Journal of Neuroscience Research 31:131-135 (1992)

Substance P Immunoreactivity of Rat Brain Stem Neurons in Primary Culture T.D. Jacquin, J.P. Denizot, and M. Denavit-Saubie LPNl, Laboratoire de Biologie Fonctionnelle du Neurone, CNRS (T.D.J., M.D.-S.) and LPN, DCpartement de Neurophysiologie Sensorielle, CNRS (J.P.D.), Gif sur Yvette Cx, France Substance P (SP)-ergic neurons from 16/17 day-embryonic rat brain stem in primary culture were identified by immunocytochemistry using biotinylated avidine and phosphatase alcaline methods with affinity-purified anti-SP antibodies. An average of 84% of neurons contained SP from day 9 to day 21 after plating. These in vitro data show that SP-containing neurons develop in our culture conditions. SP may act as a maturation factor as well as a neurotransmitter. Key words: immunocytochemistry, maturation factor, neurotransmitter

physiological studies in culture have to be performed on unidentified neuronal subtypes. Neurotransmitter immunostaining provides one way to identify neurons in culture. For example serotonin containing neurons have been identified in brain stem cultures (Yamamoto et al., 1981). With the aim of understanding the differenciation of SPcontaining neurons in primary culture, we show in this preliminary study that such neurons develop in our culture conditions when plated after SP synthesis initiation.

MATERIAL AND METHODS Tissue Culture Culture technique was derived from methods deINTRODUCTION scribed previously (Gruol et al., 1991). We kept the glial Ontogenetic studies of neurotransmitters contain- component in our cultures since glia favours neuronal ing neurons require both histochemical and electrophys- attachment, survival, neuronal shape, and neuritic iological approaches to demonstrate synthesis and activ- growth. Briefly, 16- or 17-day embryos were obtained ity of the neurotransmitters. We are studying here the from timed mated pregnant rats (albino Sprague Dawley, undecapeptide SP immunostaining of rat brain stem cells Iffa Credo). The embryos were removed from the mother in primary culture. (anesthetized with dry ice, followed by cervical dislocaThere is evidence to suggest that SP acts as a neu- tion) under aseptic condition. Dissection of brain stems rotransmitter within the adult brain stem in vivo: immu- was performed at 4°C in phosphate-buffered saline (PBS) nostaining of SP has been shown (Ljungdahl et al., 1978; until the dura was removed. Brain stem were transferred Palkovits, 1984) and iontophoretic application of SP al- to an empty culture dish and finely minced with iridecters activity of neurons involved in specific functions, tomy scissors. Two milliliters of plating medium was such as respiratory or cardiovascular functions with ex- added to the minced tissue before gentle trituration using citatory or depressant actions (Haeusler and Osterwalder, a fire-polished pasteur pipette. The resulting suspension 1980; Morin-Surun et al., 1984; Mueller et al., 1982). was divided into poly-1-ornithine (15 pglml; MW Ontogeny of SP-immunoreactive neurons in brain 40,000)-coated tissue culture dishes (35 mm, Falcon, stem has been described anatomically. SP neurons ap- Oxnard, CA). Dishes were prefilled with a 1:l mixture pear as early as day 14 of embryonic stage (EJ (Inagaki of Dulbecco’s modified Eagle’s medium (Gibco, Grand et al., 1982; Jonakait et al., 1990; Pickel et al., 1982; Island, NY) and Ham F-12 nutrient (Gibco, Grand IsSakanaka et al., 1982). This stage corresponds to the land, NY) supplemented with glucose (6 g/l), glutamine period of neuronal differentiation in the brain stem, ex- (2 mM), sodium bicarbonate (3 mM), HEPES buffer ( 5 tending from days E,, to E,, and before establishment of mM), and antibiotics (Gibco, Grand Island, NY) [penisynaptic contacts (Altman and Bayer, 1980). cillin (100 U/ml) and streptomycin (100 U/ml)]. This Simplified neuronal circuitry and increased accessibility of central neurons are obtained in dissociated cell culture under carefully controlled conditions that are un- Received February 8, 1991; revised May 25, 1991; accepted June 12, attainable in vivo. With most regions of the central ner- 1991. vous system, the difficulty involved in identifying dis- Address reprint requests to Dr. Thieny Jacquin, LPNl CNRS 91198 crete populations of neurons after plating has meant that Gif sur Yvette Cx, France. 0 1992 Wiley-Liss, Inc.

