Cholinergic neurons expressing substance P receptor (NK1) in the basal forebrain of the rat: a double immunocytochemical study

Brain Research 904 (2001) 161–166 www.elsevier.com / locate / bres

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Cholinergic neurons expressing substance P receptor (NK 1 ) in the basal forebrain of the rat: a double immunocytochemical study a, b a a c Liang-Wei Chen *, Li-Chun Wei , Hui-Ling Liu , Yong Qiu , Ying-Shing Chan b

a Institute of Neuroscience, The Fourth Military Medical University, Xi’ an, 710032, PR China Department of Radiotherapy, Xijing Hospital, The Fourth Military Medical University, Xi’ an, PR China c Department of Physiology, The University of Hong Kong, Hong Kong, Hong Kong

Accepted 27 March 2001

Abstract Cholinergic neurons expressing substance P receptor (SPR, NK 1 ) were examined in the rat brain using double immunofluorescence. The distribution of SPR-like immunoreactive (SPR-LI) neurons completely overlapped with that of choline acetyltransferase (ChAT)-LI neurons in the medial septal nucleus, the nucleus of diagonal band of Broca, the magnocellular preoptic nucleus, the substantia innominata of basal forebrain, the caudate-putamen, and the ventral pallidum of the basal ganglia. In the mesopontine tegmentum and the cranial motor nuclei of the brainstem, the distribution of SPR-LI and ChAT-LI neurons was partially overlapping. Neurons showing both SPR-like and ChAT-like immunoreactivities, however, were predominantly found above basal forebrain regions and 82–90% of these ChAT-LI neurons displayed SPR-like immunoreactivity, in addition to the confirmatory observation that 100% of the ChAT-LI neurons exhibit SPR-like immunoreactivity in the basal ganglia. In contrast, neurons double-labeled for SPR-like and ChAT-like immunoreactivities were hardly detected in aforementioned regions of the brainstem. The present study has provided morphological evidence for direct physiological modulation of cholinergic neurons by tachykinins through substance P receptor in the basal forebrain of the rat.  2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Peptide receptor structure and function Keywords: Choline acetyltransferase; Substance P receptor; Co-localization; Immunocytochemistry; Basal forebrain; Rat

The cholinergic neurons are widely distributed in the certain nuclei or regions of mammalian brain, which include four major cell groups, e.g. the basal forebrain nuclei (medial septal nucleus, nucleus of diagonal band of Broca and magnocellular preoptic nucleus), the basal ganglia (caudate-putamen, ventral pallidum), the mesopontine tegmentum and the cranial motor nuclei [4,12]. The central cholinergic neurons subserve diverse biological functions through their connections with cerebral cortex, diencephalon, brainstem, spinal cord and peripheral organs [12,15]. Previous studies have shown that these cholinergic neurons are involved in regulation or modulation of learning, memory, motor control, autonomic function, and behavioral activities [4,12,15]. Growing evidence has *Corresponding author. Tel.: 186-29-328-5726; fax: 186-29-3246270. E-mail address: [email protected] (L.-W. Chen).

indicated that atrophy or loss of the basal forebrain cholinergic neurons may result in the neurodegenerative disorders such as Alzhemer’s disease in humans [17]. Tachykinin family is composed of three major members, e.g. substance P (SP), substance K (SK, neurokinin A, neurokinin a), and neuromedin K (NK, neurokinin B, neurokinin b) in the mammalian brain. These tachykinins are widely distributed in both central nervous system and peripheral tissues, and function as neurotransmitter, neuromodulator, or neurotrophic-like factors via mediation of SP receptor (SPR, NK 1 ), neurokinin A receptor (NK 2 ), and neuromedin K receptor (NKR, NK 3 ), respectively [2,14,19,21,23,24,28]. The distribution of these tachykinin receptors has been studied by in situ hybridization and immunocytochemistry in the mammalian brain [11,13,22]. Recent evidence has shown that cholinergic neurons in the brain are potently regulated by tachykinins [3,18,26]. Morphological studies have found that tachykinin-con-

