Neuroscience

Interneuron FGF13 regulates seizure susceptibility via a sodium channel-independent mechanism

Susan Lin
Aravind R. Gade
Hong-Gang Wang
James E. Niemeyer
Allison Galante
Isabella DiStefano
Patrick Towers
Jorge Nunez
Theodore H. Schwartz
Anjali M. Rajadhyaksha
Geoffrey S. Pitt author has email address

Cardiovascular Research Institute, Weill Cornell Medicine, New York, NY
Department of Neurological Surgery and Brain and Mind Research Institute, Weill Cornell Medicine of Cornell University, New York Presbyterian Hospital, New York, NY
Department of Pediatrics, Division of Pediatric Neurology, Weill Cornell Medicine, New York, NY; Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY

https://doi.org/10.7554/eLife.98661.1

Open access
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Figures and data

Whole brain knockout of Fgf13 results in premature death and seizure susceptibility.
A. Breeding scheme to generate Nestin^Fgf13 ^KO, Nestin^Fgf13 ^HET, and wildtype littermates. B. Western blot of whole brain Nestin^Fgf13 ^KO (KO) and wildtype (WT) littermates at P2 validates Fgf13 knockout. C. Fluorescent immunohistochemistry of hippocampal tissue validates Fgf13 knockout (scale bar, 100 μm). D. Survival curve of Nestin^Fgf13 ^KO mutant mice shows decreased survival at one month of age (log-rank test, ****, p< 0.0001). E. Body mass at P14 shows Nestin^Fgf13 ^KO are smaller in size (t-test, **, p< 0.01). F. Nestin^Fgf13 ^KOare susceptible to hyperthermia induced seizures (log-rank test, ****, p< 0.0001), unlike wildtype littermates.

Mutant mice are born in Mendelian ratios
Mutant mice were born in expected Mendelian ratios. Despite embryonic targeted deletion of Fgf13, mutant suffered no prenatal mortality related to developmental deficits.

Excitatory neuronal knockout of Fgf13 does not result in premature death and seizure susceptibility.
A. Western blot shows partial loss of Fgf13 from Emx1^Fgf13 ^KOhippocampal tissue, compared to full knockout in Nestin^Fgf13 ^KO hippocampus. B. Fluorescent immunohistochemistry of hippocampal tissue validates Fgf13 knockout (scale bar, 100 μm). C. Emx1^Fgf13 ^KO mutant mice survive past one month of age (log-rank test, p=ns). D. Body mass at postnatal day 14 (P14) shows that Emx1^Fgf13 ^KO are not different in size (scale bar, 2 cm) (t-test, p=ns). E. Emx1^Fgf13 ^KO are not susceptible to hyperthermia induced seizures (log-rank test, p=ns). F. EEG recordings during hyperthermia protocol show Emx1^Fgf13 ^KO do not exhibit heat-induced seizures. G. Emx1^Fgf13 ^KOneurons exhibit diminished long-term inactivation (two-way ANOVA, *, p< 0.05), though the deficit is not sufficient to cause seizures (WT, N=2, n = 15; KO, N=2, n = 15). Example traces for WT and Emx1^Fgf13 ^KO neurons are shown on the left.

Inhibitory neuronal knockout of Fgf13 recapitulates premature death and seizure susceptibility.
A. Quantification of hippocampal interneuron histology reveals 31% of Gad2⁺interneurons co-express FGF13. B. Fluorescent immunohistochemistry of hippocampal tissue validates Fgf13 knockout in sparse inhibitory interneurons (scale bar, 50 μm). C. Western blot validates partial loss of Fgf13 from Emx1^Fgf13 ^KO and Gad2^Fgf13 ^KO hippocampal tissue, and full knockout in Nestin^Fgf13 ^KO mice. D. Gad2^Fgf13 ^KO mutant mice have survival deficits around one month of age (log-rank test, ****, p< 0.0001). E. Body mass at P14 shows Gad2^Fgf13 ^KO are smaller in size than wildtype littermates (t-test, ****, p< 0.001; scale bar, 2 cm) and brains from Gad2^Fgf13 ^KO mice are smaller (t-test, ***, p< 0.001; scale bar, 0.5 cm). F. Gad2^Fgf13 ^KO mice are susceptible to hyperthermia induced seizures (log-rank test, ***, p< 0.001). G. Example EEG recording of wildtype and Gad2^Fgf13 ^KO mice during hyperthermia protocol. Insets show i.) example interictal spike, ii.) and iii.) tonic-clonic seizures.

Inhibitory neuronal knockout of Fgf13 results in deficits of synaptic transmission.
A. Example traces from spontaneous excitatory postsynaptic currents (sEPSCs). B. Cumulative fraction (Kolmogorov Smirnov test, ****, p<0.0001) frequency, and current amplitude for sEPSCs (t-test, ****, p<0.0001, WT N=7, n=24; KO N=4, n=22.) C. Example traces from spontaneous inhibitory postsynaptic currents (sIPSCs). D. Cumulative fraction (Kolmogorov Smirnov test, ****, p<0.0001), frequency, and current amplitude for sIPSCs (t-test, ****, p<0.0001, WT N=3, n=26; KO N=4, n=25).

