Developmental Brain Research, 55 (1990) 293-297 Elsevier

293

BRESD 60373

Hippocampal neurotrophic factors influence the perikaryal size of septal acetylcholinesterase-containing neurons in culture Kunihiko Harada 1, Ryuzo Shingai 2 and Haruhide

Ito 1

Departments of lNeurosurgery and 2pharmacology, Yamaguchi University School of Medicine, Ube 755 (Japan) (Accepted 15 May 1990) Key words: Neurotrophic factor; Cell size; Acetylcholinesterase; Septal cholinergic neuron; Cell culture; Nerve growth factor

The septal neurons were cultured under the following conditions: (1) treated with 7S nerve growth factor (NGF) (50 ng/ml); (2) grown with hippocampal cell-conditioned medium supernatant; (3) cocultivated with hippocampal cells; (4) cocultivated with cerebellar cells; (5) no treatment. Acetylcholinesterase histochemistry was used to identify cholinergic cells after pretreatment with diisopropylfluorophosphate. The mean values of the perikaryal major axis and minor axis at day 14 of culture were significantly larger in septal cells cocultivated with hippocampal cells than in septal cells grown under other conditions. NGF-treated septal cells showed a smaller, but significant, increase in the mean value of the major axis of these neurons. It has become clear that neurotrophic factors are deeply involved in the formation, maintenance and regeneration of synapses in the central nervous system. Nerve growth factor (NGF) is the most fully characterized neurotrophic factor 4A6'24, and it is retrogradely transported to neurons of the basal forebrain 12'2°'21. Exogenous N G F stimulates choline acetyltransferase (CHAT) activity of cholinergic neurons in the brain of adult rats with partial lesions of the cholinergic pathway 8, in adult rats bearing explanted fetal septal tissue 25, and in cultured fetal rat forebrain 3"9'm. N G F also promotes the survival of basal forebrain cholinergic neurons after fornix-fimbria transection in adult rats 7A3'26 and in cultured postnatal rat septal neurons 5"6. Other neurotrophic factors different from N G F are also present in the hippocampus 1'17'19'27. Hsiang et al. n studied the influence of hippocampal cells on the development of cholinergic septai neurons and suggested that hippocampal cells influenced the development and survival of cholinergic neurons. Cell size and other morphological characteristics of neurons containing acetylcholinesterase (ACHE) in the cat basal forebrain have been studied both in vivo ls, and in vitro 22. With regard to the relationship between cell size and neurotrophic factors in peripheral nerves, N G F plays a role of increasing in cell size within ganglia aS. However, to our knowledge, there have been no studies on the effects of neurotrophic factors on cell size in the central nervous system. Although many effects of hippocampal neurotrophic factors on septal cholinergic neurons have been found, no relationship between such

factors and the perikaryal size of septal neuron has yet been demonstrated. In the present study, we investigated the morphology of septal neurons in culture using a sensitive histochemical method for A C h E 23, and examined the effects of hippocampal neurotrophic factors on septal neurons in terms of the cell size of AChE-positive neurons. We compared the effects of these factors with those of NGF. The methods of primary cell culture were the same as described previously 22. The initial culture medium consisted of Dulbecco's Modified Eagle's medium plus 7% fetal calf serum (FCS; GICO), 1.0 g/liter glucose, 38 mM bicarbonate and 20 mg/liter penicillin. The following cell cultures were prepared: septal cells alone, hippocampal cells alone, cerebellar cells alone, septohippocampal cell coculture, and septocerebellar cell coculture. The dissociated cells were plated in poly-e-lysine (Sigma)-coated 35-mm Falcon plastic culture dishes containing the culture medium. Plating density was 1 x 106 cells per dish for septai cells alone or hippocampal cells alone, and 1 x 106 septal cells and 1-3 x 105 hippocampal cells or cerebellar cells per dish for septohippocampal cocultures or septocerebellar cell cocultures. On the fifth day of culture, cytosine arabinofuranoside (Sigma) was added to the medium at a concentration of 10 ktM. On the 7th day, 5% horse serum (GIBCO) was substituted for the FCS. All media, irrespective of composition, were changed every 2-3 days. 7S N G F (50 ng/ml, Sigma) was added every 3 days and 0.5 ml of supernatant from the hippocampal cell culture medium was added every other day.

