- Email: [email protected]
Choline acetyltransferase in organotypic cultures of rat septum and hippocampus
Neuroscience Letters, 42 (1983) 273-278 Elsevier Scientific Publishers Ireland Ltd.
273
CHOLINE ACETYLTRANSFERASI~ IN ORGANOTYPIC CULTURES OF RAT SEPTUM AND HIPPOCAMPUS
FLAVIO KELLER, KARIN RIMVALL and PETER G. WASER
PhartnakOlogisches lnstitut der Universitiit Ziirich, Gloriastrasse 32, CH-8006 Ziirich (Switzerland) (Received July I lth, 1983; Revised version received and accepted September 28th, 1983)
Key words: septum
- hippocampus - organotypic cultures - choline acetylcholinesterase - histochemistry - development - target
acetyltransferase
-
Choline acetyltransferase (CAT) activity in the hippocampus originates almost exclusively in axons from neurons located in the medial septum. In the rat, the development of CAT in the hippocampus takes place during the first 3 Weeks after birth. The development of CAT was studied in organotypic cultures of fetal rat septum and early postnatal rat hippocampus. In some septal explants, enzyme activity increased up to 10-fold daring the first 3-4 weeks in vitro. Acetylcholinesterase (ACHE)histochemistry showed the presence of AChE-positive cells and fibers in many ex~lants. Thus it appears that septal cholinergic neurons develop CAT and AChE activity even without making contact with their target cells. However, the development of CAT was accelerated by the presence of hippocampal tissue No CAT activity was found in the hippocampal cultures, confirming that there are few, if any, intrinsic cholinergic cell bodies in the hippocampus.
The medial part of the septum contains the somata of cholinergic neurons whose axons reach the hippocampus along three routes: a supracallosal pathway, a subcallosal pathway (through the fimbria/fornix), and a ventral pathway (via the piriform lobe) [6]. From lesion experiments, it appears that the choline acetyltransferase (CAT) activity detected in the hippocampal formation originates almost exclusively from this septo-hippocampal projection (see ref. 16 for references). The cells of origin of the septo-hippocampal projection are generated between embryonic day (ED) 13 and ED 17 in the rat [1]. The development of cholinergic innervation in the hippocampal formation of the rat brain has been studied in situ by Matthews et al. [10] and Nadler et al. [13]. CAT activity was seen to increase mainly during two periods of development, from birth to 7 days of age and from 11 to 21 days of age. In our study we have looked at the development of CAT in organotypic explant cultures of fetal rat septum, in the presence or the absence of early postnatal rat hippocampus. Septal explants, taken from SIV 50 strain rats shortly before birth, at ED 20 or 21", or hippocampal explants from 7-day-old rats, were placed onto glass cover.
.
.
.
.
.
.
.
.
* The day of sperm-positivity was day one of embryonic life. 0304-3940/83/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
274
slips and cultured by the roller tube technique (see ref. 7 for details). Co-cultures of septum and hippocampus were prepared by placing on each cover-slip one septal explant between two hippocampal explants, at a distance of about 1 ram. The nutrient medium consisted of Eagle's Basal Medium with Hank's salts (50°70), Earle's BSS (25070), heat-inactivated horse serum (25070), and was supplemented with glucose to a concentration of 6.5 mg/ml. Normally, no antibiotics were required. When antibiotics were needed, then 100 U penicillin and 100 ~g streptomycin per ml medium were added. The cultures were incubated at 36°C and the medium was changed once or twice weekly. After varying lengths of time in vitro the explants were washed in Gey's BSS and removed from the cover-slips by small stainless steel spatulae. Care was taken to remove also the growth zone of the culture, containing the neurites growing out from the explants. CAT activity was assayed by the radiochemicai method of Fonnum [5], adapted by Woodward et al. [17] for explant cultures. The assays were conducted on homogenates prepared from 1-3 cultures. The proteins were measured by the method of Lowry et al. [9] with bovine serum albumin as standard. Some septal cultures were stained for acetylcholinesterase (ACHE) by a modified Karnovsky technique [4]. The unspecific cholinesterase was inhibited by 4 mM tetraisopropylpyrophosphoramide. No CAT activity could be detected when the septal homogenate was st~bstituted by buffer alone, or when the specific CAT inhibitor