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mixture was supplemented with 10% horse serum and 10% fetal calf serum (Gibco, Grand Island, NY) during the first 3 days of plating. Dishes were kept undisturbed in a 37°C incubator with constant, moist air-CO, environment. The mixture was then replaced and supplemented with 10% horse serum twice weekly.

Immunocytochemistry Cultures were fixed in 0.1 M phosphate buffer (pH 7.4) containing paraformaldehyde (2-4%) and picric acid (0.2%) for 1 hr and then washed three times with phosphate-buffered saline (PBS) (pH 7.4). Cultures were incubated in successive steps with PBS containing 1) 3% H,02 for 30 min to suppress endogenic peroxydase, 2) 20% normal porcine serum for 30-60 min to decrease non specific binding, and 3 ) the first antiserum with anti-SP (1/2,000) or anti-glialfibrillary acidic protein (GFAP) and 3% normal porcine serum with 0.3% Triton XlOO for 24 hr. We have used antibodies to formaldehyde-linked SP (Miles, Incstar) or GFAP (gift of G. Tramu, Inserm 5156) obtained from rabbit. GFAP-immunostaining cells had large somata and numerous specific processes which are characteristic of astrocytes (Bottenstein and Michler-Stuke, 1989). Therefore, these cells were easily differentiated from the other cells in culture. Four control stainings were made using the same procedure as for the experimental with the following modifications: a) without the first antiserum containing anti-SP antibody, b) by saturating the first antiserum containing anti-SP antibody ( 1/2,000) with purified SP (CRB; 500 pg/ml of undiluted antiserum), c) by replacing the first antiserum containing antiSP antibody with normal goat serum (1/1,000), or d) with normal rabbit serum (1/2,000). For SP- or controlstainings cultures were then rinsed four times with PBS for 10-20 min before incubating with biotinylated antirabbit or anti-goat IgG (1/1,000, 2 hr) (Vector or Dako) and later with the streptavidin biotinylated horseradish peroxidase complex (1/2,000, 2 hr, Amersham, Arlington Heights, IL). After washing once in PBS for 10 min, cultures were incubated for 5 min in 0.05% 3-3-diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co., St. Louis, MO) activated with 0.002% H,O, in 0.05 M Tris-HC1 and rinsed in 0.05 M Tris-HC1 buffer (brown coloration). Phosphatase alcaline (Sigma Chemical Co., St. Louis, MO) method has also been tested to compare SP staining specificity with biotinylated avidine method. After fixation, cultures were incubated with Tris salt buffer containing successively 1) 20% normal porcine serum for 30-60 min, 2) the first anti-SP (1/2,000) containing serum with 3% normal porcine serum, and 0.3% triton XlOO for 24 hr, and 3) the 1% antirabbit IgG alkaline phosphatase conjugate with 3% normal porcine

serum and 0.3% triton XlOO for 24 hr. Alkaline phosphatase was revealed with a mixture of naphtol AS-MX phosphate (0.2 mgiml, Sigma Chemical Co., St. Louis, MO), fast red TR salt (1 mg/ml, Sigma Chemical Co., St. Louis, MO), and Tetramisole (1 mM, Sigma Chemical Co., St. Louis, MO) in Tris salt buffer (0.1 M; pH 8.2) (red coloration).