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02460-X

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taining axon terminals are distributed in these cholinergic neuron-containing regions, for example, the SP-ergic terminals make synapses on cholinergic neurons in the basal forebrain and basal ganglia as well [2,7]. Physiological and pharmacological evidence has indicated that tachykinin or agonist for tachykinin receptor affects activity of the cholinergic neurons and modulates acetylcholine release [1,3,18,26]. Furthermore, these peptides may play important roles in the pathological mechanisms of neurodegenerative disease as well, for example, tachykinins protect striatial cholinergic neurons from quinolinic acid excitotoxicity [6,8,14,16,17,23,28]. Thus, it is possible that cholinergic neurons express tachykinin receptors in the forebrain. On the other hand, in situ hybridization and immunocytochemical studies have revealed that among tachykinin receptors, both SPR and NKR are densely distributed in certain regions of the forebrain, whereas NK 2 is only detected in limited areas [11,21,22,25,27]. We recently revealed the selective expression of NKR in cholinergic neurons of the basal forebrain [9]. In addition, previous studies have shown that the cholinergic neurons in striatum express SPR [5,25]. No study has, however, dealt with the actual cellular localization of SPR in cholinergic neurons of the basal forebrain though SPR mRNA was reported in this area [13]. Hence, by using double immunofluorescence we have examined the distribution of cholinergic neurons expressing SPR in the basal forebrain, and in other aforementioned regions of diencephalon and brainstem containing cholinergic neurons as well. A total of eight adult male rats (Sprague–Dawley) weighing 220–230 g were used in the present study. All animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23), and approved by the Committee of Animal Use for Research and Education of the Fourth Military Medical University, PR China. The animals were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (100 mg / kg body wt.), and then transcardially perfused with a volume of 100 ml of saline, followed by a volume of 500 ml of phosphate buffer (pH 7.4) containing 4% paraformaldehyde within 30 min. Brains were removed immediately and placed in 0.1 M phosphate buffer containing 30% sucrose overnight at 48C. After that, serially coronal sections 25 mm in thickness were cut on a frozen microtome. Double immunofluorescence staining was performed on the brain sections to visualize the co-localization of choline acetyltransferase (ChAT) and SPR in neurons of the brain. Briefly, the sections were incubated for 48 h at 48C with a mixture of mouse anti-ChAT IgG (Boehringer Mannheim 1:500 dilution) and 0.5 mg / ml of rabbit anti-SPR IgG in 0.01 M phosphate buffered saline (PBS, pH 7.4) containing 1% normal donkey serum and 0.1% Triton X-100. The rabbit anti-SPR serum was raised against a fusion

protein containing SPR C-terminus peptide and characterized by Western blotting [22]. Subsequently, the sections were rinsed in 0.01 M PBS, and then incubated for 24 h at 48C with a mixture of dichorotriazinylamino-fluorescein (DTAF)-conjugated donkey anti-mouse IgG (Chemicon, 1:100 dilution) and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated donkey anti-rabbit IgG (Chemicon, 1:100 dilution). After being washed, the sections were mounted on gelatin-coated glass slides, and coverslipped in 0.01 M PBS containing 50% glycerin and 2.5% triethylenediamine, and then examined under an Olympus epifluorescence microscope (BX-60). Blue filter or green filter was used to visualize DTAF-labeled neurons or TRITC-labeled neurons, respectively. For semi-quantification, ChAT single-, SPR single-, and ChAT / SPR doublelabeled neuron somata were counted on 10 serial sections per region of eight rat brains and data were expressed as the mean6S.E.M. (n58). For the control experiments, the primary antibody was replaced with normal mouse serum (for ChAT immunocytochemistry) or normal rabbit serum (for SPR immunocytochemistry). The ChAT-like immunoreactive (ChATLI), or SPR-like immunoreactive (SPR-LI) neurons were not found in the control sections. Both ChAT-LI and SPR-LI neurons were found in the dually immunostained sections. Their distribution was well consistent with that of previous reports [4,12,22]. Generally, ChAT-like was preferentially localized within the neuronal cytoplasm, while SPR-like immunoreactivity was predominantly seen in the cell membrane of neuronal somata and dendrites. A large number of ChAT-LI or SPR-LI neurons were observed in the basal forebrain regions. The distribution of ChAT-LI neurons completely overlapped with SPR-LI neurons in the medial septal nucleus (MS), the nucleus of diagonal band of Broca (DBB, horizontal and vertical limbs), magnocellular preoptic nucleus (MCPO) and substantia innominata (SI). Numerous neurons showing both ChAT- and SPR-like immunoreactivities were seen in the MS (Fig. 1A, A9), DBB (Fig. 1B, B9), MCPO (Fig. 1C, C9) and SI. It was found that 82–90% of ChAT-LI neurons expressed SPRlike immunoreactivity in these regions, while 100% of SPR-LI neurons showed ChAT-like immunoreactivity (Table 1). The majority of double-labeled neuronal somata were oval, fusiform, or elongated multipolar in shape and medium- or large-size with their diameters ranging 20610 mm (n530). In addition, double-labeled neurons were hardly detected in the basal nucleus of Meynert where many medium-sized ChAT-positive neurons were also distributed. ChAT-LI neurons and SPR-LI neurons were also widely distributed in the basal ganglia, e.g. the caudate and putamen (CPU) and ventral pallidum (VP). Their morphology and distribution were in agreement with that of previous reports [4,22]. It was found that 100% of ChATLI neurons expressed SPR-like immunoreactivity in these

