OPTICAL MEASUREMENTS OF SYNCHRONIZED ACTIVITY IN ISOLATED MAMMALIAN CEREBELLUM D. COHEN and Y. YAROM* Department of Neurobiology, Life Sciences Institute and Center for Neural Computation, The Hebrew University, Jerusalem 91904, Israel Abstract —An experimental system that combines optical imaging of voltage-sensitive dyes with an in vitro isolated cerebellum preparation is described. The optical imaging system is based on a photodiode array and two rather simple amplification stages. The isolated cerebellum preparation preserves the integrity of the neuronal circuitry, thus allowing the exploration of the path of information flow. In this study, we characterize the nature and sources of the optical signal evoked in the cerebellar cortex by surface stimulation. We show that this signal reflects inhibitory and excitatory synaptic potentials generated by cell bodies and dendrites of cortical neurons, whereas action potentials of the parallel fibers are not detected by the system. The spatial distribution of the optical signals agrees with the classical view of cerebellar activity following surface stimulation. Hence, this experimental system provides a powerful means to explore the functional organization, in time and space, of the cerebellar cortex.  1999 IBRO. Published by Elsevier Science Ltd. Key words: cerebellum, optical imaging, voltage-sensitive dyes. The cerebellar cortex is a modular lattice-like structure composed of five cell types organized in three layers: the granule cell layer (the inner layer); the molecular layer (the outer layer); and, in between, the Purkinje cell body layer. 19 The granule cell layer operates as a divergence relay station supplying the major inputs to the molecular layer. The granu- lar cell axons ascend to the molecular layer, where they bifur- cate and form the parallel fiber system. The parallel fibers in turn activate both the inhibitory interneurons in the cerebellar cortex and the Purkinje cells, whose axons are the sole output of the cortex. 6,16 Although the morphological organization of this structure is well established, the functional organization has been studied mostly with single-cell recordings. These studies revealed, as expected, that surface stimulation acti- vates a large population of parallel fibers and their target neurons. However, to explore the relative contribution of the different elements, and the computational role played by the basic circuit, a multi-unit recording technique with high temporal and spatial resolution is needed. One such system is optical imaging. Optical imaging is based on monitoring changes in light (absorption, emission or scattering) following brain activity, either by measuring the intrinsic signals, 10,24 or by using external probes like voltage-dependent, 4,23 pH- dependent 3 or ion-sensitive dyes. 22 The optical imaging tech- nique has been implemented in brain slices 17,18 and neuronal culture tissue, 8,11 as well as in vivo. 1,26 The first attempt to study the cerebellum by imaging voltage-sensitive dyes was made by Konnerth et al., 14 who showed that the activity of the parallel fibers could be moni- tored by optical means. Since then, several works have focused on the study of cerebellar activity in vivo 5,13 or in slices, 25 and have found different patterns of activity follow- ing direct stimulation of the parallel fibers or stimulation of various body elements. However, none of these studies have provided suitable means for analysing the spatiotemporal flow of neuronal activity in the cerebellum, as they lacked the time resolution or damaged the neuronal circuitry. Here, we present an experimental system that harnesses imaging of voltage-sensitive dyes to the exploration of infor- mation flow in an isolated mammalian cerebellum. We use this system to study the spatiotemporal patterns of activity in the cerebellar cortex. Comparison of the optical and the elec- trical responses recorded at the same location and the use of specific neuroactive drugs demonstrates that this system detects signals which reflect changes in the membrane poten- tial of cortical cells. EXPERIMENTAL PROCEDURES Isolated cerebellar preparation In preparing the isolated cerebellum, we modified the procedure described by Llinas et al. 15 Adult albino guinea-pigs (100–200 g) were anesthetized with pentobarbitone sodium (Nembutal, 30 mg/ kg). After decapitation, the whole brain was rapidly removed and immersed in a cold, oxygenated (95% O 2 /5% CO 2 ) physiological solu- tion (composed of, in mM: 124 NaCl, 5 KCl, 1.3 MgSO 4 , 1.2 KH 2 PO 4 , 26 NaHCO 3 , 10 glucose and 2.4 CaCl 2 ). As shown in Fig. 1A, the cerebellum and the brain stem were isolated from the level of the first cervical segment to the rostral edge of the inferior colliculus. To maintain the cerebellum and related structures in viable condition, it was perfused via the cerebellar vascular system with the oxygenated physiological solution, to which 5% dextran (T70 Pharmacia Biotech) had been added. The isolated cerebellum was perfused through a cannula made of polyethylene tubing (PE100) pulled to a diameter of 200 mm and inserted into one of the vertebral arteries. To ensure an efficient perfusion, the cut ends of all the other major arteries were tied with surgical thread. A peristaltic pump was used to maintain a constant flow rate of 0.5 ml/min. During experiments, the preparation was washed continuously with aerated regular physiological solution preheated to yield a constant bath temperature of 28°C. Under these conditions, signs of edema were insignificant and the preparation maintained its viability for 6–8 h. Dye application and tissue staining The voltage-sensitive styryl dyes (RH-795 or RH-414) were first dissolved in distilled water and then diluted (1:15) in physiological Synchronized activity in isolated mammalian cerebellum 859 859 Neuroscience Vol. 94, No. 3, pp. 859–866, 1999 Copyright  1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 PII: S0306-4522(99)00348-6 Pergamon *To whom correspondence should be addressed. Tel.: + 972-2-6585172; fax: + 972-2-6586296. E-mail address: yarom@vms.huji.ac.il (Y. Yarom) Abbreviations: DNQX, dinitroquinoxaline; EGTA, ethyleneglycolbis(ami- noethyl ether)tetra-acetate; LED, light-emitting diode; TEA, tetraethyl- ammonium; TTX, tetrodotoxin.