Quantitative Estimation of Cell Number The percentage of SP-immunoreactive neurons was counted under bright field optics at 400 X magnification calculated from ten contiguous fields (8.64 X lo-* mm2 each) chosen at random in the culture dish. Counting was realized under the two staining conditions using biotinylated avidine method or phosphatase alcaline method. Results were identical in both situations. RESULTS We analyzed cultivated neurons after 9 to 21 days in vitro (DIV) as follows: 9, 12, 13, 15, 19, and 21 DIV. At all stages several neuronal types were identified based on their morphological features. Soma were round, fusiform, pyramidal, or multipolar with one or several processes (Fig. 1). None of those cells showed GFAP immunoreactivity . SP immunoreactivity using the biotinylated avidine method has been tested at all stages from 9 to 21 DIV. Phosphatase alcaline method (Fig. 1B) has been compared to biotinylated avidine method (Fig. 1A) using cultures of 12 and 15 DIV issued from the same pregnant female rat for both methods. The two methods gave similar results (see Table I). Stained neurons showed all types of morphology (Fig. 1) after any period of culturing. Thus cells of a particular morphological type (e.g., bipolar neurons in Fig. 1B) could be found SP-immunostained in some cases and not stained in other cases in the same culture dish. Both soma and processes were immunostained in all types of neurons. In control stainings immunoreactivity was either suppressed either strongly decreased. The average percentage of SP-stained neurons obtained with the different periods of culturing was 84%. The percentage of SP-immunoreactive neurons was constant from 9 to 21 days after plating (see Table I).

DISCUSSION The results of this study provide evidence that neurons removed from the embryonic rat brain stem at E16E,, survive in culture and express SP. This is in accordance with results obtained in the in vivo conditions, since CNS neurons are known to express transmitters extremely early in embryogenesis; fetal ontogeny studies of SP-

Substance P in Cultivated Brain Stem Neurons

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A

B

Fig. 1 . SP-immunoreactive neurons from embryonic rat brain stem stained using the biotinylated avidine method after 15 DIV (A, left) or the phosphatase alcaline method after 12 DIV (B, left). Control staining (right, A and B) were obtained after the same time in culture and using the same immunoreactivity

procedures except for the first anti-SP-containing serum. Note SP-immunoreactive neurons of different morphology; ovoid or pyramidal soma with one or several processes. In B, two bipolar neurons (black arrow) but not a third neuron of similar morphology (white arrow) are stained. Bar, 35 pm.

TABLE I. Percentage of SP-Immunoreactive Neurons From Embryonic Rat Brain Stem After Various Days in Culture Using Biotinylated Avidine Method (Bio. Av.) or Phosphatase Alcaline Method (Ph. Al.)

15 (Inagaki et al., 1982; Jonakait et al., 1990; Sakanaka et al., 1982). Thus, in our culture conditions especially with no pharmacological treatment such as colchicine treatment to enhance somatic SP content, plating neurons after in vivo SP-synthesis onset at El,-E,, leads to SP detection in neurons after 9 to 21 DIV. During this period of culturing SP is present in a high percentage (84%) of brain stem neurons. This developmental pattern of SPcontaining neurons can be interpreted as reflecting neuronal growth and formation of new fibers and synapses especially when 1) SP has been shown to stimulate neurite outgrowth like in embryonic chick dorsal root ganglia (Narumi and Fujita, 1978), and 2) it has been suggested that target structures facilitate the development of peptidergic neurons. Limb removal during embryogenesis could reduce the development of peptidergic neurons in

Days in culture 9

12

13

15

19

21

86

79

15

88.5

86

90

Bio. Av. %

Ph. Al. %

80

85

containing neurons in rats showed initial detection of SP in brain stem at E,, and a rapid increase of SP immunoreactivity until the stage between postnatal days 5 and

