Physiological and morphological characterization of dentate granule cells in the p35 knock-out mouse hippocampus: evidence for an epileptic circuit

Abstract

There is a high correlation between pediatric epilepsies and neuronal migration disorders. What remains unclear is whether there are intrinsic features of the individual dysplastic cells that give rise to heightened seizure susceptibility, or whether these dysplastic cells contribute to seizure activity by establishing abnormal circuits that alter the balance of inhibition and excitation. Mice lacking a functional p35 gene provide an ideal model in which to address these questions, because these knock-out animals not only exhibit aberrant neuronal migration but also demonstrate spontaneous seizures. Extracellular field recordings from hippocampal slices, characterizing the input-output relationship in the dentate, revealed little difference between wild-type and knock-out mice under both normal and elevated extracellular potassium conditions. However, in the presence of the GABA(A) antagonist bicuculline, p35 knock-out slices, but not wild-type slices, exhibited prolonged depolarizations in response to stimulation of the perforant path. There were no significant differences in the intrinsic properties of dentate granule cells (i.e., input resistance, time constant, action potential generation) from wild-type versus knock-out mice. However, antidromic activation (mossy fiber stimulation) evoked an excitatory synaptic response in over 65% of granule cells from p35 knock-out slices that was never observed in wild-type slices. Ultrastructural analyses identified morphological substrates for this aberrant excitation: recurrent axon collaterals, abnormal basal dendrites, and mossy fiber terminals forming synapses onto the spines of neighboring granule cells. These studies suggest that granule cells in p35 knock-out mice contribute to seizure activity by forming an abnormal excitatory feedback circuit.

Figure 1.

Nissl-stained sections of the dentate gyrus in wild-type (A) and p35-/- (knock-out) (B) mice show dispersion of GC somata into the molecular layer and into the hilus (black arrows). Timm-stained sections of the dentate gyrus from wild-type (C) and knock-out (D) mice illustrate the characteristic MF terminal invasion into the granular layer and IML (D; black arrows). Scale bars, 50 μm.

Figure 2.

Field recordings in slices from p35-/- mice show little increased dentate excitability when extracellular potassium concentration is increased from 3 to 6 mm. A, Top, Representative field responses to perforant path stimulation (gray arrows) (moderate stimulation intensity, 0.4 mA) in wild-type and knock-out slices in 3 mm extracellular potassium. A, Bottom, Graph plots the percentage of the maximum of the slope of the fEPSP versus the stimulus intensity (milliamperes), under control (3 mm) conditions. B, Top, Representative evoked field responses (stimulation intensity of 0.4 mA) in the presence of 6 mm extracellular potassium (overlaid on gray traces from A). B, Bottom, Graph plots the rise of the fEPSP slope as a percentage of the maximum response versus the stimulus intensity. Calibration: 0.5 mV (vertical), 5 msec (horizontal). The error bars in bottom graphs represent the SEM (n = 5 in each group).

Figure 3.

The GABA_A antagonist BMI unmasks hyperexcitability in p35-/- slices. A, Under controlconditions, stimulation of the perforant path (0.4 mA) elicits similar evoked responses in wild-type and knock-out animals. B, The addition of BMI increases the amplitude of the fEPSP and induces population spiking (black arrow) in the wild-type slices. An expansion of the early part of the response is shown beneath the slow time sweep; the bicuculline condition is superimposed over the control (gray) trace. In knock-out slices, the initial evoked response is similar to that seen under control conditions (only a small increase in the fEPSP amplitude); however, there is also a long-latency, long-duration, high-amplitude event (gray shading). Calibration: 0.5 mV (vertical), 25 msec (horizontal). C, Field potential amplitude under bicuculline conditions, measured at 150 msec after the stimulus, shows the consistent occurrence of the late discharge in p35-/- slices but not in wild-type slices.

Figure 4.

Intrinsic GC properties are similar in wild-type and knock-out dentate. A, A representative family of voltage responses to step current injections (from -0.5 to +0.5 nA; steps shown in inset) from a wild-type and a knock-out GC. Calibration: 10 mV (vertical), 25 msec (horizontal); inset, 0.2 nA (vertical), 25 msec (horizontal). B, Plots of the current-voltage relationship for the cells presented in A. C, Plots of the number of action potentials elicited at given current step amplitudes. Each point shows mean ± SEM for 20 cells in the wild-type and knock-out groups.

Figure 5.

A, Schematic shows the stimulation (in the stratum lucidum) and recording (in GC layer) configuration used in these experiments. B, Families of evoked responses, from a wild-type GC and a knock-out GC, illustrate commonly seen response patterns for these groups (19 wild-type GCs; 25 p35-/- GCs). DC current injection was used to vary the membrane potential of the GC during its response to MF stimulation in the CA3 stratum lucidum. Inversion of a short-latency component of the response, after the initial antidromic spike, was taken to reflect a GABA_A-mediated IPSP. This IPSP was absent (black arrow) in a significant proportion of p35-/- cells. Dashed lines represent the resting membrane potential of the cells; gray arrows identify the time of stimulation. Calibration: 5 mV (vertical), 25 msec (horizontal). C, Representative responses from dentate granule cells from knock-out slices, illustrating the variable patterns of evoked responses. D, Hypothetical schematic suggesting potential circuitry configurations that may explain the variability illustrated in C. 1, A cell with both an antidromic and an orthodromic action potential (6 of 25 cells); 2, a cell with no antidromic response but an orthodromically driven action potential (11 of 25 cells); 3, a cell with an antidromic response but no subsequent orthodromic response component (3 of 25 cells); 4, a cell with an antidromic action potential, followed by a relatively normal IPSP (5 of 25 cells). Calibration: 5 mV (vertical), 25 msec (horizontal).

Figure 6.

Light- and electron-microscopic analyses of an aberrant axon collateral, from a biocytin-labeled dentate GC in a p35 knock-out slice. A, Camera lucida drawing of the GC, showing a MF collateral (black arrows) projecting back into the GCL and molecular layer. Scale bar, 100 μm. B, Two-dimensional reconstruction (axon collateral identified in A, from serial section electron micrographs. Scale bar, 3 μm. The boxed regions of the reconstructed axon segment are magnified (C-F) and shown with representative electron micrographs (c-f) that illustrate the ultrastructural features of synaptic contacts (arrowheads) between the mossy fiber boutons (MFBs) and the postsynaptic spines (S) of presumed neighboring GCs (arrow identifies an axonal extension without a synapse). Three-dimensional reconstructions of two MF contacts (c2, d2) were implemented to demonstrate the tertiary structure of the axon and MFBs in relation to their postsynaptic contacts. Asterisks identify C-shaped synapses, and arrowhead in c2 points to a dendritic shaft synapse.

Figure 7.

Light- and electron-microscopic analyses of a GC basal dendrite projecting into the hilus. A, Camera lucida drawing of a p35-/- GC, intracellularly labeled with biocytin, and observed at the light-microscopic level. Black arrows identify an abnormal basal dendrite. B, Reconstructed basal dendrite segment, as identified in A. The schematic (b1) identifies those areas of the basal dendrite that contact MFBs (gray shaded boxes); the adjacent schematic (b2) shows the location of not only MFB contacts but also other dendritic spine and shaft synpases. Representative regions of the basal dendrite (B; boxed areas) are magnified to show two-dimensional reconstructions (C-F) and associated representative electron micrographs (c-f), including mossy fiber boutons (MFBs) synapsing onto the spine (S) or dendrite (D) of the biocytin-labeled GC. Black arrows in C-F identify synapses in adjacent electron micrographs (c-f). AT, Axon terminals. Scale bars, 500 nm.