trans-l,2-dihydro-2-imino-4(lnaphtylvinyl)-l-pyridine-ethanoi (a derivative of NVP, a generous gift of Dr. C. Cavallito) was present in the reaction medium (1 mM). No CAT activity was found in the hippocampal cultures at various stages (14 cultures, 19-30 days in vitro). This observation confirms that there are few, if any, intrinsic cholinergic neurons in the hippocampus, and speaks for the possibility that the residual CAT activity found in the hippocampal formation after lesions of the septo-hippocampal pathway originates from other non-septal, cholinergic projection(s). A possible origin of such a projection is discussed in ref. 12. Interestingly, a small but significant CAT activity could be detected in the septum at ED 20-21 (cf. Fig. !). This finding correlates well with the observation that cells of the medial septal nucleus and the nucleus of the diagonal band stain for AChE as early as ED 20 [12]. The development of CAT (absolute activity) in a series of septal cultures is shown in Fig. 1. The enzyme activity increased 10-fold during the first 3 weeks in vitro. The increase was steepest (5-fold) in the 3rd and at the beginning of the 4th week. The mean activity of CAT at 24 days in vitro was 34 pmol acetylcholine (ACh) formed per minute per culture. Taking into account the average protein content of a 23-day-old septal culture (51 _+5 #g (S.E.M.), n = 26), we calculated a specific activity.of about 0.7 nmol ACh/mg protein/min, a value which approaches that reported by Cheney et al. [2] for the septal nuclei in the adult rat brain (0.8-1.5 nmol ACh/mg protein/rain).
275 (61 461
/.0
LU 3O
rF 2:) I-._I 2:) .,,w,.
{6)
z
~-
20
"r" ,,¢
0
n
10
(3 19) v
O_
|
0
~
,
,
J
I
10
~
,
I
I
,I
20
l
I
I
I
I
30
DAYS IN CULTURE
Fig. I. Developmentof CAT in organotypic cultures of fetal rat septum. Abscissa, days in culture; ordinate, CAT activity in pmol ACh formed per minute per culture, Each point represents the mean and S.E.M. of the number of cultures indicated in parentheses. AChE-positive cells and fibers could be observed already by 12 days in vitro (pictures not shown). Maximal staining seemed to be reached after 4-5 weeks in vitro (cf. Fig. 2). Since histochemical and biochemical evidence suggest that the septo-hippocampal projection is formed almost exclusively postnatally in the rat [10,11,13], contact with the hippocampus seems not to be absolutely necessary for the development of CAT in the septum. (Very recently, on the basis of orthograde and retrograde tracer techniques, Milne, et al. [12] have proposed that at least a few septal fibers may enter the hippocampal formation as soon as at ED 16-20. However, it is unknown at the present time if these fibers form synapses with their target cells already prenatally.) 'Normal' development of neurons in the absence of~their target cells has been observed, e.g. in explant cultures of the locus coeruleus (norepinephrine-producing cells), of the substantia nigra (dopamine-produc~ng cells) a n d in Cultured mesencephalic neurons (dopamine uptake and synthesis) [14,15]. However, in
276
Fig. 2. Septum explant, 37 days in vitro, AChE-histochemistry. A strongly AChE-positive soma (arrow) within a network of AChE-positive fibers. Bar = 30 #m.
mesencephalic neurons, both dopamine uptake and synthesis were enhanced by the presence of striatai ceils [141. in our cultures, the presence of hippocampal tissue significantly accelerated the development of CAT in the septum, as compared with septal explants cultured without hippocampus under the same conditions (cf. Table I). The mean CAT activity in the series of cultures shown in Table I is lower than the mean activity of the series of cultures of septum alone shown in Fig. 1 after 21 DIV. This is probably best explained by the differences in the composition of serum, as both culture series were fed with a different serum batch. It is well known that TABLE 1 EFFECT OF HIPPOCAMPUS ON THE DEVELOPMENT OF CAT IN SEPTAL EXPLANTS Septal (ED 21) and hippocampal (P 7) explants were cultured on the same cover-slip for 20 days. Control septal explants were grown without hippocampus under the same conditions. The values represent the mean and S.E.M. for the number of cultures indicated in parentheses. The difference is statistically significant IP= 0.05, 2-tailed test of Mann and Whitney). CAT activity after 20 days in vitro (pmol ACh/min/culture) Septum alone Septum + hippocampus
3.3 +_0.6 (14) 9.3 +_3.0 (18)
277
