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Dentate gyrus network dysfunctions precede the symptomatic phase in a genetic mouse model of seizures.

Toader O, Forte N, Orlando M, Ferrea E, Raimondi A, Baldelli P, Benfenati F, Medrihan L - Front Cell Neurosci (2013)

Bottom Line: Neuronal circuit disturbances that lead to hyperexcitability in the cortico-hippocampal network are one of the landmarks of temporal lobe epilepsy.We made use of a high-resolution microelectrode array (4096 electrodes) and patch-clamp recordings, and found that in acute hippocampal slices of young pre-symptomatic (3-6 week-old) Syn II(-/-) mice excitatory synaptic output of the mossy fibers is reduced.Moreover, we showed that the main excitatory neurons present in the polymorphic layer of the DG, hilar mossy cells, display a reduced excitability.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Brain Technologies, Fondazione Istituto Italiano di Tecnologia Genoa, Italy ; International Max-Planck Research School for Neurosciences Göttingen, Germany.

ABSTRACT
Neuronal circuit disturbances that lead to hyperexcitability in the cortico-hippocampal network are one of the landmarks of temporal lobe epilepsy. The dentate gyrus (DG) network plays an important role in regulating the excitability of the entire hippocampus by filtering and integrating information received via the perforant path. Here, we investigated possible epileptogenic abnormalities in the function of the DG neuronal network in the Synapsin II (Syn II) knockout mouse (Syn II(-/-)), a genetic mouse model of epilepsy. Syn II is a presynaptic protein whose deletion in mice reproducibly leads to generalized seizures starting at the age of 2 months. We made use of a high-resolution microelectrode array (4096 electrodes) and patch-clamp recordings, and found that in acute hippocampal slices of young pre-symptomatic (3-6 week-old) Syn II(-/-) mice excitatory synaptic output of the mossy fibers is reduced. Moreover, we showed that the main excitatory neurons present in the polymorphic layer of the DG, hilar mossy cells, display a reduced excitability. We also provide evidence of a predominantly inhibitory regulatory output from mossy cells to granule cells, through feed-forward inhibition, and show that the excitatory-inhibitory ratio is increased in both pre-symptomatic and symptomatic Syn II(-/-) mice. These results support the key role of the hilar mossy neurons in maintaining the normal excitability of the hippocampal network and show that the late epileptic phenotype of the Syn II(-/-) mice is preceded by neuronal circuitry dysfunctions. Our data provide new insights into the mechanisms of epileptogenesis in the Syn II(-/-) mice and open the possibility for early diagnosis and therapeutic interventions.

No MeSH data available.


Related in: MedlinePlus

The inhibitory output of hilar mossy cells to granule cells is reduced in both pre-symptomatic and symptomatic Syn II−/− mice. (A) Scheme of the experimental setup. (B) Representative traces of an eEPSC (−80 mV, inward) and an eIPSC (0 mV, outward) recorded in the voltage-clamp configuration from the same pre-symptomatic Syn II−/− granule cell after the stimulation of the perforant path. (C,D) Aligned dot-plots representing the amplitude of the eEPSCs (C) or eIPSCs (D) from young WT (black), pre-symptomatic (red) and symptomatic (green) Syn II−/− mice. (E,F) Plots of the normalized mean amplitude of excitatory (E) or inhibitory (F) responses vs. time showing the multiple-pulse depression during a 2-s train at 40 Hz in young WT (black), pre-symptomatic (red) and symptomatic (green) Syn II−/− mice. (G) Mean (±s.e.m.) ratio between the amplitudes of eEPSCs and eIPSCs from the same granule neurons (young WT, black; pre-symptomatic Syn II−/−, red; symptomatic Syn II−/−, green) in the case of a single stimulus (left) or for the last 10 stimuli in a 40 Hz train (right); **p < 0.001, Kruskal–Wallis test followed by Dunn's multiple comparison test.
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Figure 7: The inhibitory output of hilar mossy cells to granule cells is reduced in both pre-symptomatic and symptomatic Syn II−/− mice. (A) Scheme of the experimental setup. (B) Representative traces of an eEPSC (−80 mV, inward) and an eIPSC (0 mV, outward) recorded in the voltage-clamp configuration from the same pre-symptomatic Syn II−/− granule cell after the stimulation of the perforant path. (C,D) Aligned dot-plots representing the amplitude of the eEPSCs (C) or eIPSCs (D) from young WT (black), pre-symptomatic (red) and symptomatic (green) Syn II−/− mice. (E,F) Plots of the normalized mean amplitude of excitatory (E) or inhibitory (F) responses vs. time showing the multiple-pulse depression during a 2-s train at 40 Hz in young WT (black), pre-symptomatic (red) and symptomatic (green) Syn II−/− mice. (G) Mean (±s.e.m.) ratio between the amplitudes of eEPSCs and eIPSCs from the same granule neurons (young WT, black; pre-symptomatic Syn II−/−, red; symptomatic Syn II−/−, green) in the case of a single stimulus (left) or for the last 10 stimuli in a 40 Hz train (right); **p < 0.001, Kruskal–Wallis test followed by Dunn's multiple comparison test.

