nucleus never send projection fibers to the forebrain, but the first direct evidence has been found of ascending projections from this nucleus to the lateral septal nucleus with the use of retrograde tracers (Bittencourt et al., 1999). The present study has clearly shown another ascending pathway arising in the Edinger-Westphal nucleus. The Edinger-Westphal nucleus, classically regarded as the principal source of ocular preganglionic parasympathetic fibers, has been shown to have descending projections to various nuclei, including those expressing corticotropinreleasing factor receptors (Potter et al., 1994), such as to the olivary nucleus, parabrachial nucleus, trigeminal nuclear complex, facial nucleus, lateral reticular nucleus, rostroventral reticular nucleus, as well as the laminae I and Vof the spinal cord (Klooster et al., 1993; Loewy and Saper, 1978; Loewy et al., 1978). Some afferents to the Edinger-Westphal
nucleus have been shown to originate from the spinal cord and the hypothalamus (Swanson, 1977). Although the functional importance of the Edinger-Westphal nucleus is largely unknown, these neuroanatomical data suggest that this nucleus may play a much more complex role, for example, in autonomic regulation and some aspects of the stress response (Swanson, 1977; Weninger et al., 2000). Perhaps related is the evidence that this region, when stimulated, reproduced integrated response patterns characteristic of organized behavior evoked by stress, i.e. the defense reaction (Carrive et al., 1987; Hilton and Redfern, 1986). Also showing increases in neuronal activity to noxious stimulation was a ventral midline extension of the periaqueductal gray corresponding to the Edinger-Westphal nucleus (Lanteri-Minet et al., 1993). 4.2.2. Mesopontine tegmentum In the mesopontine tegmentum, a major source of SP projections originated from the laterodorsal tegmental nucleus. This area was previously shown as a source of SP-immunoreactive inputs to the paraventricular hypotha-
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Fig. 6. Line drawings showing the location of CT-b-labeled and/or SP-immunoreactive neurons at two levels of the pontine tegmentum (a and b). Boxed areas on the drawing of the upper left corner depict the localization of the area where cells containing immunoreactivities for either CT-b, SP or both were found. Retrogradely labeled neurons are indicated by crosses, SP-immunoreactive neurons by open circles and double-labeled neurons by filled circles.
Fig. 7. A pair of fluorescence photomicrographs showing SP- (a) and ChAT-immunoreactive (b) neurons in the laterodorsal tegmental nucleus observed on the same section. This double-labeling study revealed that the SP-immunoreactivity was co-localized with ChAT-immunoreactivities in all neurons in this region (arrows).
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Fig. 8. A set of photomicrographs showing CT-b- (a, d), SP- (b, e) and serotonin-immunoreactive (c, f) neurons in the medullary raphe nucleus on the section from the rat with (a–c) and without (d–f) CT-b injection in the PVT. This triple-labeling study revealed that a substantial number of medullary raphe neurons that project to the PVT (a) also contained both SP (b) and serotonin (c) in the experimental case (arrows). In the control case (d, e, f), SP- (e) and serotoninimmunoreactive (f) neurons were stained similarly to the experimental case (b, c), although no signal for CT-b was detected (d).
lamic nucleus (Bittencourt et al., 1991). As confirmed in the present double-labeling study, all of the SP-immunoreactive neurons in this nucleus contain ChAT. Since cholinergic fibers were sparse in the PVT (Otake and Ruggiero, 1995, for references), the laterodorsal tegmental nucleus may provide only a minor contribution to the SP-immunoreactive fibers in the PVT.
spinal cord (K. Otake, unpublished observation). The medullary raphe nucleus might modulate both the limbic thalamostriatal behavioral system and the viscero-nociceptive afferent system via SP- and/or serotonin-expressing neurons.
Acknowledgements 4.2.3. Medullary raphe nuclei In the present study, SP-immunoreactive neurons coexpressed with serotonin sent ascending projection fibers to the PVT. The previous study suggested, however, that coexpression of SP and serotonin in ascending raphe neurons does not occur in the rat brain, although SP might be coexpressed with serotonin in a proportion of dorsal raphe neurons in the human brain (Rupniak and Kramer, 1999). The precise reasons of such discrepancy cannot be elucidated, since both the present study and the previous studied employed well-established methods. One possibility is that the PVT might be a special target of the medullary raphe neurons, and thus the previous studies which did not pay special attentions to the PVT might overlook this ascending projection. Parts of the SP immunoreactive neurons co-expressed with serotonin send both ascending projection fibers to the PVT and descending fibers to the
The author thanks to Ms. Mie Taguchi for her technical assistance. This work was partly supported by Grant-in-Aid for Scientific Research for Priority Areas (A) (No. 12050217) from Japan Ministry of Education, Culture, Sports, Science and Technology.
