Fig 1.
Schematic of the peripheral sympathetic-cardiac circuit.
Within the sympathetic ganglia, pre-synaptic inputs from spinal cord preganglionic neurons form cholinergic synapses onto postganglionic sympathetic neurons, and satellite glial cells in the ganglia enwrap neuronal soma. The postganglionic neurons project to peripheral targets including the heart.
Fig 2.
Satellite glial cells increase spontaneous activity of cultured sympathetic neurons.
(a) Representative traces of spontaneous activity of neurons held in voltage-clamp at -60 mV, without hexamethonium, with 100 μM hexamethonium and after washout. (b) Average synaptic charge, normalized to control, for neurons treated with 100 μM hexamethonium (hex) and following washout (n = 10 cells per condition; paired t-test; *p<0.05). (c-j) Neurons were cultured for 14 days either alone (c, g) or in the presence of satellite glial cells starting from day 0 (d, h) or day 7 (e, i). NGF (5ng/ml) was included in all culture conditions to promote neuronal survival. (c-e) Representative voltage clamp traces showing that co-culture with satellite glial cells for the 14 days culture period, or for the last 7 days of the period increases current flow. (f) Quantification of synaptic activity. Total synaptic charge, defined as the area above the curve for neurons grown in the absence (Neurons alone, N) or presence of satellite glial cells for 14 days (NG[0]) or the last 7 days of the culture period (NG[7]), was quantified and average synaptic charge per 10 s duration was calculated. Plotted average membrane current values were quantified as averaged synaptic charge normalized to 1 ms duration. Therefore, the value of the average membrane current of e.g. -400 pA is equivalent to an average synaptic charge of 4 nC (n≥ 15 cells, Mann-Whitney U test, ***p<0.001, *p<0.05) (g-i) Representative current clamp traces showing that glial cells increase spontaneous firing in cultured sympathetic neurons. (j) Quantification of neuronal firing rate in the absence (N) or presence of satellite glial cells for 14 (NG[0]) or 7 (NG[7]) days. (n≥10 cells, Mann-Whitney U test ***p<0.001, *p<0.05). Bars represent mean ± s.e.m.; dots represent data for individual cells.
Table 1.
Neuronal characteristics of neurons co-cultured with or without glia.
Fig 3.
Satellite glial cells do not alter neuronal intrinsic excitability.
(a) Illustration of the stimulus pattern applied to determine neuronal firing threshold. (b) Quantification of neuronal firing threshold between neurons alone (N) and neurons co-cultured with glia for 14 days (N+G) conditions. (Unpaired t-test, n ≥ 10 cells, n.s. not statistically different). (c) Illustration of the stimulus pattern applied to evoke action potential firing and representative neuronal traces in response to 400 pA current pulse for neurons grown for 14 days alone (brown, upper trace) or in the presence of glia (blue, lower trace). (d) Average number of action potentials evoked by current steps of increasing amplitude. (n≥16 cells, Mann-Whitney U test pairwise comparison for 400 pA current step, n.s. not statistically different). Results are represented as mean ± s.e.m.
Fig 4.
Satellite glial cells enhance cholinergic synapse formation.
Neurons cultured in the absence (Neurons alone, N) or presence of satellite glial cells (N+G) were fixed, stained for synaptic markers, and analyzed by confocal microscopy. (a) Representative images of cells stained for the pre-synaptic marker VAChT (red), the post-synaptic marker Shank (green), and the dendritic marker MAP2 (blue). Boxed regions in left panels are magnified in the right panels to show colocalized puncta (arrows). Scale bar represents 10 μm in the left panels; 2 μm in the right panels. (b) Quantification of VAChT and Shank puncta on sympathetic neuronal soma and proximal dendrites showing a glia-dependent increase in the expression of VAChT, but not Shank-containing puncta (n ≥ 61 cells, unpaired t-test ***p<0.001 and n.s., respectively). Quantification of co-localized synaptic puncta density (c) and size (d) on neuronal soma and proximal dendrites showing that glia induce structural synapse formation (n ≥ 61 cells, unpaired t-test, ***p<0.001, **p<0.01, respectively).
