Carbon dioxide uncouples dye-coupled neuronal aggregates in neocortical slices

Letters, 42 (1983) 197-200 Elsevier Scientific Publishers Ireland Ltd.

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AGGREGATES IN NEOCORTICAL SLICES . r

M.J. GUTNICK and R. LOBEL,YAAKOV

Unit o f Physiology, Corob Center for Medical Research. Faculty of Health Sciences. Ben-Gurion University o f the Negev, P.O. Box 653, 84105 Beer Sheva (Israel) (Received September 5th, 1983; Accepged September 14th. 1983)

Key words: brain slices - neocortex - Lucifer Yellow - dye coupling - carbon dioxide - gap junctions

Lucifer Yellow was injected intracellularly into neurons in slices of guinea pig visual cortex. Dye coupling incidence was significantly decreased in slices that were incubated in a high concentration of carbon dioxide. This effect was probably due to intracellular acidification, since exposure to impermeant acid was not effective. ~ e data are consistent with the hypothesis that carbon dioxide interferes with dye coupling in neocortex through its known action as an uncoupler of electronic coupling through gap junctions.

Gutnick and Prince [7] demonstrated that in brain slices of guinea pig sensorimotor neocortex, intracellular injection of the highly fluorescent dye, Lucifer Yellow CH (LY), into a single neuron often results in staining of more than one cell. Electrophysiological observations led them to suggest that dye-coupled neuronal aggregates are also electrotonicaUy coupled. A similar experimental approach has provided evidence for the presence of direct electrical connections between neurons in rat neocortex [41 and in hippocampus [l, 10-121, In several invertebrate systems, dye coupling has been shown to reflect movement of small molecules through gap junctions [2, 9, 17]; however, it is not known if the same mechanism underlies dye coupling in mammalian forebrain preparations. We now report that the incidence of dye coupling in neocortical slices is si~ificantly reduced by exposing the tissue to fluid containing a high concentration of CO2, a procedure known to decrease intracellular pH [18], and consequently to uncouple gap junctions [6, 16, 19]. Experiments were performed in slices of guinea pig visual cortex, which were cut in the parasagittal or coronal plane at a thickness of 450 ~m and maintained in vitro at 36°C, as previously described [5, 7]. Normal bathing medium (pH 7.4) contained 124 mM NaCI, 5 mM KCI, 1.25 mM NaH2PO4, 2 m M MgSO4, 26 mM NaHCO3, 2 mM CaCI2, and 10 mM glucose, and was bubbled with a gas mixture of 950/0 02 and 5o70 CO2. CO2 exposure was accomplished by incubating the slice in Trisbuffered Ringer solution (150 mM NaCl, 5 mM KCI, 2 mM MgCI2, 2 mM CaCI2, 0304-3940/83/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.

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I0 mM glucose, and I0 mM Tris-HCl), while continuing to bubble with a gas mixture that contained 5% COz. Under these conditions, pH of the bathing medium fell to between 5 and 6. After CO2 exposure for 20 min, the slice was returned to normal bathing medium at pH 7.4 for intracellular recording and dye injection within the next hour. Glass micropipettes were filled with a 5°/o solution of the lithium salt of LY (Sigma) in water, and had resistances of 125-250 M~. After stable impalement of a neuron (resting potential > 60 mV), LY was injected iontophoretically for 5-10 min with 1-2 nA, 200 msec, hyperpolarizing pulses at 3 Hz. The slice was removed from the recording chamber within 10 min following injection, and was then fixed in 4% phosphate-buffered formalin, dehydrated in alcohols, cleared in xylene, whole mounted, and examined and photographed with a fluorescence microscope. Only one neuron was injected in each slice. All dye injections were made in the superficial cortical layers (< 400 #m beneath the pial surface). Results are summarized in Table I. Seventy-nine LY injections were made under control conditions, and 35 (44.3070) of these resulted in staining of more than one cell. As indicated by action potential amplitudes, there was no evidence that dye coupling was associated with neuronal damage. In general, the features of dye coupling in slices of visual cortex were quite similar to those previously reported for the somatosensory area [7]. Most dye-coupled aggregates consisted of only two neurons (Fig. 1), and in no case did the dye spread to more than 5 neurons. Cells in dye-coupled groups were always located in the superficial cortical layers, and considerable dendro-dendritic and/or dendro-somatic overlap was usually evident. Within a group, somata could be up to 300 um apart, and they were always clearly separated by a space of at least 10 ttm. In 24 experiments, L Y injections were made in slices that had been exposed to CO2; only two of these resulted in dye coupling. A corrected x2-test revealed that the difference between incidence of dye coupling under these conditions (8.3070) and the control incidence (44.3°7o) was highly significant (x" = 8.7, df = 1, P pocampus, Brafl: Rcs., 196 (1980) 494-497. I! MacVicar, B.A. and Dudek, F.E., Electronic coupling between pyramidal cells: a direct demonstration in rat hipp~,,:ampal slices, Science, 213 (1981) 782-785. 12 MacVicar, B.A., and Dudek, F.E., Electronic coupling between granule cells of rat dentate gyrus: physiological and anatomical evidence, J. Neurophysiol., 47 (1982) 579-592. 13 Peters, A., Morphological correlates of epilepsy: cells in the cerebral cortex. In G.H. Glaser, J.K. Penry and D.M. Woodbury (Eds.), Antiepileptic Drugs: Mechanisms of Action, Advance Neurol., Vol. 27, Raven Pres~, New York, 1980, pp. 21-48. 14 Sloper, J..I., Gap junctions bet~veen dendrites in the primate neocortex, Brain Res., 44 (1972) 64 ! -646. 15 Sloper, J.J. and Powell, T.P.S., Gap junctions between dendrites and soma of neurones in the primate sensorimotor cortex, Proc. Roy. Soc. B, 203 (1978) 39-47. 16 Spray, D.C., Harris, ~.L. and Bennett, M.V.L., Gap junctional conductance is a simple and sensitive function of intracellular pH, Science, 211 (1981) 712-715. 17 Stewart, W.W., Functional connections between cells as revealed by dye coupling with a highly fluorescent naphthalimide tracer, Cell, 4 (1978) 741-759. 18 Thomas, R.C., lntracellular pH of snail neurons measured with new pH-sensitive glass microelectrodes, J. Physiol. (fond.), 238 (1974), 159-180. 19 Turin, L. and Warner, A., Carbon dioxide reversibly abolishes ionic communication between cells of early amphibian embryo, Nature (Lond.), 270 (1977) 56-57.