Appendix A. Abbreviations used in figures Amb Arc cp CPu Cu DC Dk
ambiguus nucleus arcuate hypothalamic nucleus cerebral peduncle caudate putamen cuneate nucleus dorsal cochlear nucleus nucleus of Darkschewitsch
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Fig. 9. Line drawings showing location of CT-b-, SP-, and/or serotonin-immunoreactive neurons in the medullary raphe nuclei. Boxed areas in (a)–(d) are enlarged and shown in (a0 )–(d0 ).
DMC Ecu EW f ic icp IO IP LDTg LHb LPBC LPBV me5 MG MHb ml mlf mt MVe opt
dorsomedial hypothalamic nucleus, compact part external cuneate nucleus Edinger-Westphal nucleus fornix internal capsule inferior cerebellar peduncle inferior olive interpeduncular nucleus laterodorsal tegmental nucleus lateral habenular nucleus lateral parabrachial nucleus, central part lateral parabrachial nucleus, ventral part mesencephalic trigeminal nucleus medial geniculate nucleus medial habenular nucleus medial lemniscus medial longitudinal fasciculus mammillothalamic tract medial vestibular nucleus optic tract
PnR Pr PVT py R RLi RMg ROb RPa scp SNR sol spVe sp5 VMH VPL VPM VTA 3 4n
pontine raphe nucleus prepositus nucleus paraventricular thalamic nucleus pyramidal tract nucleus ruber rostal linear nucleus of the raphe raphe magnus nucleus raphe obscurus nucleus raphe pallidus nucleus superior cerebellar peduncle substantia nigra, reticular part solitary tract spinal vestibular nucleus spinal trigeminal tract ventromedial hypothalamic nucleus ventral posterolateral thalamic nucleus ventral posteromedial thalamic nucleus ventral tegmental area oculomotor nucleus trochlear nerve
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7 12
facial nucleus hypoglossal nucleus
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Cholecystokinin and substance P immunoreactive projections to the paraventricular thalamic nucleus in the rat Kazuyoshi Otake* Section of Neuroanatomy, Department of Systems Neuroscience, Division of Cognitive and Behavioral Medicine, Tokyo Medical and Dental University Graduate School, Tokyo 113-8519, Japan Received 28 August 2004; accepted 9 December 2004 Available online 15 January 2005
Abstract Cholecystokinin (CCK) and substance P (SP) are thought to play an important role in a variety of stress responses. Both CCK- and SPpositive fibers innervating the thalamus are found principally in the midline nuclei, including the paraventricular thalamic nucleus (PVT), which has strong reciprocal connections with the medial prefrontal cortex. In the present study, we determined the source of the CCK- and SPimmunoreactive fibers to the PVT, employing combination of retrograde neuronal tracing and immunohistochemistry in the rat. The PVTprojecting neurons showing CCK immunoreactivity were detected in the dorsomedial nucleus of the hypothalamus, and ventral mesencephalic periaqueductal gray, including the Edinger-Westphal nucleus and the dorsal raphe nucleus. Sources of SP afferents to the PVT were detected in the Edinger-Westphal nucleus, the mesopontine tegmentum and the medullary raphe nucleus. CCK- and SP-immunoreactive fibers may exert modulatory influence on the prefrontal cortical activity via the PVT and regulate behavioral components of stress-adaptation responses. # 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Midline thalamus; Cholecystokinin; Substance P; Retrograde tracing; Immunohistochemistry; Dorsomedial hypothalamic nucleus; EdingerWestphal nucleus; Stress
1. Introduction The densities of neuropeptide-immunoreactive cells and fibers in the thalamus have been reported as being relatively low with a few notable exceptions. Among these exceptions, the midline region of the thalamus, whose functions are not well understood, is heavily innervated by various peptidergic fibers, e.g. substance P (SP), cholecystokinin (CCK), a corticotrophin-releasing factor, neuropeptide Y, and somatostatin (Bentivoglio et al., 1991). The paraventricular thalamic nucleus (PVT), the most dorsal component of the thalamic midline, is known to be strongly activated following a variety of stressors and thus might be suggested to play a role in stress-related information targeted for viscerolimbic areas in the brain.
