Cocultured, but not isolated, cortical explants display normal dendritic development: a long-term quantitative study

https://doi.org/10.1016/S0165-3806(96)00163-0Get rights and content

Abstract

Dendritic growth has been studied in long-term organotypic neonatal rat occipital neocortex explants grown either apart as isolated explants or in tandem as cocultures. Quantitative light microscopic measurement of dendritic and axonal branching patterns within the cortical slice was accomplished using rapid Golgi stained materials. In both isolates and cocultures the overall cellular organization of the slice was maintained over 4 weeks in vitro with morphologically distinguishable pyramidal and nonpyramidal neurons located within the same layers and with the same orientations as observed in situ. Long-term increases in the total length of basal dendrites, apical dendrite and axons were observed only in cocultures and were similar to growth patterns reported for in situ materials. Dendritic growth was mainly due to elongation of terminal dendritic segments. Surprisingly, isolated explants showed no long-term increases in total (basal) dendrites, apical dendrites or axons with time in vitro. A transient decrease in the number of basal dendritic segments and increase in terminal segment lengths at the end of the first week in vitro, however, was observed in nonpyramidal neurons. It is hypothesized that (i) afferent inputs and/or efferent targets develop only in cocultures and provide a crucial condition for the continued growth of dendritic/axonal arborization for neocortical neurons in vitro, (ii) intrinsic interconnectivity within isolated explants is not sufficient to maintain long-term growth of neuritic arbors, and (iii) remodelling of dendritic arbors within isolated explants occurs at the same time as these explants are showing noticeable increases in the level of spontaneous bioelectric activity, which suggests that dendritic growth and network formation may be function dependent.

Introduction

During development neurites grow and make synaptic connections in the formation of functional networks. Critical stages occur during embryogenesis and in the immediate postnatal period during which a neuron finds its place within the network and, in synergy with its emerging bioelectric properties, finalizes its detailed morphological characteristics (e.g., [11,37,[45], [46], [47], [48]]). While the genetic program of the neuron is capable of allowing for the development of distinctive neural phenotypes (see [23]), epigenetic factors exert powerful influences on the ultimate morphology attained. Of these, emerging spontaneous bioelectric activity (SBA) and trophic factors appear to be most influential [3,11,12,16,20,22,33,34,38,47].

In studying the role of SBA on morphological development and synapse formation in intact animals, one is faced with experimental difficulties: surgery is often complicated, limited accessibility to tissues of interest, and, more importantly, compensatory anatomical and/or functional changes occur making interpretation of the results difficult and tenuous (see [39]). A variety of long-term in vitro model systems have now been developed and provide reliable systems for studying mechanisms underlying the morphological development of neurons within an emerging neural network. Detailed morphological studies are feasible because the anatomical organization of long-term rodent brain slices grown under a variety of culturing conditions retains its cytoarchitectural characteristics over 3–4 weeks in vitro [1,[6], [7], [8],[13], [14], [15],17,21,24,25,32,35,36]. Additionally, neocortical slices exhibit lamina-specific connectivity when two such explants are grown together as cocultures [50], or when innervated by various thalamic nuclei [5,9,26,49,50].

In spite of the increased interest in the development of in vitro models for studying neuronal development, surprisingly little is known about the quantitative nature of morphological changes that occur under a variety of culturing conditions. Quantitative measurement of dendriticraxonal arbors has been reported in isolated organotypic neonatal mouse neocortex (at 7 DIV) [1]. Intrinsic connections within the slices are sufficient to allow for the early differentiation of both morphological and functional characteristics in presumptive layers V and VI. This maturation shows correlation with emerging SBA on neuronal structure during early stages of network formation. Recent work has also shown that proper Purkinje cell dendritic differentiation relies on receiving innervation from granule cells [3]. Thus, selective afferent input to a given cell type may be essential to the regulation of neuritic growth, in effect, requiring the presence of such input for in vivo levels of maturation to occur under in vitro growth conditions.

