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Development of vasoactive intestinal polypeptide (VIP)-containing neurons in organotypic slice cultures from rat visual cortex
6
Neuroscience Letters, 107 (1989) 6-11 Elsevier Scientific Publishers Ireland Ltd.
NSL 06479
Development of vasoactive intestinal polypeptide (VIP)-containing neurons in organotypic slice cultures from rat visual cortex M a g d a l e n a G 6 t z and Jiirgen Bolz Friedrich-Miescher-Labor der Max-Planck-Gesellsehaft Tiibingen ( F,R.G. ) (Received 27 June 1989; Revised version received 27 July 1989; Accepted 31 July 1989)
Key word~': Visual cortex; Development; Slice culture; Transmitter maturation; Neuropeptide; lmmunohistochemistry Using immunohistochemistry we have been studying the postnatal maturation of vasoactive intestinal polypeptide (VIP)-positive neurons in organotypic slice cultures from rat visual cortex. The development in vitro is compared with the occurrence of VIP-containing cells in vivo, where they are first observed around postnatal day 5. A further increase in number and morphological maturation occurs within the following 3 weeks. In cultures prepared from 1- or 2-day-old rats, i.e. before VIP is expressed in vivo, VIP-containing neurons appear after about 5 days and gradually increase in number over the next 2 weeks. Thus the time course of postnatal expression of VIP in vitro and the morphology of VIP-immunoreactive neurons in culture closely matches the situation in vivo. These observations suggest that the maturation of VIP-containing neurons occurs independently of cortical afferents and that the intrinsic connectivity and activity is sufficient for their postnatal maturation. Therefore organotypic slice cultures should be a suitable system to study mechanisms of neurochemical maturation in the cortex.
Cortical neurons are immature at birth; several of their specific morphological, physiological and neurochemical properties develop postnatally. For example, different types of non-pyramidal cells in rat visual cortex contain neuropeptides, but it is not until 4-5 days after birth that vasoactive intestinal polypeptide (VIP) is first detected in these neurons [I 6]. In an attempt to learn more about the factors responsible for the neurochemical differentiation of cortical neurons, we studied as a first step the expression of VIP in an in vitro culture system from the visual cortex. As demonstrated by Gfihwiler [5, 6] slices from different brain areas can be cultivated for several weeks by means of a roller culture technique and preserve many of their morphological and physiological features. Caeser et al. [3] showed that slice cultures from rat visual cortex remain organotypically organized for up to 12 weeks in vitro and that postnatal morphological maturation of pyramidal cells continues Uorrespondence. J. Boll Friedrich-Miescher-Labor der Max-Planck-(Jesellschaft, Spemannstr. 37 39, 7400 Tfibingen, (F.R.G.). 0304-3940/89/$ 03.50 (~) 1989 Elsevier Scientific Publishers Ireland Ltd.
in these cultures. We were interested in whether potential VIP-containing neurons differentiate in cortical slice cultures prepared from young rat pups after the slices have been cultured for some days. Slice cultures were prepared as described previously [3, 5]. Briefly, 1- to 2-day-old rat pups were decapitated and small blocks of visual cortex from both cortical hemispheres were aseptically prepared in ice-cold Geys balanced salt solution (GBSS) with 6.5 mg/ml glucose, and then frontally cut into 300-~tm-thick slices with a McIlwain tissue chopper. After storing for 30-45 min at 4°C in GBSS, each slice was embedded on a cleaned coverslip in a plasma clot of 20/11 heparinized Difco chicken plasma coagulated by 20/A thrombin solution (0.2 mg/ml, 20 NIHU/ml Hoffman-LaRoche). Slice cultures were maintained with 0.75 ml medium consisting of 50% Eagles' basal medium, 25% Hanks balanced salt solution and 25% horse serum containing 0.1 mM glutamine and 6.5 mg/ml glucose (all from Gibco) in a plastic culture tube (Nunc) in a roller drum incubator at 36°C (dry air without CO2/O2-control). After 4 days in culture mitotic inhibitors (5-fluoro-2-deoxyuridine, cytosine-fl-D-arabino-furanoside, uridine, Sigma) were added to the culture medium