* Tel.: +81 3 58035149; fax: +81 3 58035151. E-mail address: [email protected].
Among neuropeptides innervating the PVT, SP and CCK are of particular interest, due to the roles ascribed to these peptides as neuromodulators in the integrated hypothalamic stress response, mediating stress–neuroendocrine interaction (Siegel et al., 1987). SP controls vomiting and various behavioral, neurochemical and cardiovascular responses to stress, besides its best-known role as a pain neurotransmitter. For example, recent clinical trials have confirmed the efficacy of NK1 receptor (i.e., SP receptor) antagonists to alleviate depression and emesis (Rupniak and Kramer, 1999). CCK has been shown to be involved in numerous physiological functions such as feeding behavior, central respiratory control and cardiovascular tonus, vigilance states, memory processes, nociception, and emotional and motivational responses (Fink et al., 1998; Noble and Roques, 1999) and has been associated with several neuropsychiatric diseases such as schizophrenia as well as anxiety and panic attacks (Bourin et al., 1998; Bradwejn et al., 1995).
0168-0102/$ – see front matter # 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2004.12.009
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Although it seems important to identify the origins of the SP-ergic and CCK-ergic innervation to the PVT in the contexts mentioned above, only limited researchers have investigated this issue so far. For example, Bhatnager et al. (2000) identified the origins of CCK innervations mainly in brainstem areas, including the lateral parabrachial, periaqueductal gray and dorsal raphe. However, previous findings of altered CCK levels in various hypothalamic regions during acute stress exposure suggested the importance of hypothalamic CCK, related to the negative feedback actions of glucocorticoids (Siegel et al., 1987). Intracerebroventricular administration of CCK produced a wide spectrum of endocrine effects, likely via a hypothalamic site of action (Vijayan et al., 1979). The origin of SP innervation of the PVT has not been so far documented. The aim of the present study was therefore to determine the detailed sites of origin of CCK- and SP-projection fibers to the PVT by immunohistochemistry for CCK and SP combined with retrograde tract tracing. A preliminary report of this study was presented in abstract form (Otake and Nakamura, 2003).
2. Materials and methods 2.1. Animals and tissue preparation Sprague–Dawley rats (male; 200–250 g; Nippon BioSupp. Center, Tokyo, Japan) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). All efforts were made to minimize the number of animals used and their suffering. Injections of a 1% solution of cholera toxin B-subunit (CT-b, Research Biochemicals International, Natick, MA, USA) diluted with 0.2 M sodium phosphate buffer (PB, pH 7.5) were delivered from glass micropipettes in the dorsal midline thalamus using coordinates from the atlas of Paxinos and Watson (Paxinos and Watson, 1998). Iontophoretic ejections were made using positive pulses (7 mA, 7 s on/off) from a Midgard precision current source (Stoelting, Wood Dale, IL, USA) for 15– 20 min. Three to four days later, these rats were reanesthetized and colchicine (150 mg/10 ml saline) was slowly injected unilaterally into the lateral ventricle to build up transmitter concentration in cell bodies, and then, after 1 more day, the animals were re-anesthetized and perfused transcardially with 200 ml heparinized saline followed by 500 ml of Zamboni’s fixative. The surgical procedures and pre- and post-operative care of the animals were approved by the Tokyo Medical and Dental University Animal Care Committee and were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication 80-23, revised 1996). The brains were removed and immersed overnight at 4 8C in 0.1 M PB containing 10% sucrose. They were sectioned in the transverse plane at 40-mm on a freezing microtome and
each 1-in-4 series was immunostained by a double or triple labeling method as follows. All histological procedures were carried out at room temperature. 2.2. Double immunohistochemistry for CT-b and CCK/CT-b and SP Two 1-in-4 series were processed for CT-b and SP or CCK reactivity. The sections were immersed for 1 h in PB containing 1% bovine serum albumin (Wako, Osaka, Japan) and incubated overnight in a mixture of goat anti-CT-b antiserum (1:20,000; List Biological, Inc., Campbell, CA, USA) and rabbit anti-SP (1:200; Peninsula Labs, Belmont, CA, USA) or rabbit anti-CCK antibody (1:200; Chemicon, Temecula, CA, USA). The sections were rinsed in PB and then incubated for 2 h in biotinylated horse anti-goat IgG (1:1000; Vector, Burlingame, CA, USA). After additional rinsing, sections were immersed for 2 h in a mixture of avidin-TRITC (1:250; Vector) and FITC-conjugated swine anti-rabbit IgG (1:20; DakoCytomation, Glostrup, Denmark). Sections were mounted in a glycerol-mounting medium containing 0.1% p-phenylenediamine (Sigma, Saint-Louis, MO, USA), examined in a Nikon fluorescent microscope (Eclipse E600) equipped with appropriate filter combinations, and photographed by using a cooled CCD camera (Keyence VB 6000/6010, Osaka, Japan) attached to a microscope. The distribution of single- and multi-labeled neurons was mapped onto drawings prepared from digital photomontage of the total area of the representative sections. 