Gap junction mediated signaling between satellite glia and neurons in trigeminal ganglia

Abstract

Peripheral sensory ganglia contain the somata of neurons mediating mechanical, thermal, and painful sensations from somatic, visceral, and oro-facial organs. Each neuronal cell body is closely surrounded by satellite glial cells (SGCs) that have properties and functions similar to those of central astrocytes, including expression of gap junction proteins and functional dye coupling. As shown in other pain models, after systemic pain induction by intra-peritoneal injection of lipopolysaccharide, dye coupling among SGCs in intact trigeminal ganglion was enhanced. Moreover, neuron-neuron and neuron-SGC coupling was also detected. To verify the presence of gap junction-mediated coupling between SGCs and sensory neurons, we performed dual whole cell patch clamp recordings from both freshly isolated and short term cultured cell pairs dissociated from mouse trigeminal ganglia. Bidirectional gap junction mediated electrical responses were frequently recorded between SGCs, between neurons and between neurons and SGCs. Polarization of SGC altered neuronal excitability, providing evidence that gap junction-mediated interactions between neurons and glia within sensory ganglia may contribute to integration of peripheral sensory responses, and to the modulation and coordinaton of neuronal activity.

Keywords: connexin; dual whole cell recording; dye coupling; electrical coupling; glia-neuron communication.

Figure 1.. Lucifer yellow (LY) injection into neurons in intact TG from LPS-treated mice reveals homocellular and heterocellular gap junction-mediated coupling.

A. Injected neuron in TG isolated from control animal is typically not dye-coupled to any cell. B. In a very small number of cases an injected neuron in a control TG was found to be coupled to a surrounding SGC and to the SGC sheath surrounding a neighboring neuron (arrow indicates the SGC envelope coupled to the injected neuron). C. TG from a mouse that had been injected with LPS (2.5 mg/kg) eight days earlier. This is an example of the larger proportion of cases in which N-G coupling was present. The injected neuron (N1) was dye-coupled to SGCs around at least six other neurons (N2–N7). Some of the coupled SGCs are indicated by arrows. Calibration, 20 μm. D. Summary of frequency of observing LY dye transfer to SGC (G) when a neuron was injected. In control conditions such coupling was rare (about 5%), but after LPS injection, N-G coupling increased four-fold. The addition of CBX (100 μM) to the medium during LY injection into cells in ganglia from LPS injected mice reduced coupling incidence to about 3% (* p<0.01 between control and LPS treatment and between LPS treatment and LPS + CBX). E. Effects of LPS injection on coupling between neurons in intact TG. In controls the prevalence of such coupling was low (<2%); however, at eight days after LPS injection we observed neuron-neuron coupling in about 13% of the cases. The gap junction blocker CBX blocked the intercellular spread of LY, confirming that gap junctions were involved. (* p<0.05: Fisher’s exact test).

Figure 2.. Neurons and SGCs are coupled to themselves and each other in dissociated cultures from trigeminal ganglion.

A-C: Strength of gap junction mediated coupling between representative pairs of satellite glial cells (SGCs, labeled as G1, G2: A1, A2), SGC-neuron pairs (N and G in B1, B2, D1–3, E1–3) and pairs of sensory neurons (N1 and N2 in C1, C2). Accompanying photographs were taken immediately prior to the electrophysiological recordings. Recordings were selected from series of depolarizing or hyperpolarizing steps applied to each cell of the pair. In A2, +40 mV step was applied to G1, current recorded in G2 (IG2) is termed junctional current, and represents the current passed by the voltage clamp circuit on that cell to hold its voltage constant; junctional conductance is calculated as –IG2/V, in this case about 2.5 nS. In B2, +40 mV step was applied to N, in C2, +40 mV step was applied to N2. D2–3 illustrate current clamp recording of bidirectional coupling between SGC and neuron; in D2 10 nA hyperpolarizing steps are applied to G; whereas the same stimulation paradigm is applied to the neuron in D3. E2–3 illustrate bidirectional coupling under voltage clamp conditions, with the neuron depolarized in E2 and the SGC depolarized in E3. F. Summary of results obtained for cell pairs of each type. NN, N-G and GG designate neuron-neuron, N-G and G-G cell pairs respectively. Subgroups a and b represent recordings made of cultures 2–3 days old during two time periods. Group c represent cells pairs recorded within 18 hr after dissociation. There were no significant differences in coupling strength between recording epochs or with regard to whether cells were freshly isolated or maintained in culture and thus groups were combined to compare coupling strength between homocellular and heterocellular pairs (all N-N, all N-G, all G-G), each of which differed significantly from the other groups (p<0.0001).

Figure 3.. In freshly dissociated (<18 hours) TG cultures neuron pairs are bidirectionally electrically coupled, and junctional conductance is generally weak.

