Nerve growth factor promotes survival of retrogradely labeled hippocampus-projecting neurons in the rat basal forebrain in vitro

DevelopmentalBrain Research, 45 (1989) 297-301 Elsevier

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BRD60298

Nerve growth factor promotes survival of retrogradely labeled hippocampus-projecting neurons in the rat basal forebrain in vitro Yasuyoshi Arimatsu, Mami Miyamoto, Hiroko Tsukui* and Hiroshi Hatanaka* Department of Neuroscience, Mitsubishi Kasei Institute of Life Sciences, Tokyo (Japan) (Accepted 11 October 1988) Key words: Nerve growth factor; Neuronal survival; Identified projection neuron; Septal cholinergic neuron; Basal forebrain; Cell culture

The effect of nerve growth factor (NGF) on the survival of neurons projecting to the hippocampus from developing medial septum and vertical limb of the diagonal band was studied in vitro. The neurons had previously been labeled retrogradely in vivo with fluorescent latex microspheres. The microspheres were injected bilaterally into the hippocampus of 5-day-old rats. Twenty to 24 h after the injection, cells from the basal forebrain were dissociated with papain and cultured for 3-5 days. The number of microsphere-labeled neurons in the culture with supplementation of NGF was much greater than that without NGF. The result clearly indicates a survivalpromoting effect of NGF on these projection neurons. It has been proposed that nerve growth factor (NGF) enhances the survival of the cholinergic neurons in the basal forebrain of mammalian species. Intraventricular administration of N G F was reported to promote the survival of neurons in the medial septurn and diagonal band which would die after fimbriafornix transection without N G F 7,t3'2°. In culture systems, N G F also p r o m o t e d the survival of cholinergic neurons isolated from the medial septum 5'6, diagonal band 6 and nucleus basalis 3 of postnatal rats. In these studies, the survival-promoting effect of N G F was concluded from the observations that the cholinergic neurons, identified by histochemical staining for acetylcholinesterase and by immunocytochemical demonstration for choline acetyltransferase, were greater in number under the influence of exogenous NGF. However, the possibility is not ruled out that N G F might have increased the number of detectable cholinergic neurons through enhancing the expression of these cholinergic enzymes instead of promoting the survival of the neurons. We here support the possibility of neuronal survival by applying N G F in vitro to the neurons of postnatal rat basal forebrain which

neurons had been retrogradely labeled with rhodamine fluorescent latex microspheres n in vivo. Five-day-old rats of Wistar strain (Sankyo Lab., Shizuoka) were injected under sodium pentobarbital anesthesia with rhodamine fluorescent latex microspheres (Luma Fluor, New City, NY) into the hippocampus by the aid of a stereotaxical positioner. The day when newborn pups were found in the morning was designated postnadal day 1. Several 0.1 ktl injections (at the concentration supplied by the company) were made through a glass micropipet (150-200 ~m in tip diameter) connected to a 10 ktl Hamilton syringe and a microinjection pump (CMA/100; 0.1 ~tl/min). Twenty to 24 h later, the rats were sacrificed by cervical dislocation and the brains were sliced in a frontal plane at 400/~m thickness by a vibration microslicer (DTK-3000W). Tissue fragments were dissected out of the septum and vertical limb of the diagonal band under a dissecting microscope. Caution was taken not to include ependymal cells which might be a source of non-specific labeling due to a diffusion of fluorescent microspheres via the 4th ventricle. The isolated fragments were collected in L15 medium

* Present address: Institute for Protein Research, Osaka University, Suita-shi, Osaka 565, Japan. Correspondence: Y. Arimatsu, Department of Neuroscience, Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machidashi, Tokyo 194, Japan. 0165-3806/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. 1. A: fluorescence photomicrograph of a frontal section through the medial septum (MS) and vertical limb of the diagonal band (vDB). Two II. 1 ~1 injections of fluorescent latex microspheres were made unilaterally into the hippocampus (1.0 mm posterior (P) from bregma, 1.5 mm lateral (L) from midline, 2.5 mm deep (D) from pia; 2.0 mm P, 1.5 mm L, 2.5 mm D) of 5-day-old rat. Twentyfour h later, the rat was perfused with 4% paraformaldehyde and 0.1% glutaraldehyde, and the brain was sectioned in a cryostat at 30 ,urn thickness. The section was mounted on glass slide, cleared in xylene and coverslipped with Enteran (Merk). The photomicrograph was taken under a fluorescence microscope with a filter set for rhodamine. A number of labeled neurons are seen in the ipsilateral side of the medial septum and vertical limb of the diagonal band. A few cells in the contralateral side are also labeled. Arrow indicates midline. Bar = 100 rum. Approximate location of the photomicrograph is schematically shown in B (shaded area). Regions from which tissue fragments were taken for culture are shown in C (dotted areas).