2.3. Triple immunohistochemistry for CT-b, SP and serotonin Since many SP-containing neurons also contained serotonin in the raphe nuclei in cat (Arvidsson et al., 1994; Lovick and Hunt, 1983) as well as in human (Baker et al., 1991), coexistence of serotonin with SP and CT-b immunoreactivity was analyzed by use of the triple-labeling technique. A 1-in-4 series through the medulla was first processed for SP and serotonin using the same procedure. In brief, the sections were incubated overnight in a mixture of rat anti-serotonin (1:100; Chemicon) and rabbit anti-SP antiserum (1:200; Peninsula). The sections were placed for 2 h in biotinylated goat anti-rat antibody (1:1000; Vector), and then for 2 h in a mixture of avidin-TRITC (1:250; Vector) and FITC-conjugated swine anti-rabbit IgG (1:20; DakoCytomation). Sections were mounted in a glycerolmounting medium. After examining and photographing the relevant areas, the sections were then immunostained for CT-b. To eliminate potential cross-reaction between avidin/ biotin system reagents, the sections first went through a blocking procedure (Ferri et al., 1999); the sections were incubated in the free avidin solution (Avidin/Biotin Blocking Kit, #SP-2001, Vector) for 40 min, in 10% formaldehyde for 40 min, and then in a free biotin solution
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(Avidin/Biotin Blocking Kit, #SP-2001, Vector) for 40 min. The sections were then incubated overnight in goat anti-CTb antiserum (1:40,000; List), for 45 min in biotinylated horse anti-goat IgG (1:2000) and for 30 min in avidin/biotin peroxidase complex (ABC Elite kit; Vector). Enzymatic development was done in a solution of 0.02% diaminobenzidine (Sigma) in 0.1 M Tris–buffer containing 0.003 and 0.6% nickel ammonium sulfate. The control experiment was conducted to verify the absence of cross-reactivity by processing sections from a rat without CT-b injection in the same triple-labeling protocol. 2.4. Double immunohistochemistry for SP and ChAT Almost all of the SP-containing neurons in the rat mesopontine tegmentum have been shown to be immunoreactive for choline acetyltransferase (ChAT) (Standaert et al., 1986; Sutin and Jacobowitz, 1988; Vincent et al., 1983, 1986). We conducted double immunohistochemistry for SP and ChAT to confirm the co-localization of these two substances. The sections were incubated overnight in a mixture of mouse anti-ChAT (1:100; Chemicon) and rabbit anti-SP antiserum (1:200; Peninsula). The sections were placed for 2 h in biotinylated horse anti-mouse antibody (1:1000; Vector), and then for 2 h in a mixture of avidinTRITC (1:250; Vector) and FITC-conjugated swine antirabbit IgG (1:20; DakoCytomation). Sections were mounted in a glycerol-mounting medium.
3. Results 3.1. Injection sites and patterns of retrograde transport In the dorsal part of the diencephalon, CCK- (Fig. 1) and SP-immunoreactive varicose fibers and punctate processes (Fig. 2a) were heavily concentrated in the PVT. Five cases were selected on the basis that the injection sites of CT-b were limited to the PVT and overlapped the area displaying the heavy CCK or SP innervation (Fig. 2b). The pattern of retrograde labeling was similar to that reported in our previous studies (Otake et al., 1994, 2002; Otake and Ruggiero, 1995). 3.2. CCK-immunoreactive afferents Principal sources of CCK-immunoreactive PVT afferents were detected in the hypothalamus and mesencephalon. The caudal part of the hypothalamus was the main forebrain region containing double-labeled neurons. CCKcontaining neurons heavily concentrated in the lateral region of the compact part of the dorsomedial hypothalamus at its most caudal level (Paxinos and Watson, 1998). Circumscribed subsets of CT-b-labeled neurons were found in this hypothalamic nucleus which overlaps the distribution of the CCK-immunoreactive neurons. Almost
Fig. 1. CCK innervation of the PVT: (a) the region of the PVT is indicated by the box in a section of rat forebrain and (b) photomicrograph showing axonal fibers and terminal-like varicosities immunoreactive for CCK in the PVT, corresponding to the boxed area in (a).