at a final concentration of 10/~M to prevent excessive growth of glia cells and fibroblasts. Medium was changed 24 h later and thereafter twice a week until the cultures were used for immunohistochemistry. For immunohistochemical staining, the slice cultures were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), washed in PB and incubated at 4°C overnight with a polyclonal VIP- or GABA antiserum (Immuno Biological Laboratories), diluted l:1000 in PB with 0.5% Triton X-100. Thereafter the slice cultures were processed using the avidin-biotin (ABC) kit (Vektor) and stained for 20 min following the glucoseoxidase-DAB-nickel method [17]. Tissue was then rinsed in PB, dehydrated in alcohols, cleared in xylene and embedded in Entellan (Merck). Specificity of the immune reaction was assessed by absorbing the primary antiserum with 10 /~M VIP (Cambridge Research Biochemicals) overnight. VIP immunoreactivity was not apparent after this treatment. Also no staining was observed when either VIP antiserum, the secondary antiserum or the ABC Complex was omitted. These controls indicate a low unspecific binding of the antisera. The addition of Triton X-100 was used to allow the penetration of the antibody. With this treatment the plasma clot of the slice cultures did not interfere with the immunostaining. Some slice cultures were counterstained in a 1% aqueous solution of Cresyl violet, differentiated in acetic acid alcohol, dehydrated in alcohol, cleared in xylene and finally embedded in Entellan. We used a well-ordered layered structure as an easily detectable criterion for 'healthiness' of the slice cultures. To make a direct comparison between the development of VIP-positive neurons in vitro and in vivo, sections of the visual cortex from rats of different ages were also immunostained for VIP. Rats were deeply anesthetized with ether and perfused with 4% paraformaldehyde in 0.1 M PB. The brain was removed, postfixed for 1 h and cryoprotected in 30% sucrose PB overnight. Frontal sections from the visual cortex (60/~m) were cut on a freezing microtome, mounted on subbed slides and processed as described above.
Fig. 1 depicts examples of VIP-containing cells from rat visual cortex at different ages. N o VIP-immunoreactive cells were detected in vivo before postnatal day 4 (P4). The earliest time we found some VIP-positive cells was at P5. On P8, VIP-containing cell bodies with short processes were stained. During the second and third postnatal weeks more VIP neurons with more elongated processes became visible. The adult pattern was reached at P26 where VIP immunoreactivity was found mostly in bipolar cells and in some bitufted and multipolar neurons. These results are in accordance with an earlier study of McDonald et al. [16], who reported that the first VIP-containing neurons in rat visual cortex appear 4-5 days after birth; the cells increase in number and reach the adult morphology by the middle of the fourth postnatal week.
organotypic
cultures
in
vivo
P8
P2,6DIV
P2,1ODIV
P12
P26
P2,23DIV •
),
,/,'
I! '¸'f,(~'~
/
adu It
;f
Fig. 1. Camera lucida drawings of V1P-immunoreactive neurons in slice cultures left) and in vivo (right). All cultures were prepared from rat pups at postnatal day 2 (P2) and cultivated for various days in vitro (DIV) as indicated. Scale bar: 100 p m
To study the development of VIP neurons in vitro, cortical slice cultures prepared from 1- and 2-day-old rat pups were stained for VIP immunoreactivity after varying numbers of days in vitro (DIV). In slice cultures prepared from l-day-old rats, a small number of immunoreactive cell bodies were first detected after 5 DIV (Fig. 2a), and in cultures from 2-day-old rats VIP-positive cells had short processes after 5 DIV (Figs. 1 and 2b). At the same developmental age, staining with GABA antiserum revealed many GABA-positive neurons in these cortical slice cultures. This corresponds to the early expression of GABA in cortical cells in vivo [20] and demonstrates that there is no general loss of non-pyramidal cells in cultures of that age.
Fig. 2. Micrographs of VIP-containing neurons (some indicated by arrows in a and b) in cortical slice cultures of different days in vitro (DIV). a: PI, 5 DIV. b: P2, 5 DIV. c: PI, 8 DIV. d: P2, 23 DIV. Scale bar: 50 gm.