The objective of the present study is to establish baseline morphological criteria for studies involved with activity-dependent neurite outgrowth, overgrowth and retraction during the formation of neural networks. The present study is the first to quantitatively examine the development of dendritic and axonal arborization within isolated and co-cultured organotypic neocortex explants for up to 4 weeks in vitro. We expected that cellular differentiation and morphological maturation in both types of explants would be similar to that reported for in vivo tissues at similar stages of development [4,19,24,25,29,42], much as has been reported for the functional maturation of these explants within similar time frames in vitro [2]. Unexpectedly we discovered that only in cocultured cortical explants were dendritic outgrowth similar to that reported in situ. This suggested that some facet of coculturing was able to compensate for whatever factor was missing within the isolated explant. Based on these findings in vitro models will allow us to differentiate between the roles connectivity, function and/or trophic factors play in the proper maturation of neural networks, studies which are now in progress.

Section snippets

PN6 material

Pieces of occipital cortex (caudal to the splenium] Were removed from 6 day old rat pups (PN6), fixed in a freshly made solution of 2% glutaraldehyde and kept in the dark overnight at room temperature. The tissue blocks were then briefly rinsed in distilled water and placed in 2% potassium dichromate-1% osmium tetroxide (4 parts to 1 part, respectively) for 3-6 days in the dark at room temperature. The pieces were then washed with 0.75% silver nitrate until the red precipitate could no longer

Organization of the organotypic explant

The cytoarchitecture of both isolates and cocultures used in the current study did not differ from those described previously [14,30,32]. Unlike serum-grown explants, tissues grown under our serum-free culturing conditions maintained their original organotypic shape and three-dimensionality throughout the in vitro period with no discernable cellular migration away from the explant borders. Only an initial thinning of the explant from the original 360 μm thick slice occurred over the first

Discussion

The present study is the first to quantitatively document the development of neuritic growth in long-term neocortical explants maintained in vitro as isolated explants or grown as cocultures. The major findings of our investigations are that (i) the overall organization of the explant (recognizable major cell types with layering and orientation of major neuritic processes as observed in vivo) remained stable throughout at least 4 weeks in vitro; (ii) the growth patterns of total (basal)

Acknowledgements

The authors wish to thank Dr. Harry Uylings for his many comments and discussions throughout this work, and Mr. Gerben van der Meulen for his photographic assistance. We also wish to thank Ms. Annette Munster of MPI for Biological Cybernetics (Tubingen, Germany) for her helpful suggestions on improving the Golgi stains used in this study.

References (50)

  • J.M. Ruijter et al.

    Chronic blockade of bioelectric activity in neonatal rat cortex grown in vitro: morphological effects

    Int. J. Dev. Neurosci.

    (1991)
  • K. Schilling et al.

    Electrical activity in cerebellar cultures determines Purkinje cell dendritic growth patterns

    Neuron

    (1991)
  • A.A. Schoups et al.

    NGF and BDNF are differentially modulated by visual experience in the developing geniculocortical pathway

    Dev. Brain Res.

    (1995)
  • F.J. Seil et al.

    Anatomical organization of cerebral neocortex in tissue culture

    Exp. Neurol.

    (1974)
  • F.J. Seil et al.

    Morphological correlates of altered neuronal activity in organotypic cerebellar cultures chronically exposed to anti-GABA agents

    Dev. Brain Res.

    (1994)
  • C.J. Shatz

    Impulse activity and the patterning of connections during CNS development

    Neuron

    (1990)
  • H.B.M. Uylings et al.

    The metric analysis of three-dimensional dendritic tree patterns: a methodological review

    J. Neurosci. Methods

    (1986)
  • H.B.M. Uylings et al.

    Geometrical and topological characteristics in the dendritic development of cortical pyramidal and non-pyramidal neurons

    Prog. Brain Res.

    (1994)
  • F. Van Huizen et al.

    Synaptogenesis in rat cerebral cortex is affected during chronic blockade of spontaneous bioelectric activity by tetrodotoxin

    Dev. Brain Res.