half of the CT-b-labeled neurons in this region (47.5 6.3%; n = 5) were also immunoreactive for CCK (Fig. 3). In the forebrain, the paraventricular hypothalamic nucleus and supramammillary nucleus occasionally contained double-labeled neurons. In the midbrain, double-labeled neurons were distributed in the midline of the ventral part of the mesencephalic periaqueductal gray (Fig. 4), corresponding to the EdingerWestphal nucleus, and the rostral linear raphe nucleus as well as the dorsal raphe nucleus. Small numbers of doublelabeled neurons were consistently found in the rostral part of the Edinger-Westphal nucleus. Although CCK immunoreactivity was present in a large proportion of the mesencephalic dopamine neurons, i.e., ventral tegmental area, no double-labeled cells were found in this area (not illustrated). In the pons, the lateral parabrachial nucleus occasionally contained double-labeled neurons (not illustrated).
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Fig. 2. The photomicrographs of the PVT and its vicinity showing axonal fibers and terminal-like varicosities immunoreactive for SP (a) and a CT-b deposit (b) observed on the same section under different illumination. Some neurons retrogradely labeled from CT-b (d) are immunoreactive for SP (c) on the same section under different illumination in the mesopontine tegmentum. Double-labeled neurons are indicated with arrows in (c) and (d).
3.3. SP-immunoreactive afferents Principal sources of SP-immunoreactive PVT afferents were detected in regions in the brainstem. Besides, doublelabeled cells were occasionally detected in the medial preoptic area, ventrolateral and dorsomedial hypothalamic nuclei and medial prefrontal cortex. In the midbrain, SP-immunoreactive neurons were widely distributed in the periaqueductal gray, and doublelabeled neurons were observed especially in the midline of its ventral part (Fig. 5). This median area corresponds to the Edinger-Westphal nucleus. Double-labeled neurons in the Edinger-Westphal nucleus were detected from the level of the Bragma 5.20 to 6.04, corresponding to the whole extent of the red nucleus. In each section, more than 50% (up to 100%) of retrogradely labeled neurons were also stained with SP, although many the EdingerWestphal neurons were stained only with SP. The SP-positive cells in the area of Edinger-Westphal nucleus seem to extend further rostrally than CCK-containing neurons. In the mesopontine tegmentum, double-labeled neurons were consistently observed in the laterodorsal tegmental
nucleus and lateral parabrachial nucleus (Fig. 6). Doublelabeling experiments of ChAT and SP confirmed that all of the SP-containing neurons in this area are also immunoreactive for ChAT (Fig. 7), indicating that PVT-projecting SP-immunoreactive neurons are cholinergic. The pedunculopontine tegmental nucleus also contained double-labeled neurons. In the medulla, double-labeled neurons were distributed in the midline region corresponding to the raphe nuclei (Figs. 8 and 9). The degree of coexistence (percentage of double-labeled cells) varied with the medullary level. The nucleus raphe magnus, at more rostral locations, showed a lower proportion of double labeling. Most of the doublelabeled neurons in the raphe were found in the nucleus raphe obscurus. Few double-labeled neurons were detected in the raphe pallidus (Fig. 9). From triple-labeled sections it was concluded that many PVT projecting neurons containing SP also contained serotonin. As shown in Fig. 9, a subpopulation of serotonin-immunoreactive neurons lacked SP-immunoreactivity and a subpopulation of SPimmunoreactive neurons lacked serotonin-immunoreactivity. Quantitatively, 63.4 22.2% of PVT-projecting neurons were immunoreactive for SP, 51.0 33.8% of which
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Fig. 3. (a) Line drawing of the forebrain section containing the dorsomedial hypothalamic nucleus. CCK-immunoreactive neurons (c) and retrogradely labeled neurons (d) on the same section in the area corresponding to the box in (a) are shown under different illumination. In (b), the distribution of retrogradely labeled and/or CCK-immunoreactive neurons is illustrated based on the photomicrographs (c) and (d). Retrogradely labeled neurons are indicated by crosses, CCKimmunoreactive neurons by open circles and double-labeled neurons by filled circles.