Correspondence: K. Harada, Department of Neurosurgery, Yamaguchi University, School of Medicine, 1144 kogushi, Ube 755, Japan. 0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

294 Demonstration of AChE staining in the cultured cells was carried out by the method reported previously 22. After pretreatment with diisopropylfluorophosphate (DFP), cultures were fixed with 2% paraformaldehyde plus 0.5% glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.4) at 4 °C for 20 min. Cultures were washed 3 times with 0.1 M PB and 3 times with 0.1 M maleate buffer (MB; pH 6.0), and incubated for 30 min at room temperature with a solution containing 18-36 ¢tM acetylthiocholine iodide, 5 ~M K3Fe(CN)6, 30 ktM CuSO4, and 50 ~uM sodium citrate in 0.1 M MB. Then the cultures were washed 5 times with 50 mM Tris-HC1 (pH 6.0), and incubated for 5 min at room temperature with a solution containing 0.04% DAB and 0.3% nickel ammonium sulfate in 50 mM Tris-HC1, followed by addition of 0.003% H 2 0 2 and incubation for 10 min at room temperature. After 14 days of culture, the cell sizes of the

AChE-positive neurons were measured in randomly distributed visual fields corresponding to 20-30% of the total area. The neurons were drawn up on a monitor "IV via a videocamera attached to an inverted microscope and the major and minor axes of the somata were measured. Cultures were divided into 5 groups. Group 1 was cultured for 14 days in the presence of 7S NGF (50 ng/ml). Group 2 was treated with the supernatant of culture medium from dissociated hippocampal cell cultures cultivated for 4-14 days. In Groups 3 and 4, septal neurons were cocultured with hippocampal cells or cerebellar cells, respectively. In Group 5 (control group) septal neurons were cultured for 14 days without NGF or hippocampal supernatant. After 14 days of culture, cultured cells were fixed and stained. No AChE-positive cells were found in cultures of embryonic hippocampus,

Fig. 1. AChE staining of dissociated septal neurons cultured for 14 days. A: cultured with 7S NGF (50 ng/ml) lor 14 days (Group 1 in the text). B,C: cocultured for 14 days with hippocampal cells (Group 3). D: cultured alone without NGF or supernatant of culture media derived from hippocampal cell cultures (Group 5). Bar: 100/~m (A,B); 50 gm (C,D).

295 nor of cerebellar cells. The processes of AChE-positive neurons were most distinct and prominent in septohippocampal cocultures (Group 3). Group 3 displayed patterns of abundant, well-defined, thick fibers that were markedly extended (Fig. 1B,C). In Group 5 (control group) by contrast, the fibers were thin, poorly stained and small in extent (Fig. 1D). In Groups 1, 2 and 4, the cultures displayed almost the same patterns as those of Group 5 (not shown). NGF demonstrated no effects on AChE-positive cells with regard to fiber outgrowth. Table I compiles the mean values and standard deviations for the major axis ~ m ) and minor axis (~m) of neuronal somata in each group after 14 days of culture. There were significant differences between Groups 1 and 5 (control) in the size of the major axis, and between Groups 3 and 5 in all mean values, while there were no significant differences between Groups 2 and 4 and Group 5 for both of the major axis and minor axis. Thus NGF had the effect of increasing the size of a neuron along its major axis. On the other hand, the supernatant of the hippocampal culture medium had no activity in increasing the size of a neuron. Addition of supernatant from the medium used for hippocampal coculture with septal neurons also had no effect on the cell size of septal neurons (n = 2, not shown). However, when septal neurons were cocultured with hippocampal cells, factors derived from the hippocampus seemed to increase neuronal size along both the major and minor axes, whereas septal neurons cocultured with cerebellar cells decreased the mean values of both axes compared to those of control. Histograms were prepared for each group for the distribution of each of the major axis and minor axis (Fig. 2). In the case of the distribution for the major axis, the histogram pattern for Groups 2 (not shown), 4 and 5 were almost the same, showing a single sharp peak at 20-22 /~m, while those for Groups 1 and 3 showed a broader peak which ranges from 20-22/~m to about 26-28 ,um.

TABLE I

Quantitative analysis of septal A Ch E-positive neurons in culture

Group Group Group Group Group

1 2 3 4 5

Major axis (l~m)

Minor axis (l~m)

n

23.4 21.1 26.6 20.6 22.3

13.7 + 3.37 13.6 + 3.28 17.3 + 4.75* 12.7 + 2.70* 13.9 _+3.13

196 151 225 187 258

_+ 6.41"* + 5.40 + 8.23* + 4.91" + 5.36

Groups 1, 3, 4 and 5 are the same as described in the legends of Figs. 1 and 2. In Group 2, septal cells were grown with hippocampal cells conditioned medium. Each value is mean + S.D. (standard deviation). * Significantly different from the control (Group 5), P < 0.001. ** Significantly different from Group 5, P < 0.05. n = number of neurons measured.

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Fig, 2. Histograms comparing the distribution of the sizes (major axis, minor axis) of the perikarya of septal AChE-positive neurons. Groups 1, 3 and 5 are the same as described in the legend of Fig. 1. In Group 4, septal cells were cocultured with cerebellar cells. Histograms of the major axis and minor axis in Group 3 and the major axis in Group 1 show a broad-peaked pattern, while other histograms have a sharp-single-peaked pattern, n = number of neurons measured.

