Surviving mossy cells enlarge and receive more excitatory synaptic input in a mouse model of temporal lobe epilepsy

Abstract

Numerous hypotheses of temporal lobe epileptogenesis have been proposed, and several involve hippocampal mossy cells. Building on previous hypotheses we sought to test the possibility that after epileptogenic injuries surviving mossy cells develop into super-connected seizure-generating hub cells. If so, they might require more cellular machinery and consequently have larger somata, elongate their dendrites to receive more synaptic input, and display higher frequencies of miniature excitatory synaptic currents (mEPSCs). To test these possibilities pilocarpine-treated mice were evaluated using GluR2-immunocytochemistry, whole-cell recording, and biocytin-labeling. Epileptic pilocarpine-treated mice displayed substantial loss of GluR2-positive hilar neurons. Somata of surviving neurons were 1.4-times larger than in controls. Biocytin-labeled mossy cells also were larger in epileptic mice, but dendritic length per cell was not significantly different. The average frequency of mEPSCs of mossy cells recorded in the presence of tetrodotoxin and bicuculline was 3.2-times higher in epileptic pilocarpine-treated mice as compared to controls. Other parameters of mEPSCs were similar in both groups. Average input resistance of mossy cells in epileptic mice was reduced to 63% of controls, which is consistent with larger somata and would tend to make surviving mossy cells less excitable. Other intrinsic physiological characteristics examined were similar in both groups. Increased excitatory synaptic input is consistent with the hypothesis that surviving mossy cells develop into aberrantly super-connected seizure-generating hub cells, and soma hypertrophy is indirectly consistent with the possibility of axon sprouting. However, no obvious evidence of hyperexcitable intrinsic physiology was found. Furthermore, similar hypertrophy and hyper-connectivity has been reported for other neuron types in the dentate gyrus, suggesting mossy cells are not unique in this regard. Thus, findings of the present study reveal epilepsy-related changes in mossy cell anatomy and synaptic input but do not strongly support the hypothesis that mossy cells develop into seizure-generating hub cells.

Keywords: GluR2; dendrites; dentate gyrus; hypertrophy; miniature EPSC.

Figure 1

GluR2-immunoreactivity in the dentate gyrus of a control (A) and epileptic pilocarpine-treated mouse (B). Boxed regions in A1 and B1 are shown at higher magnification in A2 and B2. m=molecular layer, g=granule cell layer, h=hilus, CA3=CA3 pyramidal cell layer. C Large (soma diameter >12 μm) GluR2-positive hilar cell profiles per section. Values represent mean ± s.e.m. Number of mice per group indicated at base of bars. *p<0.001, Mann-Whitney rank sum test). D Soma area of large GluR2-positive hilar cell profiles. Markers indicate average values per mouse.

Figure 2

Examples of mossy cells recorded and biocytin-labeled in control (A) and epileptic (B) mice. Cell layers were identified by NeuN-immunoreactivity (not shown) and are indicated by dashed lines. g=granule cell layer, h=hilus, CA3=CA3 pyramidal cell layer.

Figure 3

Increased soma size of biocytin-labeled surviving mossy cells in epileptic mice. A Confocal images (A1,B1) and reconstructions (A2,B2) of representative mossy cells from a control (A) and epileptic (B) mouse. g=granule cell layer, h=hilus, CA3=CA3 pyramidal cell layer. C Soma size of mossy cells was 1.3-times larger in epileptic mice compared to controls (*p=0.007, Mann-Whitney rank sum test). Values represent mean ± s.e.m. Number of cells per group indicated at base of bars. D Cumulative dendritic length per cell was not statistically different. E Number of amputated dendrites with respect to dendritic branching order was similar in control and epileptic mice.

Figure 4

Intrinsic physiology of mossy cells in control and epileptic mice. A Representative voltage responses to hyperpolarizing (−5 pA) and depolarizing (10 pA) current pulses in mossy cells of control (top) and epileptic (bottom) mice. B Input resistance (Rin), membrane time constant (τ), resting membrane potential (RMP), action potential (AP) threshold and amplitude of mossy cells. Values represent mean ± s.e.m. Number of cells per group indicated at base of bars. Input resistance in epileptic mice was significantly reduced (*p=0.008, t test). There were no significant differences in other parameters.

Figure 5

Excitatory synaptic input to mossy cells was increased in epileptic mice. A Representative miniature EPSC recordings of mossy cells from a control (top) and epileptic (bottom) mouse. Expanded views indicated by bars. B Frequency, amplitude, rise time (10–90%), decay time (τ), charge transfer per event, and charge transfer per second of mEPSCs. Values represent mean ± s.e.m. Number of cells per group indicated at base of bars. Frequency and charge transfer per second were increased in epileptic mice (*p<0.001, Mann-Whitney rank sum test). There were no significant differences in other parameters.