were immunoreactive for serotonin. The presence of all three compounds (CT-b, SP and serotonin) in one and the same cell body could be demonstrated principally in the caudal raphe obscurus nucleus.
critical role of these neurochemicals in regulating a neural network subserving complex spontaneous and reactive behaviors via the PVT (Brown et al., 1992; Robinson and Mishkin, 1968). 4.1. CCK
4. Discussion It has been previously reported that certain extrahypothalamic limbic CCK and SP neuronal systems (mesolimbic, mesocortical, septal and hippocampal) are responsive to stress (Siegel et al., 1985). Thus, both CCK and SP may play roles as neuromodulators in the integrated function of the limbic system and may be key elements in the biology of several psychiatric disorders, including depression and schizophrenia. The present study focused on the neuroanatomical substrates involved in regulation of the limbic thalamostriatal system by SP- and CCK-containing neurons. The importance of our study is underscored by the
CCK has been associated with several neuropsychiatric diseases, including panic attack and schizophrenia. The tetrapeptide form of CCK (CCK-4) is reported to be panicogenic in man and is considered to provide a pharmacological model of panic attack (Bradwejn et al., 1990), although the efficacy of CCK antagonist in panic disorder or anxiety has not yet been reported. In the present study, principal sources of CCKimmunoreactive neurons projecting to the PVT were detected in the dorsomedial hypothalamic nucleus and Edinger-Westphal nucleus as well as in the mesencephalic dorsal raphe nucleus.
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importance for the motor behavior in several psychiatric diseases. The mesolimbic CCK-containing neurons, including those in the ventral tegmental area, do not project to the PVT (Bhatnager et al., 2000). This is consistent with the previous study showing that the mesolimbic dopaminergic neurons do not project to the PVT, in spite of rich dopaminergic innervation of the PVT (Otake and Ruggiero, 1995). Mesolimbic dopamine neurons instead supply heavy innervation to the nucleus accumbens (Wang, 1981), which constitutes the limbic thalamostriatal system together with the PVT. Thus mesolimbic neurons that co-express dopamine and CCK may influence thalamostriatal systems implicated in behavioral responses via the nucleus accumbens.
Fig. 4. Line drawings showing the location of CT-b-labeled and/or CCKimmunoreactive neurons in the mesencephalic periaqueductal gray. Boxed areas in (a) and (b), which frame the vicinity of the Edinger-Westphal nucleus, are enlarged and shown in (a0 ) and (b0 ). Retrogradely labeled neurons are indicated by crosses, CCK-immunoreactive neurons by open circles and double-labeled neurons by filled circles.
4.1.1. Dorsomedial hypothalamic nucleus The dorsomedial hypothalamic nucleus contained mutually exclusive populations of GABA and glutamate neurons (Ziegler et al., 2002), both of which supply projections to the paraventricular hypothalamic nucleus (Tasker et al., 1998). The location of CCK-containing neurons in the dorsolateral part of this nucleus overlaps that of glutamatergic neurons (Ziegler et al., 2002), whereas GABA-expressing neurons almost avoid the location of the CCK-immunoreactive neurons in the ventrolateral part. The role of the dorsomedial nucleus in the HPA integration may be subregionally differentiated; GABAergic neurons in the dorsomedial nucleus show c-fos activation following swim stress (Cullinan et al., 1996), whereas the dorsolateral part, presumably containing glutamate and/or CCK, does not show c-fos activation following immobilization stress (K. Otake, unpublished observation). Thus, many CCK-containing neurons in the dorsolateral part, presumably coexpressing glutamate, may project to the PVT as well as to the hypothalamic paraventricular nucleus, promoting ACTH secretion (Bailey and Dimicco, 2001) and activating limbic thalamo-striatal systems implicated in behavioral responses. Under stressful conditions, these neurons are inhibited, presumably by local GABAergic neurons, and thus disfacilitate neuroendocrine and behavioral responses. Large populations of mesolimbic dopaminergic neurons co-express CCK and are supposed to be of crucial