the quality of the sera profoundly affects the survival and differentiation of cultured cells. Further experiments are needed to assess if hippocampal tissue hastens the development of septal CAT activity in culture in a specific manner. In a recent work [8] it has been shown that intraventricular injections of nerve growth factor (NGF) to neonatal rats markedly increased the level of CAT in the septal area and the cortex, while no increase was observed in other brain areas. However, at the present time it is not clear if this responsiveness reflects on a physiological role for NGF in these neurons during their normal development. The finding that antibodies against NGF failed to reduce CAT activity in the neonatal brain and in dissociated septal cultures argues against this possibility. Nevertheless, the existence of a cholinergic neuronotrophic factor in the central nervous system different from the 'classical' NGF molecule is suggested by a study of Crutcher and Collins [3]. They showed that antibodies against NGF failed to inhibit the effect of hippocampal extract on neurite extension from cholinergic parasympathetic neurons in vitro. It would then be interesting to test if antibodies against NGF would counteract the effect of the hippocampus on cultured septal explants suggested by the present study. The authors sincerely thank Dr. B.H. Gfihwiler for his assistance during this work and for critical reading of the manus,.-ript, and Dr. D.M. Davis for helpful comments. ! Bayer, S.A., The development of the septal region in the rat. 1. Neurogenesis examined with ~Hthymidin¢ autoradiography, J. comp. Neurol., 183 (1979) 89-106. 2 Cheney, D.L., Racagni, G. and Costa, E., Distribution of acetylcholine and choline acetyltransferase in specific nuclei and tracts of rat brain. In A.M. Goldberg and !. Hanin (Eds.), Biology of Cholinergic Function, Raven Press. New York, 1976, pp. 655-659. 3 Crutcher, K.A. and Collins, F., in vitro evidence for two distinct hippocampal growth factors: basis of neuronal plasticity?, Science, 217 (1982)67-68. 4 EI-Badawi, A. and Schenk, E.A., Histochemical methods for :,eparate, consecutive and simultaneous demonstration of acetylcholinesterase and norepinephrine in cryostat sections, J. Histochem. Cytochem., 15 (1967) 580-588. 5 Fonnum, F., A rapid radiochemical me-~hod for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 6 Gage, F.H., Bj6rklund, A. and Stenevi, U., Reirmervation of the partially deafferented hippocampus by compensatory collateral sprouting from spared cholinergic and noradrenergic afferents, Brain Res., 268 (1983) 27-37. 7 Gfihwiler, B.H., Organotypic monolayer cultu,~es of nervous tissue, J. Neurosci. Meth., 4 (1981) 329-342. 8 Gnahn, H., Hefti, F., Heumann, R., Schwab, M.E. and Thoenen, H., NGF-mediated increase of choline acetyltransferase (CHAT) in the neonatal rat forebrain: evidence for a physiological role of NGF in the brain? Develop. Brain Res., 9 (1983) 45-52. 9 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275.
278
i
10 Matthews, D.A., Nadler, J.V., Lynch, G.S. and Cotman, C.W., Development of cholinergic innervation in the hippocampal formation of the rat. 1. Histochemical demonstration of acetylcholinesterase activity, Develop. Biol., 36 (1974) 130--141. 11 Mellgren, S.I., Distribution of acetylcholinesterase in the hippocampal region of the rat during postnatal development, Z. Zellforsch., 141 (1973) 375-400. 12 Milner, T.A., Loy, R. and Amaral, D.G., An anatomical study of the development of the septohippocampal projection in the rat, Brain Res., 284 (1983) 343-371. 13 Nadler, J.V., Matthews, D.A., Cotman, C.W. and Lynch, G.S., Development of cholinergic innervation in the hippocampal formation of the rat. 11. Quantitative changes in choline acetyltransferase and acetylcholinesterase activities, Develop. Biol., 36 (1974) 142-154. 14 Prochiantz, A., Di Porzio, U., Kato, A., Berger, B. and Glowinski, J., In vitro maturation of mesencephalic dopaminergic neurons from mouse embryos is enhanced in presence of their striatal target cells, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5387-5391. 15 Schlumpf., M., Shoemaker, W.J. and Bloom, F.E., Explant cultures of catecholamine-containing neurons from rat brain: biochemical, P,istofluorescence, and electron microscopic studies, Proc. nat. Acad. Sci. U.S.A., 74 (1977)4471-4475. 16 Storm-Mathisen, J., Localization of transmitter candidates in the brain: the hippocampal formation as a model, Progr. Neurobiol., 8 (1977) 119-181. 17 Woodward, W.R., Blank, N.K. and Sell, F.J., Choline acetyltransferase activity in mouse cerebellar cultures, Brain Res., 241 (1982) 323-327.