10
At the beginning of the second postnatal week in culture, VIP-containing neurons increased in number and showed elongated and branched processes (Fig. 2c). The processes of such cells became more and more elaborated up until the fourth week in culture (Figs. 1 and 2d). Thereafter, no further growth in size and extent of these processes could be observed. The morphology of VIP-immunoreactive neurons that developed in culture matches very well the morphology of VIP neurons in vivo (Fig. 1). All VIP neurons were non-pyramidal cells with bipolar, bitufted or multipolar shape, which ar also found in adult visual cortex of the rat [4, 8]. After 2 weeks in culture, when VIP neurons no longer increase in number, about 0.7 1.7% of all neurons in Nissl-stained slice cultures are VIP-positive. This is ill good agreement with the in vivo data of Hajos et al. [8], who report that VIP cells constitute 1.5% of the total number of neurons in rat visual cortex. Moreover, the time course of the appearance of VIP-containing neurons in vitro is comparable to the development in vivo. The first VIP-positive cells are present after 5 days in vitro, gradually increase in number up to the middle of the second postnatal week and show morphological maturation until about the fourth week in culture. It has been suggested that arriving afferents contribute to the final differentiation of cortical neurons [9, 15]. Thalamocortical fibers invade the cortical plate between postnatal days 2 and 5 [14], just around the same time as VIP is first expressed. Our experiments show that the postnatal expression of VIP occurs in a culture system without thalamocortical afferents. This rules out the possibility that invading afterents trigger the expression of VIP in cortical neurons. However, there are studies showing that activity of afferents plays a role in the regulation of the transmitter amount of neurons in the early postnatal as well as in adult visual cortex [10, 12]. In the macaque retina [19] and in rat spinal cord [18] an increase in VIP-like immunoreactivity was tk)und after lid fusion and deafferentation respectively. This modulation of transmitter content by missing afferent activity could be mediated via a change of the intrinsic activity in vivo. We know from our own electrophysiological data cortical slice cultures show high intrinsic activity [3]. Therefore further experiments will examine the relation between peptide development and neuronal activity. The appearance of neuropeptide Y, somatostatin and cholecystokinin has also been observed in dissociated cell cultures from neonatal rat cerebral cortex [I]. These results seem to suggest that either a fixed genetic program or undetermined components in the culture medium are responsible for peptide expression. However, many neurons in dissociated cultures unfold an abnormal morphology [11, 13], thus it is not clear in this system, whether neuropeptttle expression occurs in the appropriate cells. On the other hand, dissociated cells are not completely isolated, as new synaptic connections are formed when the cells reaggregate, so that activity or transmitter actions could still influence the peptide expression. This makes it difficult to draw firm conclusions from experiments in dissociated cell cultures about factors regulating transmitter expression. This is why we choose a culture system with an organotypic environment. In these cultures the activity evoked by cortical afferents is abolished, whereas connectivity and intrinsic activity are largely intact [2, 3, 7]. It is now possible to block selectively
11
the intrinsic activity or the action of neurotransmitters to examine their role in VIP differentiation. Thus, organotypical slice cultures seem to be well suited to study mechanisms of neurotransmitter expression in the cortex. We thank Iris Kehrer and Renate Thanos for excellent technical assistance and their patient help during the ups and downs of immunohistochemistry. Special thanks to Tim Allsopp for helpful comments on the manuscript. 1 Alho, H., Ferrarese, C., Vicini, S. and Vaccarino, F., Subsets of GABAergic neurons in dissociated cell cultures of neonatal rat cerebral cortex show co-localization with specific modulator peptides, Dev. Brain Res., 39 (1988) 193-204. 2 Baker, R.E., Bingmann, D. and Ruijter, J.M., Electrophysiological properties of neurons in neonatal rat occipital cortex slices grown in a serum-free medium, Neurosci. Lett., 97 (1989) 310-315. 