4.1.2. Edinger-Westphal nucleus Neurons in a well-defined cell group in the midline of the rostral, ventral periaqueductal gray, including the Edinger-Westphal nucleus, contained both SP- and CCKlike peptides (Skirboll et al., 1982). Recently it has been shown that virtually all of the neurons in the EdingerWestphal nucleus contain a newly discovered neuropeptide urocortin (Skelton et al., 2000), suggesting that SP and CCK might co-localize with urocortin within this nucleus. Urocortin is a corticotropin-releasing hormone-related neuropeptide and, when injected into the brain, it has been demonstrated to cause an increase in anxiety-like behavior (Moreau et al., 1997). The multiple peptidergic systems in this nucleus might be implicated in the delicate balance critical for maintenance of mental and physical homeostasis under stressful conditions. Following immobilization stress, the Edinger-Westphal neurons projecting to the PVT, parts of which presumably contain SP and/or CCK, express Fos protein (Otake et al., 2002) (see also Section 4.2.1). 4.2. SP Although SP has been best known as a pain neurotransmitter, it also plays an important role in the regulation of emotional states and the development of anxiety disorders and depression. Recent clinical trials have confirmed the efficacy of NK1 receptor antagonists to diminish anxious behavior and stress-related responses (Stout et al., 2001). It has also been shown that selective deletion of the Tac1 gene, which encodes SP and neurokinin A, yielded an anxiolytic profile in animal models of anxiety and depression (BilkeiGorzo et al., 2002). SP and its preferring NK1 receptor are widely expressed throughout the CNS implicated in fear processing, including the PVT (Mantyh et al., 1984; Ribeiro-da-Silva and Hokfelt, 2000). Our present study demonstrated that principal sources of SP-immunoreactive neurons projecting to the PVT were located in the three brainstem regions, i.e., the Edinger-Westphal nucleus, the mesopontine tegmentum and the medullary raphe nuclei.
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Fig. 5. Line drawings showing the location of CT-b-labeled and/or SP-immunoreactive neurons in the mesencephalic periaqueductal gray. Boxed areas in (a), (b) and (c), frame the vicinity of the Edinger-Westphal nucleus, are enlarged and shown in (a0 ), (b0 ) and (c0 ). Retrogradely labeled neurons are indicated by crosses, SP-immunoreactive neurons by open circles and double-labeled neurons by filled circles.
4.2.1. Edinger-Westphal nucleus In this study, several SP- and/or CCK-immunoreactive neurons were demonstrated to send ascending projection fibers from the Edinger-Westphal nucleus to the PVT. It has long been believed that the Edinger-Westphal nucleus never send projection fibers to the forebrain, but the first direct evidence has been found of ascending projections from this nucleus to the lateral septal nucleus with the use of retrograde tracers (Bittencourt et al., 1999). The present study has clearly shown another ascending pathway arising in the Edinger-Westphal nucleus. The Edinger-Westphal nucleus, classically regarded as the principal source of ocular preganglionic parasympathetic fibers, has been shown to have descending projections to various nuclei, including those expressing corticotropinreleasing factor receptors (Potter et al., 1994), such as to the olivary nucleus, parabrachial nucleus, trigeminal nuclear complex, facial nucleus, lateral reticular nucleus, rostroventral reticular nucleus, as well as the laminae I and Vof the spinal cord (Klooster et al., 1993; Loewy and Saper, 1978; Loewy et al., 1978). Some afferents to the Edinger-Westphal
nucleus have been shown to originate from the spinal cord and the hypothalamus (Swanson, 1977). Although the functional importance of the Edinger-Westphal nucleus is largely unknown, these neuroanatomical data suggest that this nucleus may play a much more complex role, for example, in autonomic regulation and some aspects of the stress response (Swanson, 1977; Weninger et al., 2000). Perhaps related is the evidence that this region, when stimulated, reproduced integrated response patterns characteristic of organized behavior evoked by stress, i.e. the defense reaction (Carrive et al., 1987; Hilton and Redfern, 1986). Also showing increases in neuronal activity to noxious stimulation was a ventral midline extension of the periaqueductal gray corresponding to the Edinger-Westphal nucleus (Lanteri-Minet et al., 1993). 4.2.2. Mesopontine tegmentum In the mesopontine tegmentum, a major source of SP projections originated from the laterodorsal tegmental nucleus. This area was previously shown as a source of SP-immunoreactive inputs to the paraventricular hypotha-
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Fig. 6. Line drawings showing the location of CT-b-labeled and/or SP-immunoreactive neurons at two levels of the pontine tegmentum (a and b). Boxed areas on the drawing of the upper left corner depict the localization of the area where cells containing immunoreactivities for either CT-b, SP or both were found. Retrogradely labeled neurons are indicated by crosses, SP-immunoreactive neurons by open circles and double-labeled neurons by filled circles.
