A Constraint on the Formation of Electrical Coupling in Neurons
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The Journal of Neuroscience, March 1994, 14(3): 1477-l 485 Self-Recognition: A Constraint on the Formation of Electrical Coupling in Neurons Peter B. Guthrie, Robert E. Lee, Vincent Rehder, “Marc F. Schmidt, and bStanley B. Kater Program in Neuronal Growth and Development, Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523 Electrical coupling between specific neurons is important erologous connexon proteins (Swenson et al., 1989; Traub et for proper function of many neuronal circuits. Identified cul- al., 1989; Barrio et al., 199 1). Gap junctions can also be found tured neurons from the snail Helisoma show a strong cor- between different processesof a single cell (called reflexive gap relation between electrical coupling and presence of gap junctions) (Iwyama, 1971; Herr, 1976; Majack and Larsen, 1980). junction plaques in freeze-fracture replicas. Gap junction These observations suggestthat processeswhose membranes plaques, however, were never seen between overlapping have connexons present(or nearby) might normally be expected neurites from a single neuron, even though those same neu- to couple with other, equally competent, processes.Relatively rites formed gap junctions with neurites from another es- few studies have investigated the mechanismsregulating the sentially identical identified neuron. This observation sug- specificity of gap junction formation. What is not clear is what gests that a form of self-recognition inhibits reflexive gap the mechanismsare that might prevent inappropriate gap junc- junction formation between sibling neurites. When one or tion formation between competent processes. both of those growth cones had been physically isolated Studiesof the specificity of electrical synapseformation in the from the neuronal cell body, both electrical coupling and gap pond snail Helisoma have shown that synapse specificity can junction plaques, between growth cones from the same neu- be different in vivo than in vitro. In vivo, regenerating buccal ron, were observed to form rapidly (within 30 min). Thus, neurons will reconnect to their appropriate targets and will also inhibition of electrical coupling between sibling neurites ap- form novel electrical connections (Hadley et al., 1982). Not parently depends on cytoplasmic continuity between neu- every neuron will be connected, however, and many of the rites, and not the molecular composition of neurite mem- electrical connections are transient (Cohan et al., 1987). In con- brane. The formation of gap junctions is not likely due to the trast, regulation of electrical coupling in vitro appearsto depend isolation process; rather, the physical isolation appears to solely on the growth state of the neurons (Hadley and Kater, release an inhibition of reflexive gap junction formation. These 1983; Hadley et al., 1983, 1985). Electrical coupling is observed data demonstrate the existence of a previously unknown between every pair of neurons that overlap when both are in an constraint on the formation of electrical synapses. active growth state, suggestingthat every neuron, in culture, is [Key words: gap junctions, Helisoma, autapse, electrical competent to form gapjunctions. Coupling is almost completely coupling, synapse specificity, freeze fracture, fura- absent when one of the neuron ceasesactive outgrowth before overlap of neurites occurs. Gap junctions are widespreadthroughout the animal kingdom We report here that a specific inhibition of gap junction for- and have been proposed to serve a number of functions, in- mation occurs between sibling neurites from a single neuron. cluding metabolically linking cells, controlling morphogenesis, Theseneurites, which are quite competent to form gapjunctions and serving as an integral part of neuron circuits (Larsen, 1983; with neurites from other growing neurons, fail to establishgap Bennett et al., 1991). A number of studies have examined the junctions with equally competent sibling neurites from the same development of coupling (Liu and Nicholls, 1989; Swensonet neuron. This form of self-recognition is unlikely to occur be- al., 1989; Barrio et al., 1991) regulation of coupling (Murphy causeof molecular recognition of sibling membrane, since ul- and Kater, 1980; Hadley et al., 1982; Bulloch, 1985; Janse et trastructurally and functionally defined gap junctions can be al., 1986; Pelletier, 1988; Carrow and Levitan, 1989; Matsu- formed between sibling neurites after they have been physically moto et al., 1991), and the regulation of gap junction activity isolated from the parent neuron. These results point to a novel (Liu and Nicholls, 1989; Swenson et al., 1989; Barrio et al., mechanism regulating the formation of gap junctions