3 Caeser, M., Bonhoeffer, T. and Bolz, J., Cellular organization and development of slice cultures from rat visual cortex, Exp. Brain Res., in press. 4 Connors, J.R. and Peters, A., Vasoactive intestinal polypeptide-immunoreactive neurons in rat visual cortex, Neuroscience, 12 (1984) 1027-1044. 5 G/ihwiler, B.H., Organotypic monolayer cultures of nervous tissue, J. Neurosci. Methods, 4 (1981) 329 342. 6 Gahwiler, B.H., Slice cultures of cerebellar, hippocampal and hypothalamic tissue, Experientia, 40 (1984) 235 243. 7 Gfihwiler, B.H., Organotypic cultures of neural tissue, Trends Neurosci., 11 (1988) 484489. 8 Hajos, F. and Zilles, K., Quantitative immunohistochemical analysis of VIP-neurons in the rat visual cortex, Histochemistry, 90 (I 988) 139-144. 9 Hamre, K.M., Cassell, M.D. and West, J.R., The development of laminar staining for neuron-specific enolase in the rat somatosensory cortex, Dev. Brain Res., 46 (1989) 213-220. 10 Hendry, S.H.C. and Jones, E.G., Activity-dependent regulation of GABA-expression in the visual cortex of adult monkeys, Neuron, 1 (1988) 701 712. 11 Huettner, J.E. and Baughman, R.W., Primary culture of identified neurons from the visual cortex of postnatal rats, J. Neurosci., 6 (1986) 3044-3060. 12 Jeffery, G. and Parnavelas, J.G., Early visual deafferentation of the neocortex results in an asymmetry of somatostatin labelled cells, Exp. Brain Res., 67 (1987) 651~555. 13 Kriegstein, A.R. and Dichter, M.A., Morphological classification of rat cortical neurons in cell culture, J. Neurosci., 3 (1983) 1634-1647. 14 Lund, R.D. and Mustari, M.J., Development of the geniculocortical pathway, J. Comp. Neurol., 242 (1985) 611~13. 15 Marin-Padilla, M., Early ontogenesis of the human cerebral cortex. In A. Peters and E.G. Jones (Eds.), Cerebral Cortex, Vol. 7, Plenum, New York, 1988, pp. 1 30. 16 McDonald, J.K., Parnavelas, J.G., Karamanlidis, A.N. and Brecha, N., The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. II. Vasoactive intestinal polypeptide, J. Neurocytol., 11 (1982) 825437. 17 Siyun, S., Gong, J. and Lingzhi, F., The glucose oxidas~DAB-nickel method in peroxidase histochemistry of the nervous system, Neurosci. Lett., 85 (1988) 169 171. 18 F. Shebab, S.A.S. and Atkinson, M.E., Vasoactive intestinal polypeptide increases in areas of the dorsal horn of the spinal cord from which other neuropeptides are depleted following peripheral axotomy, Exp. Brain Res., 62 (1986) 422~430. 19 Stone, R.A., Laties, A.M., Raviola, E. and Wiesel, T.N., Increase in retinal vasoactive intestinal polypeptide after eyelid fusion in primates, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 257-260. 20 Wolff, J.R., Brttcher, H., Zetsche, T., Oertel, W.H. and Chronwall, B.M., Development of GABAergic neurons in rat visual cortex as identified by glutamate decarboxylase-like immunoreactivity, Neurosci. Lett., 47 (1984) 207-212.
Neuroscience Letters, 107 (1989) 6-11 Elsevier Scientific Publishers Ireland Ltd.
NSL 06479
Development of vasoactive intestinal polypeptide (VIP)-containing neurons in organotypic slice cultures from rat visual cortex M a g d a l e n a G 6 t z and Jiirgen Bolz Friedrich-Miescher-Labor der Max-Planck-Gesellsehaft Tiibingen ( F,R.G. ) (Received 27 June 1989; Revised version received 27 July 1989; Accepted 31 July 1989)
Key word~': Visual cortex; Development; Slice culture; Transmitter maturation; Neuropeptide; lmmunohistochemistry Using immunohistochemistry we have been studying the postnatal maturation of vasoactive intestinal polypeptide (VIP)-positive neurons in organotypic slice cultures from rat visual cortex. The development in vitro is compared with the occurrence of VIP-containing cells in vivo, where they are first observed around postnatal day 5. A further increase in number and morphological maturation occurs within the following 3 weeks. In cultures prepared from 1- or 2-day-old rats, i.e. before VIP is expressed in vivo, VIP-containing neurons appear after about 5 days and gradually increase in number over the next 2 weeks. Thus the time course of postnatal expression of VIP in vitro and the morphology of VIP-immunoreactive neurons in culture closely matches the situation in vivo. These observations suggest that the maturation of VIP-containing neurons occurs independently of cortical afferents and that the intrinsic connectivity and activity is sufficient for their postnatal maturation. Therefore organotypic slice cultures should be a suitable system to study mechanisms of neurochemical maturation in the cortex.