In each case illustrated, neuronal identity was validated by large inward currents in response to depolarizing pulses in addition to appearance of the cells. In frames A-E, top and bottom traces are recordings from two neurons, generally depolarizing pulses applied first to N1 (middle traces) and then to N2 (right-hand traces). Current recorded in the non-pulsed cells reflects junctional current, from which junctional conductance is calculated. A. Traces shown for each cell correspond to no pulse and depolarization from −60 to 0 mV. B. Traces shown correspond to steps from −60 to −60, −40,−20, 0, 20, 40 mV. C. Traces shown correspond to steps from −60 to 0 and +40 mV. D. Traces shown correspond to steps from −60 to −60, 0 and +40 mV. E. Traces shown correspond to steps from 0 to −60, −40, −20, 0 and +20 mV. F. In these recordings, N2 was depolarized from −60 to −60, −20 and 0 mV in voltage clamp mode (middle traces) and 0 or 0.1 nA pulse was applied to N2 under current clamp conditions in the right-hand panel. Scale bar in A (20 μm) applies to all photographs.

Figure 4.. Properties of gap junctions recorded in pairs of SGCs and in SGC-neuron pairs.

A,B. Illustrated examples of voltage dependence of gap junction-mediated coupling in pairs of SGCs and C, D in SGC-neuron pairs; in A, C voltage ramps from −100 mV to +100 mV were applied to one cell (that cell’s current is not shown), and illustrated currents were recorded in the coupled cell. In B, D pulses from 0 to ±100 mV were applied in 20 mV increments to estimate steady state voltage dependence. Note the moderate and symmetric bidirectional rectification in SGC pairs, which is much less in SGC-neuron pairs. E,F illustrate experiments in which SGC-neurons pairs were treated with gap junction uncoupling agents heptanol (2 mM) and CBX (0.2 mM) while hyperpolarizing pulses were delivered repeatedly to cell 2. Note that uncoupling is virtually complete within a short time in these recordings, and is quickly reversible. G. Illustrates junctional currents recorded in an SGC-neuron pair after substantial reduction of junctional conductance by heptanol. The uppermost trace represents the transjunctional voltage applied to cell 1, the middle trace is the input current evoked in cell 1 and the lower four traces are selected from the junctional current recordings in which stepwise fluctuations reveal individual gap junction channels opening and closing. From the slopes of the current / trans-junctional voltage relations, junctional conductance for a single opening is about 65 pS and for two channels open is about 130 pA. H. From recordings such as those illustrated in G performed on SGC cell pairs (N=7) and SGC-neuron pairs (N=7), we measured unitary currents and calculated single channel conductance. Results are plotted in the graphs, demonstrating that in SGC pairs (G-G) gj was 100.5±3 pS and in G-N pairs it was substantially lower (56±4 pS).

Figure 5.. Lucifer Yellow (LY) injections performed in dissociated cultures of trigeminal ganglion demonstrate gap junction mediated coupling between neurons and SGCs.

A1–4 and B1–4 illustrate time course of dye spread into SGCs (arrows) during LY injection into neurons. C1–D3 illustrate two experiments in which LY was injected into neurons in cultures which were acutely treated with the nuclear indicator Hoechst 33342. Overlays in C3, D3 demonstrate LY presence in small SGCs that are closely apposed to the neuron surface. E, F. Summary of experiments in which incidence of LY dye coupling was quantified after injection into neurons (E) or SGC (F). Drugs applied were 200 μM CBX, 100 μM flufenamic acid (FFA), 2 mM heptanol, 100 μM 2-APB, 1 mM probenecid. Intracellular acidification (low pH) was obtained through application of solution bubbled with 100% CO_2, LY Dextran (MW 10 Da), a large fluorescent gap junction impermeant molecule, was completely retained in the injected cell. * indicates p<0.05, ** p< 0.01 and *** p< 0.005 (Fisher’s exact test). The numbers in the bars indicate the number of cells injected. In the case of both SGC and neuron injections, most recipients of the dye injections were SGCs.

Figure 6.. SGC-neuron coupling strength is sufficiently high that glial polarization can modify neuron excitability.

A1–3. Supra-threshold current pulses were applied repeatedly to the neuron (lower trace in A2) while glial resting membrane potential (upper trace) was changed by injecting hyperpolarizing and then depolarizing current. Note that at resting potential (A2) responses are variable in amplitude, reflecting intermittent action potential failure. When SGC was hyperpolarized (middle portion of recording epoch) firing was blocked, whereas depolarizing the SGC resulted in 1:1 firing of neuron. A3. Neuronal responses to current pulses in each of the recording epochs are enlarged to show impact of glial depolarization on neuron responses to the same amplitude current step. B1–4. Comparison of effects of SGC polarization on coupled (B1–2) and uncoupled (B3–4) SGC-neuron pair. Note that same current pulse applied to neuron results in markedly different firing patterns when SGC is depolarized or hyperpolarized in the coupled pair but has no effect when coupling is absent (red, green and black traces are coincident). C1–3. Series of current steps applied to neuron under conditions where SGC was hyperpolarized (C1), not polarized (C2) and depolarized (C3). Note profound effects on firing threshold and activity patterns in response to this 4-pulse paradigm depending on SGC polarization. Photographs of cells from which recordings were made were taken immediately before dual whole cell recordings.(Magni et al. 2015)