within 1991). Gap junctions between cells have been found to form neurons. across species(Epstein and Gilula, 1977) and even from het- Methods and Materials Received Jan. 25, 1993; revised June 30, 1993; accepted Aug. 26, 1993. Cellculture. Individual identified neurons were isolated from adult Hel- We acknowledge the assistance of Dennis Giddings with photography and il- isoma nervous system and cultured as previously described (Mattson lustrations. This work and its publication were supported by the National Institutes of Health: NS-24683 (S.B.K.) and NS-29747 (P.B.G.). and Kater, 1987). For the ultrastructural and optical experiments, neu- Correspondence shduld be’addressed to Dr. Peter B. Guthrie, Department of rons were plated onto glass coverslips on the bottom of 35 mm culture Anatomy and Neurobiology, Colorado State University, Fort Collins, CO 80523. dishes. B5 and B 19 neurons were used. Different cultures were used for = Present address: Department of Biology, Georgia State University, P.O. Box ultrastructural, electrophysiological, and optical experiments to avoid 4010, Atlanta, GA 30302-4010. ultrastructural damage that might occur from the electrophysiological h Present address: Division of Biology 216-76, California Institute of Technol- or optical recording procedures. ogy, 120 1 East California, Pasadena, CA 9 1125. Growthcone isolation. Growth cones were isolated from the rest of Copyright 0 1994 Society for Neuroscience 0270-6474/94/141477-09$05.00/O the neuron by severing the neurite immediately proximal to the growth 1478 Guthrie et al. * Self-Recognition Regulates Gap Junction Formation cone, using a glass electrode as a microknife (Rehder et al., 199 1). Such containing 34 mM K+ (Rehder and Kater, 1992). Four neurons were isolated growth cones have been observed to resume normal elongation immediately fixed after the 30 min depolarization; four additional neu- within 30 min of isolation, and to exhibit normal branching and turning rons were washed with normal defined medium, and cultured for an behaviors, electrophysiological properties, and responsiveness to neu- additional 24 hr before fixation. The cultures were fixed and processed rotransmitters for several days following isolation (Davis et al., 1992b). for freeze-fracture examination as described above. Growth cones that showed unusual swelling or other signs of trauma Calcium measurements. Free cytoplasmic calcium concentrations were were not used in these experiments. measured as described (Rehder et al., 199 1). Briefly, the cell body was Freeze-fracture procedures. Freeze-fracture procedures were as de- penetrated with a microelectrode containing fura- (pentapotassium scribed previously (Guthrie et al., 1989). Briefly, neurons were grown salt, IO mM in distilled H20; Molecular Probes, Eugene, OR). The fura- on glass coverslips and fixed with 3% glutaraldehyde in 0.1 M phosphate was injected with hyperpolarizing current for a maximum of 5 min, buffer (pH 7.2) for 1 hr at 4°C. The cultures were then photographed to yielding intracellular fura- concentrations estimated at less than 60 PM. permit identification of individual neurites in the freeze-fracture rep- The preparation was allowed to recover for 15 min before manipulations licas. The coverslips were washed twice in 0. I M phosphate buffer. The began. Fura- fluorescence was measured on a Zeiss ICM microscope material was cryoprotected by infiltration of 25% glycerol in 0.1 M using a quartz collector on the HBO 50 mercury lamp and a 40 x oil phosphate buffer at 4°C for 30 min. Pieces ofglass containing the neurons Nikon Fluorite objective. The excitation light was directed through a were obtained by scoring and breaking the coverslip. The neurons were computer-controlled shutter/filter wheel system for the two fura- ex- frozen according to the method of Pauli et al. (1977), which consists of citation wavelengths (350 -+ IO nm and 380 ? IO nm). Neutral density placing a drop of a solution containing polyvinyl alcohol, phosphate- filters were inserted in the excitation path as necessary to reduce bleach- buffered saline, glycerol, and dimethyl sulfoxide onto a 3-mm-diameter ing and saturation of the fluorescence. The image was directed to an gold specimen support with a central 1 mm hole. The glycerol solution intensified CCD camera (Quantex Carp, Sunnyvale, CA). The output was drained from the piece of glass coverslip with the attached neuron, of the camera was fed to a Quantex QX7 Image Acquisition/Analysis and the glass was placed neuron-side down onto the polyvinyl alcohol System (Quantex Corp, Sunnyvale, CA). Calcium concentrations were solution drop on the specimen support. The specimen was then frozen estimated from the ratio of emission intensities (495 nm long-pass) at by plunging the gold support into partially