About 70% of the cells in Groups 4 and 5, and about 60% in Group 1 measured less than 24/~m along the major axis, while only 44% of AChE-positive cells lay in this small size range and 25% of the cells were larger than 30 /~m in Group 3. With regard to the size distribution of the minor axis, Groups 1, 2 (not shown), 4 and 5 showed almost the same histogram pattern, i.e., a single sharp

296 peak between 12 and 16 ~m, while Group 3 showed a broad peak, which ranges from 12 to 20/~m. In Groups 1, 4 and 5, 70-80% of AChE-positive cells were found at 10-16 ktm, while in Group 3 only 44% of cells lay within the same size range, and the incidence of neurons with a size of 16/~m or more was high. We employed AChE histochemistry to study the neurotrophic effects of hippocampal trophic factors from the aspect of cell size and compared the results with those obtained by administration of NGF alone. AChE histochemistry after pretreatment with DFP, as performed in the present study, has been considered to be a reliable marker for cholinergic neurons, since 80-90% of all AChE-positive neurons have been found to be co-stained for ChAT in the septum 2'14. Moreover, the processes of cholinergic neurons are more clearly stained by AChE histochemistry in comparison with direct immunostaining using anti-ChAT antibodies In the present study, the mean length of the major axis of septal cholinergic neurons in culture was 22.3 + 5.36 S.D. j~m, and the histogram for the major axis showed a sharp peak at 20-22 ~m. The range of sizes of the septal neurons in the present" cultures is comparable to that demonstrated previously s, where it was 20-30/~m. In septo-hippocampal cocultures, the mean length of the major axis was significantly larger than that of the control group and the histogram showed a broad peak. Since in septo-cerebellar cocultures, the mean length of the major axis is significantly smaller than that in the control group, and the histogram shows almost the same pattern as that of the control, the difference in results between septohippocampal coculture and the control (Group 5) is not due to the effect of cell densities in culture. Administra-

tion of NGF alone also produced a slight increase in cell size along the major axis, and the histogram pattern for the major axis also showed a broad-peaked pattern. However, the peak was smaller in size than that of cocultures, and the mean value of the minor axis showed no significant difference from that of the control group. Moreover the histogram for the minor axis in septal cell cultures treated with NGF showed a single sharp peak, while that of cocultures with hippocampal cells showed a broad-peaked pattern. Thus, the increase in cell size seen in septohippocampal cocultures was not only due to the effects of NGF, but also chiefly mediated by the other neurotrophic factors derived from the hippocampal cells. On the other hand, administration of supernatant of the media derived from hippocampal cell cultures did not increase the size of neurons. As to the reason for this, the following can be speculated: (1) the amount of neurotrophic factors added from the supernatant of hippocampal cultures was too small to affect the cell size; (2) the neurotrophic factor was stable only for a short period in the culture medium, and this factor in the supernatant had already been degraded when it was transferred to the septal cultures, or was degraded shortly after the administration. It remains also unclear from the present study why the histograms demonstrated that the number of large cells increased in septohippocampal cocultures and in septai neurons treated with N G E The following reasons are suggested: (1) some population of smaller cells increased their cell size, and the proportion of large cells became higher in these groups than among septal cells alone; (2) the hippocampal neurotrophic factors produced a more marked effect on the survival of large neurons than that of small neurons.

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22 Shingai, R., Itoh, E., Harada, K. and Kimura, H., Acetylcholinesterase-containing neurons in the striatum, septum and hippocampus of the rat in embryonic culture and adult in situ, Dev. Brain Res., in press. 23 Tago, H., Kimura, H. and Maeda, T., Visualization of detailed acetylcholinesterase fiber and neuron staining in rat brain by a sensitive histochemical procedure, J. Histochem. Cytochem., 34 (1986) 1431-1438. 24 Thoenen, H. and Barde, Y.A., Physiology of nerve growth factor, Physiol. Rev., 60 (1980) 1284-1335. 25 Toniolo, G., Dunnett, S., Hefti, F. and Will, B., Acetylcholinerich transplants in the hippocampus: influence of intrinsic growth factors and application of NGF on choline acetyltransferase activity, Brain Res., 345 (1985) 141-146. 26 Williams, L.R., Varon, S., Peterson, G.M., Wictorin, K., Fischer, W., Bjorklund, A. and Gage, EH., 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. 27 Yoshida, K., Kohsaka, S., Ide, T., Nii, S., Otani, M., Toya, S. and Tsukada, Y., Septal deafferentation enhances the neurotrophic effects of rat hippocampus on cultured neural cells from the central nervous system, Neurosci. Lett., 66 (1986) 181-186.

Hippocampal neurotrophic factors influence the perikaryal size of septal acetylcholinesterase-containing neurons in culture.

The septal neurons were cultured under the following conditions: (1) treated with 7S nerve growth factor (NGF) (50 ng/ml); (2) grown with hippocampal ...
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