Fig 5.
Postnatal development of neurons and satellite glia in the Superior Cervical Ganglion (SCG).
(a-b) Representative confocal images of (a) P2 and (b) P21 SCG. Sympathetic neurons were stained for the neuron-specific marker MAP2 (red), satellite glial cells for the glial cell marker S100β (green) and cell nuclei using DAPI (blue). Scale bar = 100 μm. (c-d) Quantification of (c) neuron soma size, measured as average cell area in the section and (d) neuronal and glial cell densities from sections of P2 (n = 3; mean ± s.e.m.) and 3 wks (n = 3; mean ± s.e.m.). ***p<0.001, **p<0.01, *p<0.05 determined by ANOVA followed by pairwise post hoc (Tukey’s HSD) comparison test.
Fig 6.
Glial cell-conditioned medium recapitulates the effect of satellite glial cells on cultured sympathetic neuron activity.
(a-b) Neurons grown in control medium—CM (a) or glial cell-conditioned medium—GCM (b) for the last 7 days of the 14-day culture period. NGF (5ng/ml) was included in both culture conditions to promote neuronal survival. Cells were fixed and stained for Tuj-1 (neuronal marker, in red) and DAPI (nuclear staining, in blue). Scale bar represents 50 μm. (c) Neuronal cell number was not altered by the cell culture condition. (n = 6 independent cell culture experiments, unpaired t-test, n.s.). (d-e) Representative voltage-clamp traces of neurons cultured in CM (d) or GCM (e). (f) Quantification of average membrane current showing increased spontaneous activity in GCM (n ≥ 12 cells, non-parametric Mann-Whitney U test, ***p<0.001). Results are represented as mean ± s.e.m., dots represent data for individual cells.
Fig 7.
Satellite glial cells support neuronal survival.
Satellite glial cells partly prevent sympathetic neuronal death upon NGF deprivation. (a-b) Establishment of sympathetic neuron-satellite glia co-cultures. Neurons (N) were cultured alone (a) or in the presence of satellite glia (b) in the presence of 5 ng/ml NGF in serum-containing medium. Cultures were fixed at 12 days in vitro (div) and stained for Tuj-1 (neuronal marker, in red), S100β (glial cell marker, in green) and DAPI (nuclear staining, in blue). Scale bar represents 50 μm. Under these growth conditions, glia did not alter the number of sympathetic neurons (c). (d) Neurons alone (N), (e) Neurons and glia (N+G), and (f) N+G with anti-NGF antibody (1:1000, final concentration 1 μg/ml). (g) Quantification of cell survival upon NGF deprivation. Data are shown as percent neuronal survival compared to comparable cultures (neurons alone or neurons + glia) grown in the presence of 5 ng/ml NGF in serum-free medium (n = 3 independent cell culture experiments, One-way ANOVA, ***p<0.001). All data are represented as mean ± s.e.m.
Fig 8.
Satellite glial conditioned medium supports neuronal survival via an NGF-dependent mechanism.
Representative images of neurons grown for 3 days in (a) 5 ng/ml NGF (+NGF), (b) glial conditioned medium without added NGF (GCM-NGF), and (c) control conditioned medium without added NGF (CM-NGF). Cultures were fixed at 3 div and stained with Tuj1 (red) an DAPI (blue). Scale bar: 200 μm. (d) Quantification of percent survival compared to the NGF condition for neurons cultured for 3 days in no NGF, GCM, GCM+TrkA-IgG, CM, CM+TrkA-IgG (n ≥ 4 independent cell culture experiments, **p<0.01, ***p<0.001, compared to GCM; #p<0.001, compared to no NGF condition; ANOVA followed by pairwise post hoc (Tukey-Kramer) comparison test. All data are represented as mean ± s.e.m.). Note that the greater survival effect of GCM compared to the co-culture with the glial cells (Fig 7), is likely to reflect the analysis of the co-cultures at a later time point than the GCM-treated cultures.