Inhibitory inputs to hippocampal interneurons are reorganized in Lis1 mutant mice

Abstract

Epilepsy and brain malformation are commonly associated with excessive synaptic excitation and decreased synaptic inhibition of principal neurons. However, few studies have examined the state of synaptic inhibition of interneurons in an epileptic, malformed brain. We analyzed inhibitory inputs, mediated by gamma-aminobutyric acid (GABA), to hippocampal interneurons in a mouse model of type 1 lissencephaly, a neurological disorder linked with severe seizures and brain malformation. In the disorganized hippocampal area CA1 of Lis1(+/-) mice, we initially observed a selective displacement of fast-spiking, parvalbumin-positive basket-type interneurons from stratum oriens (SO) locations to s. radiatum and s. lacunosum-moleculare (R/LM). Next, we recorded spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) onto visually identified interneurons located in SO or R/LM of Lis1(+/-) mice and age-matched littermate controls. We observed significant, layer-specific reorganizations in GABAergic inhibition of interneurons in Lis1 mutant mice. Spontaneous IPSC frequency onto SO interneurons was significantly increased in hippocampal slices from Lis1(+/-) mice, whereas mIPSC mean amplitude onto these interneurons was significantly decreased. In addition, the weighted decay times of sIPSCs and mIPSCs were significantly increased in R/LM interneurons. Taken together, these findings illustrate the extensive redistribution and reorganization of inhibitory connections between interneurons that can take place in a malformed brain.

FIG. 1.

Parvalbumin-positive interneurons exhibit an altered distribution in Lis1 mutant mice. A, E, and I: hippocampal sections were divided into 400 × 50-μm bins (A, inset), and immunoreactive somata were counted in each bin (A, calretinin [CR]; E, parvalbumin [PV]; I, somatostatin [SOM]). Black bars indicate percentage of wild-type (WT) cells in a given bin; gray bars indicate Lis1^+/− cells. The overall distributions of CR and SOM cells appear normal, whereas PV cells are shifted from SO into stratum radiatum/s. lacunosum-moleculare (R/LM). (SO: P = 0.005; R/LM: P = 0.01, Student's t-test), with no change in the pyramidal cell layer (P). B, F, and J: bins are grouped together to represent hippocampal layers. C and D: WT and Lis1^+/− section, respectively, stained for CR and focused on area CA1. G and H: WT and Lis1^+/− section, respectively, stained for PV and focused on area CA1. K and L: WT and Lis1^+/− section, respectively, stained for SOM and focused on area CA1.

FIG. 2.

Lis1^+/− fast-spiking (FS) interneurons exhibit an altered distribution. A: sample trace from a WT regular-spiking nonpyramidal (RSNP) interneuron. B: Lis1^+/− RSNP interneuron. C: WT FS interneuron. D: Lis1^+/− FS interneuron. E: WT burst-spiking (BST) interneuron. F: Lis1^+/− BST interneuron. G: the relative proportions of each subtype encountered were similar between WT and Lis1^+/− animals. Black bars: WT cells. Gray bars: Lis1^+/− cells. H: the layer distribution of RSNP cells recorded was normal in mutant mice. I: the layer distribution of FS cells was abnormal in Lis1^+/− mice, with cells shifted from SO into R/LM (P < 0.001, χ² test).

FIG. 3.

Spontaneous inhibitory postsynaptic current (sIPSC) frequency is increased in Lis1^+/− SO interneurons. A, top trace: sIPSC recording from a WT SO interneuron. Bottom trace: current-clamp recording from the same interneuron. B, top trace: sIPSC recording from a Lis1^+/− SO interneuron showing an increased event frequency. Bottom trace: current-clamp recording from the same interneuron. C: frequency of sIPSCs on SO interneurons is significantly increased in Lis1^+/− mutant mice (P = 0.01, Student's t-test). D: amplitude of sIPSCs is comparable between WT and Lis1^+/− SO interneurons. E: decay time of sIPSCs is also unchanged in Lis1^+/− SO interneurons. Black bars: WT. Gray bars: Lis1^+/−.

FIG. 4.

Miniature (m)IPSC amplitude is decreased in Lis1^+/− SO interneurons. A, top trace: mIPSC recording from a WT SO interneuron. Bottom trace: current-clamp recording from the same interneuron. B, top trace: mIPSC recording from a Lis1^+/− SO interneuron showing decreased mean mIPSC amplitude. Bottom trace: current-clamp recording from the same interneuron. C: frequency of mIPSCs on SO interneurons is not significantly different in WT and Lis1^+/− mutant mice. D: amplitude of mIPSCs is significantly reduced in Lis1^+/− SO interneurons (P = 0.04, Student's t-test). E: amplitude histogram showing a leftward shift toward smaller mIPSC amplitudes in Lis1 mutant animals. F: decay time of mIPSCs is unchanged in Lis1^+/− SO interneurons. Black bars: WT. Gray bars: Lis1^+/−.

FIG. 5.

sIPSC weighted decay time is increased in Lis1^+/− R/LM interneurons. A, top trace: sIPSC recording from a WT R/LM interneuron. Bottom trace: current-clamp recording from the same interneuron. B, top trace: sIPSC recording from a Lis1^+/− R/LM interneuron. Bottom trace: current-clamp recording from the same interneuron. C: frequency of sIPSCs on R/LM interneurons not significantly different between WT and Lis1^+/− mutant mice. D: amplitude of sIPSCs is unchanged in Lis1^+/− R/LM interneurons. E: averaged sIPSC traces from representative WT (black) and mutant (gray) interneurons showing lengthened decay in Lis1 mutants. F: weighted decay time is significantly increased in Lis1 mutant R/LM interneurons (P = 0.03, Student's t-test). G: decay time histogram showing a trend toward longer decay events in Lis1^+/− R/LM interneurons. Black bars: WT. Gray bars: Lis1^+/−.

FIG. 6.

mIPSC weighted decay time is increased in Lis1^+/− R/LM interneurons. A, top trace: mIPSC recording from a WT R/LM interneuron. Bottom trace: current-clamp recording from the same interneuron. B, top trace: mIPSC recording from a Lis1^+/− R/LM interneuron. Bottom trace: current-clamp recording from the same interneuron. C: frequency of mIPSCs on R/LM interneurons not significantly different between WT and Lis1^+/− mutant mice. D: amplitude of mIPSCs is unchanged in Lis1^+/− R/LM interneurons. E: averaged mIPSC traces from representative WT (black) and mutant (gray) interneurons showing lengthened decay in Lis1 mutants. F: weighted decay time is significantly increased in Lis1 mutant R/LM interneurons (P = 0.01, Student's t-test). G: decay time histogram showing a shift toward longer decay events in Lis1^+/− R/LM interneurons. Black bars: WT. Gray bars: Lis1^+/−.