Inhibitory neuronal knockout of Fgf13 results in deficits of interneuron excitability.
A. Examples of FGF13-stained neurons from primary hippocampal neuron cultures generated from Gad2-Cre wildtype (top) and Gad2^Fgf13 ^KO (bottom) mice (scale bar, 20 μm). B. Example traces of AP spike trains from wildtype (left) and Gad2^Fgf13 ^KO interneurons. Input-output curve shows decreased firing of evoked action potentials from Gad2^Fgf13 ^KO interneurons (two-way ANOVA, **, p< 0.01, WT N=4, n=28; KO N=4, n=26). C. Gad2^Fgf13 ^KO interneurons enter depolarization block at earlier current injections than wildtype interneurons (log-rank test, **, p< 0.01). D. Rheobase and threshold potential for cultured wildtype and Gad2^Fgf13 ^KO interneurons. E. Action potential wave forms and phase plots (mean ± s.e.m.) for the first elicited action potential of the spike train for wildtype and Gad2^Fgf13 ^KO interneurons. F. Action potential wave forms and phase plots for the second and third action potentials of the spike train of wildtype and Gad2^Fgf13 ^KO interneurons. G. Analysis of the first three action potentials in the spike train show a decrease in the dV/dt max, a decrease in action potential (AP) peak, a decrease in AP amplitude, and an increase in APD50 by the third AP of Gad2^Fgf13 ^KO interneurons (two-way ANOVA, *, p<0.05). H. Analysis of the first three action potentials in the spike train show an increase in membrane potential at the end of the AP for Gad2^Fgf13 ^KO interneurons (two-way ANOVA, **, p<0.01, ****, p<0.0001). I. Example traces of K⁺ currents from wildtype and Gad2^Fgf13 ^KOinterneurons (left). I-V curve for K⁺ currents (right), (two-way ANOVA, *, p<0.05, WT N=3, n = 26; KO N=5, n=29).

AAV-mediated expression of FGF13 isoforms rescues excitability deficits in Gad2^Fgf13 ^KO neurons.
A. Examples of FGF13-stained neurons from primary hippocampal neuron cultures generated from Gad2^Fgf13 ^KO mice transduced with AAV8-DIO-GFP only, AAV8-DIO-GFP and AAV8-DIO-Fgf13-S, or AAV8-DIO-GFP and AAV8-DIO-Fgf13-VY (scale bar, 20 μm). B. Gad2^Fgf13 ^KO neurons expressing FGF13-VY or FGF13-S were not different in terms of threshold potential or rheobase, and were not different from wildtype (black line, from Figure 5) and Gad2^Fgf13 ^KO neurons (red line, from Figure 5) (t-test, p=ns, KO+VY N=3, n=14; KO+S N=3 n=17). C. Evoked action potential traces from Gad2^Fgf13 ^KO interneurons expressing FGF13-VY or FGF13-S. Input-output curve shows increased firing of evoked action potentials from the FGF13-VY or FGF13-S expressing interneurons, relative to Gad2^Fgf13 ^KO interneurons (red line, from Figure 5; black line = wild type, from Figure 5) (two-way ANOVA, *, p<0.05). D. Gad2^Fgf13 ^KO interneurons expressing FGF13-VY and FGF13-S do not enter depolarization block as early as Gad2^Fgf13 ^KO interneurons (Red line [Gad2^FGF13 ^KO] and black line [wild type] are from Figure 5) (log-rank test, *, p<0.05). E. Action potential wave forms and phase plots for the initial three action potentials of the spike train for FGF13-VY and FGF13-S rescued Gad2^Fgf13 ^KOinterneurons. The black and red lines are from Figure 5. F. For the first three action potentials in the spike train, Gad2^Fgf13 ^KO neurons re-expressed with FGF13-VY show no difference from Gad2^Fgf13 ^KO neurons in terms of dV/dt max, AP peak, and AP amplitude, and AP50. Gad2^Fgf13 ^KO neurons re-expressed with FGF13-S show difference from Gad2^Fgf13 ^KO neurons only for the first action potential for AP peak and AP amplitude, but not dV/dt max or AP50. (two-way ANOVA, ***, p <0.001, KO+S vs. KO; **, p <.01, KO+S vs. KO). G. FGF13-S rescued neurons show a significant decrease in membrane voltage from Gad2^Fgf13 ^KO neurons by the third action potential in the spike train (two-way ANOVA, *, p <0.05, KO+S vs. KO). H. Example traces of K⁺ currents from Gad2^Fgf13 ^KO neurons expressing FGF13-VY and FGF13-S. K⁺ currents are rescued by expression of FGF13-S in Gad2^Fgf13 ^KOinterneurons (two-way ANOVA, *, p<0.05, KO+VY N=5, n=21, KO+S N=5, n=21).

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