273
CHOLINE ACETYLTRANSFERASI~ IN ORGANOTYPIC CULTURES OF RAT SEPTUM AND HIPPOCAMPUS
FLAVIO KELLER, KARIN RIMVALL and PETER G. WASER
PhartnakOlogisches lnstitut der Universitiit Ziirich, Gloriastrasse 32, CH-8006 Ziirich (Switzerland) (Received July I lth, 1983; Revised version received and accepted September 28th, 1983)
Key words: septum
- hippocampus - organotypic cultures - choline acetylcholinesterase - histochemistry - development - target
acetyltransferase
-
Choline acetyltransferase (CAT) activity in the hippocampus originates almost exclusively in axons from neurons located in the medial septum. In the rat, the development of CAT in the hippocampus takes place during the first 3 Weeks after birth. The development of CAT was studied in organotypic cultures of fetal rat septum and early postnatal rat hippocampus. In some septal explants, enzyme activity increased up to 10-fold daring the first 3-4 weeks in vitro. Acetylcholinesterase (ACHE)histochemistry showed the presence of AChE-positive cells and fibers in many ex~lants. Thus it appears that septal cholinergic neurons develop CAT and AChE activity even without making contact with their target cells. However, the development of CAT was accelerated by the presence of hippocampal tissue No CAT activity was found in the hippocampal cultures, confirming that there are few, if any, intrinsic cholinergic cell bodies in the hippocampus.
The medial part of the septum contains the somata of cholinergic neurons whose axons reach the hippocampus along three routes: a supracallosal pathway, a subcallosal pathway (through the fimbria/fornix), and a ventral pathway (via the piriform lobe) [6]. From lesion experiments, it appears that the choline acetyltransferase (CAT) activity detected in the hippocampal formation originates almost exclusively from this septo-hippocampal projection (see ref. 16 for references). The cells of origin of the septo-hippocampal projection are generated between embryonic day (ED) 13 and ED 17 in the rat [1]. The development of cholinergic innervation in the hippocampal formation of the rat brain has been studied in situ by Matthews et al. [10] and Nadler et al. [13]. CAT activity was seen to increase mainly during two periods of development, from birth to 7 days of age and from 11 to 21 days of age. In our study we have looked at the development of CAT in organotypic explant cultures of fetal rat septum, in the presence or the absence of early postnatal rat hippocampus. Septal explants, taken from SIV 50 strain rats shortly before birth, at ED 20 or 21", or hippocampal explants from 7-day-old rats, were placed onto glass cover.
.
.
.
.
.
.
.
.
* The day of sperm-positivity was day one of embryonic life. 0304-3940/83/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
274
slips and cultured by the roller tube technique (see ref. 7 for details). Co-cultures of septum and hippocampus were prepared by placing on each cover-slip one septal explant between two hippocampal explants, at a distance of about 1 ram. The nutrient medium consisted of Eagle's Basal Medium with Hank's salts (50°70), Earle's BSS (25070), heat-inactivated horse serum (25070), and was supplemented with glucose to a concentration of 6.5 mg/ml. Normally, no antibiotics were required. When antibiotics were needed, then 100 U penicillin and 100 ~g streptomycin per ml medium were added. The cultures were incubated at 36°C and the medium was changed once or twice weekly. After varying lengths of time in vitro the explants were washed in Gey's BSS and removed from the cover-slips by small stainless steel spatulae. Care was taken to remove also the growth zone of the culture, containing the neurites growing out from the explants. CAT activity was assayed by the radiochemicai method of Fonnum [5], adapted by Woodward et al. [17] for explant cultures. The assays were conducted on homogenates prepared from 1-3 cultures. The proteins were measured by the method of Lowry et al. [9] with bovine serum albumin as standard. Some septal cultures were stained for acetylcholinesterase (ACHE) by a modified Karnovsky technique [4]. The unspecific cholinesterase was inhibited by 4 mM tetraisopropylpyrophosphoramide. No CAT activity could be detected when the septal homogenate was st~bstituted by buffer alone, or when the specific CAT inhibitor trans-l,2-dihydro-2-imino-4(lnaphtylvinyl)-l-pyridine-ethanoi (a derivative of NVP, a generous gift of Dr. C. Cavallito) was present in the reaction medium (1 mM). No CAT activity was found in the hippocampal cultures at various stages (14 cultures, 19-30 days in vitro). This observation confirms that there are few, if any, intrinsic cholinergic neurons in the hippocampus, and speaks for the possibility that the residual CAT activity found in the hippocampal formation after lesions of the septo-hippocampal pathway originates from other non-septal, cholinergic projection(s). A possible origin of such a projection is discussed in ref. 12. Interestingly, a small but significant CAT activity could be detected in the septum at ED 20-21 (cf. Fig. !). This finding correlates well with the observation that cells of the medial septal nucleus and the nucleus of the diagonal band stain for AChE as early as ED 20 [12]. The development of CAT (absolute activity) in a series of septal cultures is shown in Fig. 1. The enzyme activity increased 10-fold during the first 3 weeks in vitro. The increase was steepest (5-fold) in the 3rd and at the beginning of the 4th week. The mean activity of CAT at 24 days in vitro was 34 pmol acetylcholine (ACh) formed per minute per culture. Taking into account the average protein content of a 23-day-old septal culture (51 _+5 #g (S.E.M.), n = 26), we calculated a specific activity.of about 0.7 nmol ACh/mg protein/min, a value which approaches that reported by Cheney et al. [2] for the septal nuclei in the adult rat brain (0.8-1.5 nmol ACh/mg protein/rain).