Fig. 7. A pair of fluorescence photomicrographs showing SP- (a) and ChAT-immunoreactive (b) neurons in the laterodorsal tegmental nucleus observed on the same section. This double-labeling study revealed that the SP-immunoreactivity was co-localized with ChAT-immunoreactivities in all neurons in this region (arrows).
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Fig. 8. A set of photomicrographs showing CT-b- (a, d), SP- (b, e) and serotonin-immunoreactive (c, f) neurons in the medullary raphe nucleus on the section from the rat with (a–c) and without (d–f) CT-b injection in the PVT. This triple-labeling study revealed that a substantial number of medullary raphe neurons that project to the PVT (a) also contained both SP (b) and serotonin (c) in the experimental case (arrows). In the control case (d, e, f), SP- (e) and serotoninimmunoreactive (f) neurons were stained similarly to the experimental case (b, c), although no signal for CT-b was detected (d).
lamic nucleus (Bittencourt et al., 1991). As confirmed in the present double-labeling study, all of the SP-immunoreactive neurons in this nucleus contain ChAT. Since cholinergic fibers were sparse in the PVT (Otake and Ruggiero, 1995, for references), the laterodorsal tegmental nucleus may provide only a minor contribution to the SP-immunoreactive fibers in the PVT.
spinal cord (K. Otake, unpublished observation). The medullary raphe nucleus might modulate both the limbic thalamostriatal behavioral system and the viscero-nociceptive afferent system via SP- and/or serotonin-expressing neurons.
Acknowledgements 4.2.3. Medullary raphe nuclei In the present study, SP-immunoreactive neurons coexpressed with serotonin sent ascending projection fibers to the PVT. The previous study suggested, however, that coexpression of SP and serotonin in ascending raphe neurons does not occur in the rat brain, although SP might be coexpressed with serotonin in a proportion of dorsal raphe neurons in the human brain (Rupniak and Kramer, 1999). The precise reasons of such discrepancy cannot be elucidated, since both the present study and the previous studied employed well-established methods. One possibility is that the PVT might be a special target of the medullary raphe neurons, and thus the previous studies which did not pay special attentions to the PVT might overlook this ascending projection. Parts of the SP immunoreactive neurons co-expressed with serotonin send both ascending projection fibers to the PVT and descending fibers to the
The author thanks to Ms. Mie Taguchi for her technical assistance. This work was partly supported by Grant-in-Aid for Scientific Research for Priority Areas (A) (No. 12050217) from Japan Ministry of Education, Culture, Sports, Science and Technology.
Appendix A. Abbreviations used in figures Amb Arc cp CPu Cu DC Dk
ambiguus nucleus arcuate hypothalamic nucleus cerebral peduncle caudate putamen cuneate nucleus dorsal cochlear nucleus nucleus of Darkschewitsch
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Fig. 9. Line drawings showing location of CT-b-, SP-, and/or serotonin-immunoreactive neurons in the medullary raphe nuclei. Boxed areas in (a)–(d) are enlarged and shown in (a0 )–(d0 ).
DMC Ecu EW f ic icp IO IP LDTg LHb LPBC LPBV me5 MG MHb ml mlf mt MVe opt
dorsomedial hypothalamic nucleus, compact part external cuneate nucleus Edinger-Westphal nucleus fornix internal capsule inferior cerebellar peduncle inferior olive interpeduncular nucleus laterodorsal tegmental nucleus lateral habenular nucleus lateral parabrachial nucleus, central part lateral parabrachial nucleus, ventral part mesencephalic trigeminal nucleus medial geniculate nucleus medial habenular nucleus medial lemniscus medial longitudinal fasciculus mammillothalamic tract medial vestibular nucleus optic tract
PnR Pr PVT py R RLi RMg ROb RPa scp SNR sol spVe sp5 VMH VPL VPM VTA 3 4n
pontine raphe nucleus prepositus nucleus paraventricular thalamic nucleus pyramidal tract nucleus ruber rostal linear nucleus of the raphe raphe magnus nucleus raphe obscurus nucleus raphe pallidus nucleus superior cerebellar peduncle substantia nigra, reticular part solitary tract spinal vestibular nucleus spinal trigeminal tract ventromedial hypothalamic nucleus ventral posterolateral thalamic nucleus ventral posteromedial thalamic nucleus ventral tegmental area oculomotor nucleus trochlear nerve
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facial nucleus hypoglossal nucleus
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