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Fig. 1. Photomicrographs showing the neurons with ChAT-like (A, B, C) and SPR-like (A9, B9, C9) immunoreactivity in the medial septal nucleus (MS), the nucleus of diagonal band of Broca (DBB) and the magnocellular preoptic nucleus (MCPO). Co-localization of ChAT and SPR is demonstrated in the MS (A, A9), the vertical limb of the DBB (B, B9) and MCPO (C, C9). Double-labeled neurons are representatively indicated by small solid arrows, and ChAT single-labeled neurons are indicated with a hollow arrow (triangle). A large solid arrow indicates the midline of the basal forebrain. The field of A, B or C is the same as that of A9, B9 or C9, respectively. Scale bars: 60 mm in A, A9, B, B9; 30 mm in C, C9.

regions, and they constitute only 71–89% of the SPR-LI neurons (Table 1). In addition, a few double-labeled neurons were scattered in the globus pallidum and the nucleus of accumbens. Both ChAT-LI neurons and SPR-LI neurons were found in the mesopontine tegmentum, and partial overlapping of their distribution was seen in laterodorsal tegmental nu-

cleus (LDT) and pedunculopontine tegmental nucleus (PPT). Neurons exhibiting both ChAT- and SPR-like immunoreactivities, however, were not detected in the LDT and PPT of pons. In addition, ChAT-LI neurons in the brainstem were also densely distributed in the cranial motor nuclei, e.g. oculomotor nucleus (III), trochlear nucleus (IV), motor nucleus of the trigeminal nerve (V),

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Table 1 Comparison of ChAT single-, SPR single-, and ChAT / SPR double-labeled neuronal somata in distinct regions of the rat brain a

MS VDB HDB MCPO SI CPU VP LDT PPT III IV V VI VII Am X XII

ChAT

SPR

ChAT / SPR (% of ChAT)

ChAT / SPR (% of SPR)

20876189 1063689 21756315 18856176 925687 30906275 878687 1227690 9836105 775697 103628 1590656 287678 21276163 268635 7896135 9366176

1756698 8756105 18786138 17046112 8206106 43456360 9856112 285645 142625 0 0 0 0 0 0 175653 0

1756698 (8465) 8756105 (8268) 18786138 (8667) 17046112 (9064) 8206106 (8964) 30906275 (100) 878687 (100) 0 0 0 0 0 0 0 0 0 0

1756698 (100) 8756105 (100) 18786138 (100) 17046112 (100) 8206106 (100) 30906275 (7167) 878687 (8964) 0 0 0 0 0 0 0 0 0 0

a

The neurons were counted on 10 sections per region from rat brains (mean6S.E.M.; n58). Abbreviations: III, oculomotor nucleus; IV, trochlear nucleus; V, motor nucleus of the trigeminal nerve; VI, abducens nucleus; VII, facial nucleus; X, dorsal motor nucleus of the vagus; XII, hypoglossal nucleus; Am, nucleus ambiguus; CPU, caudate-putamen; HDB, nucleus of the diagonal band of Broca, horizontal limb; LDT, laterodorsal tegmental nucleus; MCPO, magnocellular preoptic nucleus; MS, medial septal nucleus; PPT, pedunculopontine tegmental nucleus; SI, substantia innominata; VDB, nucleus of the diagonal band of Broca, vertical limb; VP, ventral pallidum.