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Prochiantz A (1982): Specific binding of an immunoreactive the corresponding sensory ganglia rather than direct nerve and biologically active 1251-labeledsubstance P derivative to damage (Kessler and Black, 1981). Similarly, our in vitro mouse mesencephalic cells in primary culture. Mol Pharmacol preparation is composed of different neurons from nu22148-55. merous brain stem structures constituting a well-diversi- Bottenstein JE, Michler-Stuke A (1989): Serum-free culture of dissofied network with numerous possible synaptical targets. ciated neonatal rat cortical astrocytes. In Shahar, De Vellis, Vernadakis, and Haber (eds): “A Dissection and Tissue Cul3 ) SP-receptive sites in target neurons are present. Quirion ture Manual of the Nervous System.” New York: Alan R. Liss, and Dam (1986) have reported that upper and lower brain Inc., pp. 109-111. stem contain very high densities of SP binding sites up to Carter MS, Krause JE (1990): Structure, expression, and some reguday 14 after birth. Therefore these data suggest that unlatory mechanisms of the rat preprotachykinin gene encoding known maturational factors selectively promote SP exsubstance P, neurokinin A, neuropeptide K, and neuropeptide. J Neurosci 10:2203-2214. pression in growing neurons. Strikingly, tachykinins neurokinin A, neurokinin B, Diez-Guerra FJ, Veira JAR, Augood S , Emson PC (1989): Ontogeny of the novel tachykinins neurokinin A, neurokinin B and neuand neuropeptide K have the same ontogenic development ropeptide K in the rat central nervous system. Regul Pep 25: as SP (Diez-Guerra et al., 1989). Two different genes 87-97. encode for these tachykinins (Carter and Krause, 1990; Gruol D, Jacquin TD, Yo01 A (1991): Single channel K’ currents recorded from the somatic and dendritic regions of cerebellar Kotani et al., 1986). Neurokinin A and neuropeptide K purkinje neurons in culture. J Neurosci 11(4):1002-1015. are encoded from the same preprotachykinin gene as SP. It can be suggested that 1) both genes are directed by Haeusler G, Ostenvalder R (1980): Evidence suggesting a transmitter or neuromodulatory role for substance P at the first synapse of similar maturational signals since their products are exthe baroreceptor reflex. Naun Schmied Arch Pharm 3 14:111pressed at the same time and 2) these maturational signals 121. are very active during ontogenesis of the rat CNS. Thus Inagaki S, Sakanaka M, Shiosaka S , Senba E, Takatsuki K, Takagi H, Kawai Y, Minagawa H, Tohyama M (1982): Ontogeny of subthe culture technique can help understanding the matustance P-containing neuron system of the rat: Immunohistorational regulation of neurotransmitters and more particchemical analysis-I forebrain and upper brain stem. Neuroularly SP expression in neurons during ontogenesis. science 7(1):251-277. At the time of development from 9 to 21 DIV, SP Jonakait GM, Ni L, Walker PD, Hart RP (1990): Development of can also be considered as a good candidate as a transsubstance P (SP)-containing cells in the central nervous system: Consequences of neurotransmitter co-localization. Prog Neumitter in neurons in primary culture since 1) SP-conrobiol 36: 1-21. taining neurons are present, 2) SP-binding sites have Kessler JA, Black IB (1981): Nerve growth factor stimulates develbeen described on neurons (Beaujouan et al., 1982) and opment of substance P in the embryonic spinal cord. Br Res astrocytes (Torrens et al., 1986), and 3) SP-sensitive 208: 135-145. neurons have been recorded (Okada and Miura, 1983). It Kotani H, Hoshimaru M, Nawa H, Nakanishi S (1986): Structure and would be interesting to confirm these results when platgene organization of bovine neuromedin K precursor. PNAS 83:7074-7078. ing the cells before the time of initial SP detection in vivo. Thus, the culture of brain stem neurons can be a Ljungdahl A, Hokfelt T, Nilsson G, Goldstein M (1978): Distribution of substance P-like immunoreactivity in the central nervous good model to study differentiation of transmitters and system of the rat. I. Cell bodies and nerve terminals. Neuromore particularly SP containing neurons. science 3(2):86 1-944.

ACKNOWLEDGMENTS We express our thanks to Dr. M. Synguelakis and Dr. A. Reboul for technical advises and J. Champagnat for valuable suggestions. This work was supported by the Centre National de la Recherche Scientifique, the Ministkre de la Recherche et de la Technologie (grant 9005), the Direction des Recherches, Etudes et Techniques (grant 89.167), the Fondation pour la Recherche Medicale, and NATO.

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