Cortical neurons are immature at birth; several of their specific morphological, physiological and neurochemical properties develop postnatally. For example, different types of non-pyramidal cells in rat visual cortex contain neuropeptides, but it is not until 4-5 days after birth that vasoactive intestinal polypeptide (VIP) is first detected in these neurons [I 6]. In an attempt to learn more about the factors responsible for the neurochemical differentiation of cortical neurons, we studied as a first step the expression of VIP in an in vitro culture system from the visual cortex. As demonstrated by Gfihwiler [5, 6] slices from different brain areas can be cultivated for several weeks by means of a roller culture technique and preserve many of their morphological and physiological features. Caeser et al. [3] showed that slice cultures from rat visual cortex remain organotypically organized for up to 12 weeks in vitro and that postnatal morphological maturation of pyramidal cells continues Uorrespondence. J. Boll Friedrich-Miescher-Labor der Max-Planck-(Jesellschaft, Spemannstr. 37 39, 7400 Tfibingen, (F.R.G.). 0304-3940/89/$ 03.50 (~) 1989 Elsevier Scientific Publishers Ireland Ltd.
in these cultures. We were interested in whether potential VIP-containing neurons differentiate in cortical slice cultures prepared from young rat pups after the slices have been cultured for some days. Slice cultures were prepared as described previously [3, 5]. Briefly, 1- to 2-day-old rat pups were decapitated and small blocks of visual cortex from both cortical hemispheres were aseptically prepared in ice-cold Geys balanced salt solution (GBSS) with 6.5 mg/ml glucose, and then frontally cut into 300-~tm-thick slices with a McIlwain tissue chopper. After storing for 30-45 min at 4°C in GBSS, each slice was embedded on a cleaned coverslip in a plasma clot of 20/11 heparinized Difco chicken plasma coagulated by 20/A thrombin solution (0.2 mg/ml, 20 NIHU/ml Hoffman-LaRoche). Slice cultures were maintained with 0.75 ml medium consisting of 50% Eagles' basal medium, 25% Hanks balanced salt solution and 25% horse serum containing 0.1 mM glutamine and 6.5 mg/ml glucose (all from Gibco) in a plastic culture tube (Nunc) in a roller drum incubator at 36°C (dry air without CO2/O2-control). After 4 days in culture mitotic inhibitors (5-fluoro-2-deoxyuridine, cytosine-fl-D-arabino-furanoside, uridine, Sigma) were added to the culture medium at a final concentration of 10/~M to prevent excessive growth of glia cells and fibroblasts. Medium was changed 24 h later and thereafter twice a week until the cultures were used for immunohistochemistry. For immunohistochemical staining, the slice cultures were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), washed in PB and incubated at 4°C overnight with a polyclonal VIP- or GABA antiserum (Immuno Biological Laboratories), diluted l:1000 in PB with 0.5% Triton X-100. Thereafter the slice cultures were processed using the avidin-biotin (ABC) kit (Vektor) and stained for 20 min following the glucoseoxidase-DAB-nickel method [17]. Tissue was then rinsed in PB, dehydrated in alcohols, cleared in xylene and embedded in Entellan (Merck). Specificity of the immune reaction was assessed by absorbing the primary antiserum with 10 /~M VIP (Cambridge Research Biochemicals) overnight. VIP immunoreactivity was not apparent after this treatment. Also no staining was observed when either VIP antiserum, the secondary antiserum or the ABC Complex was omitted. These controls indicate a low unspecific binding of the antisera. The addition of Triton X-100 was used to allow the penetration of the antibody. With this treatment the plasma clot of the slice cultures did not interfere with the immunostaining. Some slice cultures were counterstained in a 1% aqueous solution of Cresyl violet, differentiated in acetic acid alcohol, dehydrated in alcohol, cleared in xylene and finally embedded in Entellan. We used a well-ordered layered structure as an easily detectable criterion for 'healthiness' of the slice cultures. To make a direct comparison between the development of VIP-positive neurons in vitro and in vivo, sections of the visual cortex from rats of different ages were also immunostained for VIP. Rats were deeply anesthetized with ether and perfused with 4% paraformaldehyde in 0.1 M PB. The brain was removed, postfixed for 1 h and cryoprotected in 30% sucrose PB overnight. Frontal sections from the visual cortex (60/~m) were cut on a freezing microtome, mounted on subbed slides and processed as described above.