275 (61 461
/.0
LU 3O
rF 2:) I-._I 2:) .,,w,.
{6)
z
~-
20
"r" ,,¢
0
n
10
(3 19) v
O_
|
0
~
,
,
J
I
10
~
,
I
I
,I
20
l
I
I
I
I
30
DAYS IN CULTURE
Fig. I. Developmentof CAT in organotypic cultures of fetal rat septum. Abscissa, days in culture; ordinate, CAT activity in pmol ACh formed per minute per culture, Each point represents the mean and S.E.M. of the number of cultures indicated in parentheses. AChE-positive cells and fibers could be observed already by 12 days in vitro (pictures not shown). Maximal staining seemed to be reached after 4-5 weeks in vitro (cf. Fig. 2). Since histochemical and biochemical evidence suggest that the septo-hippocampal projection is formed almost exclusively postnatally in the rat [10,11,13], contact with the hippocampus seems not to be absolutely necessary for the development of CAT in the septum. (Very recently, on the basis of orthograde and retrograde tracer techniques, Milne, et al. [12] have proposed that at least a few septal fibers may enter the hippocampal formation as soon as at ED 16-20. However, it is unknown at the present time if these fibers form synapses with their target cells already prenatally.) 'Normal' development of neurons in the absence of~their target cells has been observed, e.g. in explant cultures of the locus coeruleus (norepinephrine-producing cells), of the substantia nigra (dopamine-produc~ng cells) a n d in Cultured mesencephalic neurons (dopamine uptake and synthesis) [14,15]. However, in
276
Fig. 2. Septum explant, 37 days in vitro, AChE-histochemistry. A strongly AChE-positive soma (arrow) within a network of AChE-positive fibers. Bar = 30 #m.
mesencephalic neurons, both dopamine uptake and synthesis were enhanced by the presence of striatai ceils [141. in our cultures, the presence of hippocampal tissue significantly accelerated the development of CAT in the septum, as compared with septal explants cultured without hippocampus under the same conditions (cf. Table I). The mean CAT activity in the series of cultures shown in Table I is lower than the mean activity of the series of cultures of septum alone shown in Fig. 1 after 21 DIV. This is probably best explained by the differences in the composition of serum, as both culture series were fed with a different serum batch. It is well known that TABLE 1 EFFECT OF HIPPOCAMPUS ON THE DEVELOPMENT OF CAT IN SEPTAL EXPLANTS Septal (ED 21) and hippocampal (P 7) explants were cultured on the same cover-slip for 20 days. Control septal explants were grown without hippocampus under the same conditions. The values represent the mean and S.E.M. for the number of cultures indicated in parentheses. The difference is statistically significant IP= 0.05, 2-tailed test of Mann and Whitney). CAT activity after 20 days in vitro (pmol ACh/min/culture) Septum alone Septum + hippocampus
3.3 +_0.6 (14) 9.3 +_3.0 (18)
277
the quality of the sera profoundly affects the survival and differentiation of cultured cells. Further experiments are needed to assess if hippocampal tissue hastens the development of septal CAT activity in culture in a specific manner. In a recent work [8] it has been shown that intraventricular injections of nerve growth factor (NGF) to neonatal rats markedly increased the level of CAT in the septal area and the cortex, while no increase was observed in other brain areas. However, at the present time it is not clear if this responsiveness reflects on a physiological role for NGF in these neurons during their normal development. The finding that antibodies against NGF failed to reduce CAT activity in the neonatal brain and in dissociated septal cultures argues against this possibility. Nevertheless, the existence of a cholinergic neuronotrophic factor in the central nervous system different from the 'classical' NGF molecule is suggested by a study of Crutcher and Collins [3]. They showed that antibodies against NGF failed to inhibit the effect of hippocampal extract on neurite extension from cholinergic parasympathetic neurons in vitro. It would then be interesting to test if antibodies against NGF would counteract the effect of the hippocampus on cultured septal explants suggested by the present study. The authors sincerely thank Dr. B.H. Gfihwiler for his assistance during this work and for critical reading of the manus,.