abducens nucleus (VI), facial nucleus (VII, Fig. 2A, A9), dorsal motor nucleus of the vagus (X), nucleus ambiguus and hypoglossal nucleus (XII). Although ChAT-LI neurons partially overlapped with SPR-LI neurons in the dorsal motor nucleus of the vagus (Fig. 2B, B9), neurons displaying both ChAT- and SPR-like immunoreactivities were hardly detected in this region (Table 1). The distribution of neurons containing ChAT or SPR in the rat brain has been examined in previous studies [4,10,12,13,15,22]. The present results were consistent with these reports, and further demonstrated that a large proportion (82–90%) of cholinergic neurons expressed SPR in the basal forebrain of rats. In addition, it was shown that 100% of cholinergic neurons expressed SPR in the rat basal ganglia as well, which confirmed the previous observation on co-localization of SPR and ChAT in striatal neurons. On the other hand, SPR-like immunoreactivity was hardly detected in the ChAT-containing neurons in the pontine tegmentum and cranial nerve motor nuclei, though a larger number of ChAT-LI neurons were also distributed in those regions. Our previous studies showed that the mesencephalic dopaminergic and basal forebrain cholinergic neurons expressed NKR, while noradrenergic neurons in the locus coeruleus selectively expressed SPR in the rat [9,10]. The morphological evidence for the modulation of the central catecholaminergic and cholinergic neuron groups by tachykinins was, thus, provided in those regions. Though expression of SPR in the striatal neurons was well demonstrated [5,25], only SPR mRNA was detected in the basal

forebrain cholinergic neurons in the previous in situ hybridization study [13]. Our present study has revealed that about 82–90% of ChAT-LI neurons in MS, DBB, MCPO and SI of the basal forebrain displayed SPR-LI immunoreactivity. On the basis of present findings, the cholinergic neurons in the basal forebrain may be categorized into one major group with SPR and the other minor group without SPR. It can be speculated that the large population of cholinergic neurons with SPR may be directly regulated or modulated by SP in the rat basal forebrain. On the other hand, the differential expression patterns of SPR in cholinergic neurons, suggesting that variation in their physiological property exists among these distinct populations of cholinergic neurons, may contribute to cell vulnerability to excitotoxins and the selective death or loss of certain cholinergic neurons in neurodegenerative diseases. Regulation of the activity and acetylcholine release of cholinergic neurons by substance P has been well documented in the striatum and basal forebrain [1,3]. Furthermore, SP exhibits a protective effect on striatal and cerebral neurons from excitatory damage and b amyloid toxicity, indicating that SP-SPR may play neurotrophic roles in the pathological mechanisms of neurodegenerative disease as well [6,8,14,16,23,28]. It is possible that SPSPR may exhibit a neuroprotective effect on the cholinergic neurons in the basal forebrain. Previous evidence suggested that tachykinins stimulate both phosphatidylinositol hydrolysis and cyclic AMP signal cascades in transfected Chinese Hamster ovary cells via three tachykinin

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Fig. 2. Photomicrographs showing the distribution of ChAT-like (A, B) and SPR-like (A9, B9) immunoreactivity in the facial nucleus (VII, A, A9) and dorsal motor nucleus of the vagus (X, B, B9). Neurons displaying both ChAT-like and SPR-like immunoreactivity are not seen in VII and X. The field of A or B is the same as that of A9 or B9, respectively. Scale bar560 mm.

receptors [20], which might be involved in their neuromodulation and neurotrophic-like actions. As we noticed, both SPR and NKR were expressed, or possibly co-localized, in certain cholinergic neurons of the basal forebrain of rats. Further studies are still needed, therefore, to elucidate the tachykinin receptors (SPR and NKR) interacting with cholinergic neurons, intracellular signaling and their role in physiology or pathophysiology of the mammalian basal forebrain, particularly their involvement in the neuronal survival or degeneration of the central cholinergic nervous system.

Acknowledgements The authors are grateful to Professor N. Mizuno and Dr Shigemoto, Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan for their gift of antiserum to SPR. This work was supported by grants from the National Natural Science Foundation of China (30040012) and the Foundation of Fourth Military Medical University (CX01A013).

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