European Journal zyxwvutsrq of Neuroscience, Vol. zyxwvutsrq 5, pp. 915-926 zyxwvut 0 1993 European Neuroscience Association z The Isolated and Perfused Brain of the Guinea-pig z In Vitro zyxwv M. Muhlethaler’, M. de Curtis2, K. Walton3 and R. Llinds3 ’Laboratoire de Psychophysiologie, Departement de Physiologie, CMU, 1 Rue Michel Servet, 1211 Geneve 4, Switzerland ’Present address: Dipartimento di Neurofisiologia, lstituto Neurologico, via Celoria 1 1, 201 33 Milano, Italy 3Department of Physiology & Biophysics, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA zy Key words : visual system, superior colliculus Abstract We describe here an isolated and perfused in vitro adult guinea-pig whole brain preparation which is an extension of the previously described in vitro brainstem - cerebellum preparation, Viability was tested by the analysis of trans-synaptic responses along the visual pathways following the electrical stimulation of the optic nerve or the optic radiations. The evoked field potentials were recorded in the dorsal lateral geniculate, the superior colliculus and the visual cortex. The distribution of extracellular currents was studied using current source density analysis, in order to determine the amplitude, time course and spatial organization of the synaptic activity at these sites. The study indicates that field potentials were very similar to those described in vivo. These data demonstrate the survival of a complex adult sensory system in vitro and suggest that this preparation can be used for the analysis of multisynaptic circuits in the mammalian brain. Introduction zyxwvutsrqpo The technical difficulties involved in obtaining stable intracellular recordings and in accessing deep brain structures under in vivo conditions stimulated the development zyxwvutsrq of alternative in vitro techniques (reviewed in Grantyn and Ketterman, 1992). Of these the most useful and popular one thus far has been the brain slice. The early limitations of this preparation for certain types of studies prompted the development of techniques for recording from acutely dissociated single mammalian brain neurons (Kay and Wong, 1986). In addition to these ‘simplified’ preparations, it has also become clear that more complex in vitro preparations were required, ones in which multisynaptic circuits and the intracellular activity they generate were well preserved. Electrophysiological studies using preparations comprising large blocks of CNS tissue were first developed using the nervous system of lower vertebrates (Hackett and Llinas, 1973). As far as mammalian tissue is concerned, perfused tissue blocks of the brainstem, the brainstem - cerebellum or the basal hypothalamus were successfully introduced in the early and middle eighties (Llinas et al., 1981; Walton and Llinas, 1982; Bourque and Renaud, 1984; Llinas and Miihlethaler, 1988a,b). In addition the superfused spinal cord of young rats (Otsuka and Konishi, 1974; Bagust et al., 1985; Fulton and Walton, 1986) have also been successfully utilized. The feasibility of maintaining an isolated, adult mammalian brain in vitro remained to be determined. The arterially perfused in vitro adult guinea-pig brain preparation was introduced a decade ago (Walton and Llids, 1982), but it was only following the development of better perfusion techniques with the reduced isolated and perfused brain- stem-cerebellum ( L W and Miihlethaler, 1988a,b) that the possibility of maintaining the entire brain in vitro was reassessed. The present study addresses the question of the functional integrity of the extended neuronal circuits and of monosynaptic as well as polysynaptic interactions at different levels of the neuraxis in the isolated adult guinea-pig brain. For that purpose it was determined that the study of field potentials, as in the brainstem-cerebellum (Llinis et al., 1981; Llinis and Miihlethaler, 1988a,b), was the most sensitive way to study the functional integrity of large populations of neurons. Indeed, single- cell studies reflect the viability of individual elements, but not that of the larger percentage of the neuronal population. Here we have chosen to study the visual system since its anatomy and function is well known and it can be investigated in great detail using field potential analysis at several levels following optic nerve stimulation. Given that such field potential analysis has been carried out using in vivo preparations by many investigators (Freeman and Singer, 1983; Mitzdorfand Singer, 1977, 1978) it provides a clear standard for determining the viability of the isolated in vitro brain. This paper provides the first detailed and complete account of the techniques developed to isolate and maintain a perfused adult guinea- pig brain preparation in v i m . Several papers have appeared using this technique to study various regions of the brain, such as the entorhinal cortex (Alonso et al., 1990), thalamus (Miihlethaler and Serafin, 1990) and limbic system (de Curtis et al., 1991; Par6 et al., 1992). A number of abstracts have also appeared reporting intracellular or extracellular studies of the cerebral cortex (Walton and Llinds, 1982; de Curtis and Correspondence to: zyxwvutsrq R. Llinb, as above Received 16 July 1992, revised 18 January 1993, accepted 4 February 1993