Fig. 1 depicts examples of VIP-containing cells from rat visual cortex at different ages. N o VIP-immunoreactive cells were detected in vivo before postnatal day 4 (P4). The earliest time we found some VIP-positive cells was at P5. On P8, VIP-containing cell bodies with short processes were stained. During the second and third postnatal weeks more VIP neurons with more elongated processes became visible. The adult pattern was reached at P26 where VIP immunoreactivity was found mostly in bipolar cells and in some bitufted and multipolar neurons. These results are in accordance with an earlier study of McDonald et al. [16], who reported that the first VIP-containing neurons in rat visual cortex appear 4-5 days after birth; the cells increase in number and reach the adult morphology by the middle of the fourth postnatal week.
organotypic
cultures
in
vivo
P8
P2,6DIV
P2,1ODIV
P12
P26
P2,23DIV •
),
,/,'
I! '¸'f,(~'~
/
adu It
;f
Fig. 1. Camera lucida drawings of V1P-immunoreactive neurons in slice cultures left) and in vivo (right). All cultures were prepared from rat pups at postnatal day 2 (P2) and cultivated for various days in vitro (DIV) as indicated. Scale bar: 100 p m
To study the development of VIP neurons in vitro, cortical slice cultures prepared from 1- and 2-day-old rat pups were stained for VIP immunoreactivity after varying numbers of days in vitro (DIV). In slice cultures prepared from l-day-old rats, a small number of immunoreactive cell bodies were first detected after 5 DIV (Fig. 2a), and in cultures from 2-day-old rats VIP-positive cells had short processes after 5 DIV (Figs. 1 and 2b). At the same developmental age, staining with GABA antiserum revealed many GABA-positive neurons in these cortical slice cultures. This corresponds to the early expression of GABA in cortical cells in vivo [20] and demonstrates that there is no general loss of non-pyramidal cells in cultures of that age.
Fig. 2. Micrographs of VIP-containing neurons (some indicated by arrows in a and b) in cortical slice cultures of different days in vitro (DIV). a: PI, 5 DIV. b: P2, 5 DIV. c: PI, 8 DIV. d: P2, 23 DIV. Scale bar: 50 gm.
10
At the beginning of the second postnatal week in culture, VIP-containing neurons increased in number and showed elongated and branched processes (Fig. 2c). The processes of such cells became more and more elaborated up until the fourth week in culture (Figs. 1 and 2d). Thereafter, no further growth in size and extent of these processes could be observed. The morphology of VIP-immunoreactive neurons that developed in culture matches very well the morphology of VIP neurons in vivo (Fig. 1). All VIP neurons were non-pyramidal cells with bipolar, bitufted or multipolar shape, which ar also found in adult visual cortex of the rat [4, 8]. After 2 weeks in culture, when VIP neurons no longer increase in number, about 0.7 1.7% of all neurons in Nissl-stained slice cultures are VIP-positive. This is ill good agreement with the in vivo data of Hajos et al. [8], who report that VIP cells constitute 1.5% of the total number of neurons in rat visual cortex. Moreover, the time course of the appearance of VIP-containing neurons in vitro is comparable to the development in vivo. The first VIP-positive cells are present after 5 days in vitro, gradually increase in number up to the middle of the second postnatal week and show morphological maturation until about the fourth week in culture. It has been suggested that arriving afferents contribute to the final differentiation of cortical neurons [9, 15]. Thalamocortical fibers invade the cortical plate between postnatal days 2 and 5 [14], just around the same time as VIP is first expressed. Our experiments show that the postnatal expression of VIP occurs in a culture system without thalamocortical afferents. This rules out the possibility that invading afterents trigger the expression of VIP in cortical neurons. However, there are studies showing that activity of afferents plays a role in the regulation of the transmitter amount of neurons in the early postnatal as well as in adult visual cortex [10, 12]. In the macaque retina [19] and in rat spinal cord [18] an increase in VIP-like immunoreactivity was tk)und after lid fusion and deafferentation respectively. This modulation of transmitter content by missing afferent activity could be mediated via a change of the intrinsic activity in vivo. We know from our own electrophysiological data cortical slice cultures show high intrinsic activity [3]. Therefore further experiments will examine the relation between peptide development and neuronal activity. The appearance of neuropeptide Y, somatostatin and cholecystokinin has also been observed in dissociated cell cultures from neonatal