European Journal zyxwvutsrq of Neuroscience, zyxwvutsrqpo Vol. I, pp. zyxwvutsrq 678- 689. zyxwvut 0 1989 European Neuroscience Association 0953-81 6.~189 $3.00 z Organotypic Co-Cultures of Rat Locus Coeruleus and H ippocampus T. Knopfel, L. Rietschin and B. H. Gahwiler Brain Research Institute, University of Zurich, August-Forel-Strasse zyxwvut 1, CH-8029 Zurich, Switzerland Key words: slice cultures, catecholamines, fibre ingrowth, multiple axons, tyrosine-hydroxylase, immunohistochemistry 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 CAlICA3 region of the hippocampal explant. TH-positive neurons were also present in mono-cultures zyxwvuts 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 zyx 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. coeruleus, or in the co-cultures. Nerve growth factor had no apparent trophic effect on TH-positive cells in the hippocampus, the locus Introduction The brainstem nucleus locus coeruleus (LC) contains the major group of noradrenaline containing neurons sending fibres to various regions throughout the rat brain. The noradrenergic innervation of the hippocampal formation, in particular, appears to arise exclusively in the LC (Loy et al., 1980). Noradrenaline is thought to play an important role in neuronal synaptic plasticity such as long-term potentiation in the CA3 region of the hippocampus and memory function (Stanton and Sarvey, 1986; Bliss et al., 1983; Crow and Wendlandt, 1976). The postsynaptic actions on central target cells of noradrenaline released from noradrenergic neurons have, however, not yet been studied in detail. A thorough investigation zyxwvutsrqp of the mechanisms involved in central noradrenergic transmission is hampered by the difficulties in obtaining long-lasting and stable intracellular recordings in situ and the limited possibilities for selective stimulation of noradrenergic fibres in acute slice preparations. We expect to overcome these limitations in organotypic slice co-cultures of the hippocampus and the LC. where it is possible to stimulate electrically the noradrenergic neurons and to record the responses of target neurons in the hippocampal tissue using advanced electrophysiological techniques. This expectation is supported by previous studies on slice co-cultures of hippocampus and septum (Gahwiler and Hefti, 1984). These co-cultures have provided a powerful tool for the study of functional properties of central muscarinergic synaptic transmission (Gahwiler and Brown, 1985). Moreover, slice cultures could be observed during the entire period of growth and experimental conditions such as growth medium can be varied at will. In septo-hippocampal co-cultures, these circumstances facilitated the characterization of factors involved in the establishment of stable synaptic interactions between cholinergic fibres and their target neurons (Giihwiler et al., 1989). The present work was designed to evaluate the conditions under which noradrenergic neurons derived from the LC survive in tissue cultures prepared with the roller tube technique. Furthermore, we intended to describe the morphological properties of these tyrosine-hydroxylase (TH)-containing neurons and to establish whether these neurons have the capacity to innervate hippocampal tissue under our culture conditions. Additionally, we described a morphologically distinct group of TH-immunoreactive neurons located in the co-cultured hippocampal slices. This preparation will be instrumental in the investigation of the Correspondence 10: Dr T. Knopfel, as above Received 4 April 1989, revised 22 May 1989, accepted 23 May 1989