299 (Gibco) with O 2 gassing and then incubated at 37 °C for 30 min in 10 ml of oxygenated CaZ+,Mga+-free phosphate-buffered saline containing 120 units papain (Pharmacia), 2 mg O.L-cysteine h y d r o c h l o r i d e (Sigma), 2 mg bovine serum albumin ( A r m o u r , recrystallized), 50 nag glucose ( W a k o ) and 1 mg

DNase I (Sigma). The fragments were s e d i m e n t e d following an addition of a few milliliters of horse serum (Gibco) and the pellet was resuspended in a 1:1 mixture of Dulbecco's modified E a g l e ' s and H a m ' s F12 media (both Gibco) containing 5% (v/v) precolostrum newborn calf serum (Mitsubishi Kasei

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Fig. 2. Cultured basal forebrain neurons from 6-day-old rats. Four 0.1 pl injections of fluorescent latex microspheres were made bilaterally into the hippocampus (1.0 mm P, 1.5 mm L, 2.5 mm D: 2.0 mm P, 1.5 mm L, 2.5 mm D: both sides) of 5-day-old rats. After 20-24 h, cells from the basal forebrain were dissociated and cultured as described in the text. A,B: 2 h after plating on a polyethyleneimine-coated glass slide. C,D: 3 days after plating on astroglial feeder layer with an addition of NGF. A and C, fluorescence photomicrograph; B and D, phase-contrast photomicrograph. Bar = 60 pm. Fluorescent latex microsphere-labeled ceils are indicated by arrows.

300 Corp.), 5 ~ (v/v) heat-inactivated horse serum and 1% (v/v) heat-inactivated rat serum. They were then triturated gently through plastic tips of two different orifice sizes (1.2 mm, then 0.8 mm diameter). The dispersed cells were centrifuged and resuspended in the same medium, and inoculated onto astroglial feeder layers in wells of plastic 4-well-plates (Nunc) at a density of 2-3 x 10s cells/cm 2. The cells were cultured with or without addition of 100 ng/ml 2.5S NGF in humidified 5% CO2/95% air atmosphere (37 °C). Detailed procedures for cell cultures of basal forebrain neurons from postnatal rats were described elsewhere 6'~7. Three to 5 days after the innoculation, the cells were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 5 min at room temperature. Neuronal cells labeled with fluorescent latex microspheres were counted under a fluorescence microscope (Nikon) with a filter set for rhodamine. Following the injection of fluorescent latex microspheres into the hippocampus, a number of neurons in the medial septum and vertical limb of the diagonal band were labeled (Fig. 1). The pattern of distribution of the labeled neurons was similar to that reported previously using other retrograde tracers such as HRP 14, W G A - H R P 15 and Fast blue 12. Immediately after the dissociation with papain, the labeled neurons were spherical in shape without any neurite extensions (Fig. 2A,B). Although many of the labeled neurons underwent disintegration within 24 h, a significant population of cells survived even for longer periods. They extended the processes on the astroglial feeder layer (Fig. 2C,D). The pattern of the processes extension was either bipolar, bitufted or multipolar, Under phase-contrast optics, these labeled neurons were easily distinguished from astrocytes which occasionally contained fluorescent latex microspheres probably released from the disrupted neurons. An addition of NGF in the culture medium greatly promoted the survival rate of the retrogradely labeled neurons. As shown in Fig. 3, surviving neurons at 3 and 5 days in vitro were much greater in the number in culture with NGF than without NGF (P < 0.001, P < 0.005, respectively; Student's t-test). The survival-promoting effect of NGF was repeatedly observed at 3 days in vitro in 3 separate experiments. In an additional experiment, the NGF-dependent sur-