    (1985)
  • F. Van Huizen et al.

    Accelerated neural network formation in rat cerebral cortex cultures chronically disinhibited with picrotoxin

    Exp. Neurol.

    (1987)
  • A. Van Ooyen et al.

    Implications of activity dependent neurite outgrowth for neuronal morphology and network development

    J. Theor. Biol.

    (1995)
  • N. Yamamoto et al.

    Laminar specificity of extrinsic cortical connections studied in coculture preparations

    Neuron

    (1992)
  • C.M. Annis et al.

    Aspects of early postnatal development of cortical neurons that proceed independently of normally present extrinsic influences

    J. Neurobiol.

    (1993)
  • C.A. Batista et al.

    Cell cell interactions influence survival and differentiation of purified Purkinje cells in vitro

    Neuron

    (1994)
  • M.E. Blue et al.

    The formation and maturation of synapses in the visual cortex of the rat. I. Qualitative analysis

    J. Neurocytol.

    (1983)
  • Cited by (29)

    • Spontaneous neuronal burst discharges as dependent and independent variables in the maturation of cerebral cortex tissue cultured in vitro: A review of activity-dependent studies in live 'model' systems for the development of intrinsically generated bioelectric slow-wave sleep patterns

      2008, Brain Research Reviews
      Citation Excerpt :

      Such a replacement of glutamate by acetylcholine – along with a minor contribution by several other receptor types: Belousov et al. (2001, 2004) – as the major excitatory neurotransmitter when the former had been chronically inactivated was already on record for primary cultures of hypothalamus and spinal cord but not hitherto for neocortical neurons (Belousov et al., 2001, 2002). Indeed, it was only when we allowed two organotypic explants to cross-innervate one another (Baker and van Pelt, 1997) that any cholinergic plasticity at all manifested itself in occipital cortex tissue. Although addition of the selective cholinergic antagonist, atropine, to the excitatory receptor suppressant ‘cocktail’ prevented spontaneous firing rates from fully attaining control levels even after several weeks in culture, it failed to prevent an as yet unknown source of synaptic drive from in large measure compensating for even this drastic an interference with normal excitatory neurotransmission (Corner et al., 2005).

    • Physiological consequences of selective suppression of synaptic transmission in developing cerebral cortical networks in vitro: Differential effects on intrinsically generated bioelectric discharges in a living 'model' system for slow-wave sleep activity

      2008, Neuroscience and Biobehavioral Reviews
      Citation Excerpt :

      The earlier adaptations could then lead to a more or less permanent hypo- or hyperactivity that might prove to be extremely difficult to correct later on (De Lima et al., 2004; Houweling et al., 2005; Friel and Martin, 2005; Van Huizen et al., 1987a; Van Oss and van Ooyen, 1997). Quantitative dendritic measurements have established that cyto-morphological development is very poor in isolated organotypic neocortex explants despite the retention of a characteristic histotypic organization (Baker and van Pelt, 1997). In addition, SAP firing patterns in such explants show less change with maturation in culture than do dissociated cell cultures (cf. Corner and Ramakers, 1992; Ramakers et al., 1990).

    • Evidence of altered calmodulin immunoreactivity in areas 9 and 32 of schizophrenic prefrontal cortex

      2008, Journal of Psychiatric Research
      Citation Excerpt :

      These thalamic afferents aid in the development of the cortex and more specifically in the activity-dependent process of dendritic pruning (Baker and Van Pelt, 1997; Kossel et al., 1997; Van Ooyen et al., 1995; Van Pelt et al., 1996; Wise et al., 1979). Moreover, this process is dependent upon calcium (Baker and Van Pelt, 1997; Kossel et al., 1997; Van Ooyen et al., 1995; Van Pelt et al., 1996; Wise et al., 1979). Binding of glutamate to the NMDA receptor causes an influx of calcium, which triggers the release of calcium from internal stores and the release of calmodulin from neurogranin (Chakravarthy et al., 1999).

    View all citing articles on Scopus
    View full text