-ript, and Dr. D.M. Davis for helpful comments. ! Bayer, S.A., The development of the septal region in the rat. 1. Neurogenesis examined with ~Hthymidin¢ autoradiography, J. comp. Neurol., 183 (1979) 89-106. 2 Cheney, D.L., Racagni, G. and Costa, E., Distribution of acetylcholine and choline acetyltransferase in specific nuclei and tracts of rat brain. In A.M. Goldberg and !. Hanin (Eds.), Biology of Cholinergic Function, Raven Press. New York, 1976, pp. 655-659. 3 Crutcher, K.A. and Collins, F., in vitro evidence for two distinct hippocampal growth factors: basis of neuronal plasticity?, Science, 217 (1982)67-68. 4 EI-Badawi, A. and Schenk, E.A., Histochemical methods for :,eparate, consecutive and simultaneous demonstration of acetylcholinesterase and norepinephrine in cryostat sections, J. Histochem. Cytochem., 15 (1967) 580-588. 5 Fonnum, F., A rapid radiochemical me-~hod for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 6 Gage, F.H., Bj6rklund, A. and Stenevi, U., Reirmervation of the partially deafferented hippocampus by compensatory collateral sprouting from spared cholinergic and noradrenergic afferents, Brain Res., 268 (1983) 27-37. 7 Gfihwiler, B.H., Organotypic monolayer cultu,~es of nervous tissue, J. Neurosci. Meth., 4 (1981) 329-342. 8 Gnahn, H., Hefti, F., Heumann, R., Schwab, M.E. and Thoenen, H., NGF-mediated increase of choline acetyltransferase (CHAT) in the neonatal rat forebrain: evidence for a physiological role of NGF in the brain? Develop. Brain Res., 9 (1983) 45-52. 9 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275.
278
i
10 Matthews, D.A., Nadler, J.V., Lynch, G.S. and Cotman, C.W., Development of cholinergic innervation in the hippocampal formation of the rat. 1. Histochemical demonstration of acetylcholinesterase activity, Develop. Biol., 36 (1974) 130--141. 11 Mellgren, S.I., Distribution of acetylcholinesterase in the hippocampal region of the rat during postnatal development, Z. Zellforsch., 141 (1973) 375-400. 12 Milner, T.A., Loy, R. and Amaral, D.G., An anatomical study of the development of the septohippocampal projection in the rat, Brain Res., 284 (1983) 343-371. 13 Nadler, J.V., Matthews, D.A., Cotman, C.W. and Lynch, G.S., Development of cholinergic innervation in the hippocampal formation of the rat. 11. Quantitative changes in choline acetyltransferase and acetylcholinesterase activities, Develop. Biol., 36 (1974) 142-154. 14 Prochiantz, A., Di Porzio, U., Kato, A., Berger, B. and Glowinski, J., In vitro maturation of mesencephalic dopaminergic neurons from mouse embryos is enhanced in presence of their striatal target cells, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5387-5391. 15 Schlumpf., M., Shoemaker, W.J. and Bloom, F.E., Explant cultures of catecholamine-containing neurons from rat brain: biochemical, P,istofluorescence, and electron microscopic studies, Proc. nat. Acad. Sci. U.S.A., 74 (1977)4471-4475. 16 Storm-Mathisen, J., Localization of transmitter candidates in the brain: the hippocampal formation as a model, Progr. Neurobiol., 8 (1977) 119-181. 17 Woodward, W.R., Blank, N.K. and Sell, F.J., Choline acetyltransferase activity in mouse cerebellar cultures, Brain Res., 241 (1982) 323-327.