vival was recognized at the time period of 24 h after plating (data not shown). Most of the previous studies suggesting the survival-promoting effect of NGF on the central cholinergic neurons were based on the histochemical or immunohistochemical staining for cholinergic enzymes. Therefore, it should be important to settle whether the observed 'survival' due to NGF were derived really from promotion of neuronal survival or from enhancement (or maintenance) of expressions of the cholinergic enzymes. In fact, the induction or enhancement of these enzyme activities by exogenous NGF was suggested in various experimental systems in C N S 2"4'6'8'16. The vital labeling of neurons with a retrogradely transported tracer is one of the conclusive approaches to the assessment of the survival of specific projection neurons, Fluorescent latex microspheres were originally used for anatomical and physiological studies of identified projection neurons in vivo and in brain slices 1°'11 and applied later to dissociated cell cultures 9. The present result indicates the survival-promoting effect of NGF on the neurons projecting to the hippocampus from the developing

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Fig. 3. Effect of NGF on the survival of cultured hippocampus projecting neurons in the basal forebrain. Eight 5-day-old rats were injected bilaterally with fluorescent latex microspheres into the hippocampus (1.0 mm P, 1.5 mm L, 2.5 mm D; 2.0 mm P, 1.5 mm L, 2.5 mm D: both sides). Dissociated basal forebrain cells were cultured in total 16 wells with (shaded columns) or without (open columns) supplementation of NGF as described in the text. Cells were fixed at 3 and 5 days in vitro and the microsphere-labeled neurons were counted. Bars represent S.E.M. (n = 4).

301 medial septum and vertical limb of the diagonal

ported by NGF. A preliminary study suggested that

band.

N G F promotes the survival of not only cholinergic

It is known that G A B A e r g i c n e u r o n s are included in those projection n e u r o n s 11 as well as cholinergic 18

al septum and vertical limb of the diagonal band 1.

but also G A B A e r g i c projection neurons in the medi-

ones. A recent study suggested a possible loss of the G A B A e r g i c n e u r o n s in the medial septum after fimbria-fornix transection in adult rats ~9 by immunohis-

We thank Dr. Hiroshi K a w a m u r a for his generous

tochemical method using anti-glutamic acid decarboxylase antibody. In this context, it is interesting to

suggestions on the manuscript. This work was sup-

examine the species of neurotransmitter of microsphere-labeled n e u r o n s whose survival would be sup-

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encouragements, and Dr. Akihiko Ogura for his kind ported in part by a Scientific Research G r a n t of the Ministry of Education of Japan.

latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex, Nature (Lond.), 310 (1984) 498-500. 12 KOhler, C., Chan-Palay, V. and Wu, J,-Y., Septal neurons containing glutamic acid decarboxylase immunoreactivity project to the hippocampal region, Anat. Embryol., 169 (1984) 41-44. 13 Kromer, L.F., Nerve growth factor treatment after brain injury prevents neuronal death, Science, 235 (1987) 214-216. 14 Meibach, R.C. and Siegel, A., Efferent connections of the septal area in the rat: an analysis utilizing retrograde and anterograde transport methods, Brain Res., 119 (1977) 1-20.

15 Milner, T.A., Loy, R. and Amaral, D.G., An anatomical study of the development of the septo-hippocampal projection in the rat, Dev. Brain Res., 8 (1983) 343-371. 16 Mobley, W.C., Rutkowski, J.L., Tennekoon, C.I., Gemski, J., Buchanan, K. and Johnston, M.V., Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons, Mol. Brain Res., 1 (1986) 53-62. 17 Nakajima, Y., Nakajima, S., Leonard, R.J. and Yamaguchi, K., Acetylcholine raises excitability by inhibiting the fast transient potassium current in cultured hippocampal neurons, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 3022-3026. 18 Paxinos, G. and Butcher, L.L., Organizational and principles of the brain as revealed by choline acetyltransferase and acetylcholinesterase distribution and projections. In G. Paxinos (Ed.), The Rat Nervous System, Vol. 1, Forebrain and Midbrain, Academic, Sydney, 1985, pp. 487-521. 19 Peterson, G.M., Williams, L.R., Varon, S. and Gage, F.H., Loss of GABAergic neurons in medial septum after fimbria-fornix transection, Neurosci. Lett., 76 (1987) 140-144. 20 Williams, L.R., Varon, S., Peterson, G.M., Wictorin, K., Fischer, W., Bj6rklund, A. and Gage, F.H., Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 9231-9235.