rat cerebral cortex [I]. These results seem to suggest that either a fixed genetic program or undetermined components in the culture medium are responsible for peptide expression. However, many neurons in dissociated cultures unfold an abnormal morphology [11, 13], thus it is not clear in this system, whether neuropeptttle expression occurs in the appropriate cells. On the other hand, dissociated cells are not completely isolated, as new synaptic connections are formed when the cells reaggregate, so that activity or transmitter actions could still influence the peptide expression. This makes it difficult to draw firm conclusions from experiments in dissociated cell cultures about factors regulating transmitter expression. This is why we choose a culture system with an organotypic environment. In these cultures the activity evoked by cortical afferents is abolished, whereas connectivity and intrinsic activity are largely intact [2, 3, 7]. It is now possible to block selectively
11
the intrinsic activity or the action of neurotransmitters to examine their role in VIP differentiation. Thus, organotypical slice cultures seem to be well suited to study mechanisms of neurotransmitter expression in the cortex. We thank Iris Kehrer and Renate Thanos for excellent technical assistance and their patient help during the ups and downs of immunohistochemistry. Special thanks to Tim Allsopp for helpful comments on the manuscript. 1 Alho, H., Ferrarese, C., Vicini, S. and Vaccarino, F., Subsets of GABAergic neurons in dissociated cell cultures of neonatal rat cerebral cortex show co-localization with specific modulator peptides, Dev. Brain Res., 39 (1988) 193-204. 2 Baker, R.E., Bingmann, D. and Ruijter, J.M., Electrophysiological properties of neurons in neonatal rat occipital cortex slices grown in a serum-free medium, Neurosci. Lett., 97 (1989) 310-315. 3 Caeser, M., Bonhoeffer, T. and Bolz, J., Cellular organization and development of slice cultures from rat visual cortex, Exp. Brain Res., in press. 4 Connors, J.R. and Peters, A., Vasoactive intestinal polypeptide-immunoreactive neurons in rat visual cortex, Neuroscience, 12 (1984) 1027-1044. 5 G/ihwiler, B.H., Organotypic monolayer cultures of nervous tissue, J. Neurosci. Methods, 4 (1981) 329 342. 6 Gahwiler, B.H., Slice cultures of cerebellar, hippocampal and hypothalamic tissue, Experientia, 40 (1984) 235 243. 7 Gfihwiler, B.H., Organotypic cultures of neural tissue, Trends Neurosci., 11 (1988) 484489. 8 Hajos, F. and Zilles, K., Quantitative immunohistochemical analysis of VIP-neurons in the rat visual cortex, Histochemistry, 90 (I 988) 139-144. 9 Hamre, K.M., Cassell, M.D. and West, J.R., The development of laminar staining for neuron-specific enolase in the rat somatosensory cortex, Dev. Brain Res., 46 (1989) 213-220. 10 Hendry, S.H.C. and Jones, E.G., Activity-dependent regulation of GABA-expression in the visual cortex of adult monkeys, Neuron, 1 (1988) 701 712. 11 Huettner, J.E. and Baughman, R.W., Primary culture of identified neurons from the visual cortex of postnatal rats, J. Neurosci., 6 (1986) 3044-3060. 12 Jeffery, G. and Parnavelas, J.G., Early visual deafferentation of the neocortex results in an asymmetry of somatostatin labelled cells, Exp. Brain Res., 67 (1987) 651~555. 13 Kriegstein, A.R. and Dichter, M.A., Morphological classification of rat cortical neurons in cell culture, J. Neurosci., 3 (1983) 1634-1647. 14 Lund, R.D. and Mustari, M.J., Development of the geniculocortical pathway, J. Comp. Neurol., 242 (1985) 611~13. 15 Marin-Padilla, M., Early ontogenesis of the human cerebral cortex. In A. Peters and E.G. Jones (Eds.), Cerebral Cortex, Vol. 7, Plenum, New York, 1988, pp. 1 30. 16 McDonald, J.K., Parnavelas, J.G., Karamanlidis, A.N. and Brecha, N., The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. II. Vasoactive intestinal polypeptide, J. Neurocytol., 11 (1982) 825437. 17 Siyun, S., Gong, J. and Lingzhi, F., The glucose oxidas~DAB-nickel method in peroxidase histochemistry of the nervous system, Neurosci. Lett., 85 (1988) 169 171. 18 F. Shebab, S.A.S. and Atkinson, M.E., Vasoactive intestinal polypeptide increases in areas of the dorsal horn of the spinal cord from which other neuropeptides are depleted following peripheral axotomy, Exp. Brain Res., 62 (1986) 422~430. 19 Stone, R.A., Laties, A.M., Raviola, E. and Wiesel, T.N., Increase in retinal vasoactive intestinal polypeptide after eyelid fusion in primates, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 257-260. 20 Wolff, J.R., Brttcher, H., Zetsche, T., Oertel, W.H. and Chronwall, B.M., Development of GABAergic neurons in rat visual cortex as identified by glutamate decarboxylase-like immunoreactivity, Neurosci. Lett., 47 (1984) 207-212.