Organotypic Co‐Cultures of Rat Locus Coeruleus and Hippocampus

Abstract

Slices from the brainstem at the level of the locus coeruleus and from the hippocampus of 5 - 7 day old rats were co-cultured using the roller tube technique. After 2 - 6 weeks in vitro the co-cultures were examined with antibodies raised against tyrosine-hydroxylase (TH). The cultures derived from the brainstem consistently contained a bilateral cluster of TH-positive neurons with 3 - 5 long slender dendrites. These neurons typically gave rise to several fine varicose fibres reminiscent of catecholaminergic axons. A morphologically distinct group of TH-positive neurons was detected in the hippocampal slices. The vast majority of them were located in the subicular region and a smaller number in the CA1/CA3 region of the hippocampal explant. TH-positive neurons were also present in mono-cultures of hippocampus or brainstem. In the vast majority of co-cultures, a variable number of TH-immunoreactive fibres of neurons derived from the locus coeruleus grew over considerable distances to terminate finally within the co-cultured hippocampus where they branched to form a diffuse innervation plexus with club-like endings. Even after several weeks in vitro, TH-positive fibres could still be seen exploring sites which were not related to their target, including the cell-free areas surrounding the cultures. Fibres in these outgrowth areas formed whirl-like endings. TH-positive fibres arising from neurons located in the hippocampus, on the other hand, did not branch extensively and never projected over long distances. Nerve growth factor had no apparent trophic effect on TH-positive cells in the hippocampus, the locus coeruleus, or in the co-cultures.

Organotypic brain slice cultures: A review

“…Several brain areas have been cultured as organotypic brain slices: cortex (Giesing et al, 1975), striatum (Ostergaard et al, 1995), substantia nigra (Whetsell et al, 1981; Kida, 1986), raphe (Jonakait et al, 1988; Hochstrasser et al, 2011), locus coeruleus (Knöpfel et al, 1989), the basal forebrain (Robertson et al, 1997) and the suprachiasmatic nucleus (Wray et al, 1993) as well as several others, such as the hypothalamus, thalamus, supraoptic nucleus or olfactory system.…”

Organs to Cells and Cells to Organoids: The Evolution of in vitro Central Nervous System Modelling

“…In 1966, Crain cultured explants from embryonic rat spinal cord and ganglia on collagen coated glass demonstrating that grafted neural tissue possessed organotypic differentiation and bioelectric properties for electrophysiological studies (Crain, 1966). Since then, brain slices of several cerebral areas have been established as organotypic cultures, including the hippocampus, substantia nigra, locus coeruleus, striatum and basal forebrain (LaVail and Wolf, 1973; Whetsell et al, 1981; Knopfel et al, 1989; Ostergaard et al, 1995; Robertson et al, 1997).…”

In vitro Models of Neurodegenerative Diseases

“…Cells derived from embryonic rat spinal cord and ganglia were subsequently cultured on collagen-coated glass, revealing their potential for organotypic differentiation and bioelectric properties suitable for electrophysiological studies (Crain, 1966). Since then, organotypic cultures have been prepared from brain slices encompassing several cerebral areas, including the hippocampus, substantia nigra, locus coeruleus, striatum, and basal forebrain (Lavail and Wolf, 1973;Whetsell and Schwarcz, 1983;Knopfel et al, 1989;Ostergaard et al, 1995;Robertson et al, 1997). Although tissue explants and organotypic slice cultures faithfully represent the cerebral architecture, they are difficult to prepare and maintain in a viable state, and their inherent variability leads to a lack of reproducibility in experiments (Walsh et al, 2005).…”