Cultured basal forebrain cholinergic neurons in contact with cortical cells display synapses, enhanced morphological features, and decreased dependence on nerve growth factor

Abstract

Prior studies examining the dependence of basal forebrain cholinergic neurons (BFCNs) on nerve growth factor (NGF) for survival have reached differing conclusions depending on the experimental paradigm employed, suggesting the importance of environmental and developmental variables. The present study examined the NGF dependence of BFCNs and modulatory effects of target (cortical) neurons under the controlled conditions of dissociated cell cultures. Initial experiments found BFCNs (identified by using choline acetyltransferase immunocytochemistry) in pure basal forebrain (BF) cultures to be dependent on NGF between the 2nd and 4th week in vitro. During that developmental period, NGF deprivation for 3 days, induced by application of anti-NGF antibody, resulted in degeneration of over 80% of BFCNs, whereas at earlier or later times, BFCNs were largely resistant to NGF deprivation. When BF neurons were plated together with cortical neurons (as dissociated co-cultures), the BFCNs grew neuritic processes (labeled with acetylcholinesterase histochemistry) that appeared to specifically target cortical neurons; electron microscopy revealed that synapses formed between these cells. BFCNs in co-cultures were more resistant to NGF deprivation, were larger, and had much more extensive neuritic growth than BFCNs in pure BF cultures. The resistance of BFCNs to NGF deprivation provided by cortical neurons could be largely reproduced by addition of other trophic factors (brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT3; neurotrophin 4/5, NT4/5; or glial-derived neurotrophic factor, GDNF) during NGF deprivation in pure BF cultures. These results suggest that developing BFCNs undergo a critical period requiring trophic influences that may be provided by NGF or other trophic factors, as well as unknown factors derived from cortical neurons.

Basal forebrain cholinergic cell attachment and neurite outgrowth on organotypic slice cultures of hippocampal formation

“…2B). These features are similar to those described for BFCNs in situ (Amaral and Witter, 1995;Houser et al, 1983;Shingai et al, 1990;Wenk et al, 1980;Woolf, 1991), and similar E. S. Tsai et alto the features described for these cells in both dissociated cell cultures (Dreyfus, 1989;Ha et al, 1996;Shingai et al, 1990) and in slice cultures (Baratta et al, 1996a,b;Robertson, 1992, 1993;Ga «hwiler et al, 1987).…”

“…However, it remains possible that the development of the cholinergic phenotype occurs preferentially in cells that have settled down in a supportive environment or have established appropriate adhesive contacts on the slice. Other studies have reported expression of the cholinergic phenotype in dissociated cultures (Dreyfus, 1989;Hartikka and Hefti, 1988;Li et al, 1995;Nonomura et al, 1996;Shingai et al, 1990), however, the presence of exogenously applied neurotrophic factors or the presence of normal target cells is important for expression of the cholinergic phenotype and for survival (Dreyfus, 1989;Ha et al, 1996;Hartikka and Hefti, 1988;Li et al, 1995;Morse et al, 1993;Nonomura et al, 1996;Shingai et al, 1990). Grown in isolation, without target contacts or supplemental neurotrophins, the BFCNs do not thrive.…”

“…The brains were then removed and placed in cold DMEM. Under a dissection microscope the basal forebrain region, including septum and diagonal band, was exposed and removed surgically, using sharp forceps (Ha et al, 1996;Hartikka and Hefti, 1988). Basal forebrain tissue pieces were incubated in 0.09% trypsin in DMEM at 36 ‡C for 30 min.…”

“…Because the dissociated cells were taken from brains of animals at gestational day 17, this 2^3-day delay may simply re£ect the normal sequence of development of these neurons (Amaral and Witter, 1995;Brady et al, 1989;Ha et al, 1996;Milner et al, 1983;Shingai et al, 1990;Thal et al, 1991). However, it remains possible that the development of the cholinergic phenotype occurs preferentially in cells that have settled down in a supportive environment or have established appropriate adhesive contacts on the slice.…”

“…Studying these putative cell^cell recognition processes in the whole developing animal is a challenging problem that has prompted the use of more reduced in vitro approaches (Alderson et al, 1990;Amaral and Witter, 1995;Baratta et al, 1996a,b;Robertson, 1992, 1993;Dreyfus, 1989;Ha et al, 1996;Hartikka and Hefti, 1988). Recently, we have introduced a method in which dissociated cells are seeded onto organotypic slice cultures of known target tissue .…”