Mentions: The axons of hilar mossy cells project into the inner molecular layer of the DG, where they make excitatory synapses directly with granule cells, or with GABA interneurons, leading to disynaptic inhibition of granule cells (Scharfman and Myers, 2012; Jinde et al., 2013). Since Syn II is abundantly and specifically expressed in the mossy cell terminals of the inner molecular layer (Figure 3), the next step was to evaluate the net effect of the mossy cell output on granule cell activity. To this aim, we patched granule neurons from the granule cell layer and stimulated the axons of mossy cells in the region below the granule layer (Figure 7A). When granule neurons are voltage-clamped at -80 mV, close to the reversal potential of Cl−, the stimulation should result in an evoked excitatory inward response non-contaminated by inhibition. On the contrary, when the clamped voltage is shifted to 0 mV, the Cl− drive will be predominant, and the stimulation should elicit a net inhibitory, outward response representing the fast-forward inhibition resulting from the intermediate activation of GABA interneurons (Figure 7B). Single stimulation did not reveal any difference between the amplitude of both eEPSC and eIPSCs in WT and pre-symptomatic and adult Syn II−/− slices (n = 16 neurons/6 mice for WT, 10 neurons/4 mice for young Syn II−/− and 13 neurons/3 mice for adult Syn II−/−; One-Way ANOVA followed by the Bonferroni's multiple comparison test, p = 0.344 and 0.751 for eEPSCs and eIPSC, respectively) (Figures 7C,D). Instead, the application of a 40 Hz tetanic stimulation revealed that depression was significantly increased at inhibitory synapses in both young and adult Syn II−/− granule neurons (Figure 7F), while it was similar between genotypes at excitatory synapses (Figure 7E). We quantified this effect by measuring the ratio between the evoked excitatory and inhibitory responses (E/I ratio) on the same granule cell. Single pulse stimulation of the mossy cell axon produced postsynaptic inhibitory and excitatory currents whose ratio was similar between genotypes (p = 0.938; One-Way ANOVA followed by the Bonferroni's multiple comparison test) (Figure 7G, left). However, when the E/I ratio was measured for the last 10 responses in the train, it was significantly increased in both pre-symptomatic and symptomatic Syn II−/− slices, with a decrease of inhibitory responses (E/I ratio = 0.3 ± 0.04 for WT, n = 6 neurons/3 mice; 2.6 ± 0.6 for young Syn II−/−, n = 7 neurons/4 mice; 1.8 ± 0.6 for adult Syn II−/−, n = 6 neurons/3 mice; p = 0.0011; Kruskal–Wallis test followed by the Dunn's multiple comparison test) (Figure 7G, right). This change in the ratio between excitation and inhibition may be responsible for the hyperexcitability of the DG under conditions of sustained high frequency synaptic input, as seen in Figure 2.


Dentate gyrus network dysfunctions precede the symptomatic phase in a genetic mouse model of seizures.

Toader O, Forte N, Orlando M, Ferrea E, Raimondi A, Baldelli P, Benfenati F, Medrihan L - Front Cell Neurosci (2013)

The inhibitory output of hilar mossy cells to granule cells is reduced in both pre-symptomatic and symptomatic Syn II−/− mice. (A) Scheme of the experimental setup. (B) Representative traces of an eEPSC (−80 mV, inward) and an eIPSC (0 mV, outward) recorded in the voltage-clamp configuration from the same pre-symptomatic Syn II−/− granule cell after the stimulation of the perforant path. (C,D) Aligned dot-plots representing the amplitude of the eEPSCs (C) or eIPSCs (D) from young WT (black), pre-symptomatic (red) and symptomatic (green) Syn II−/− mice. (E,F) Plots of the normalized mean amplitude of excitatory (E) or inhibitory (F) responses vs. time showing the multiple-pulse depression during a 2-s train at 40 Hz in young WT (black), pre-symptomatic (red) and symptomatic (green) Syn II−/− mice. (G) Mean (±s.e.m.) ratio between the amplitudes of eEPSCs and eIPSCs from the same granule neurons (young WT, black; pre-symptomatic Syn II−/−, red; symptomatic Syn II−/−, green) in the case of a single stimulus (left) or for the last 10 stimuli in a 40 Hz train (right); **p < 0.001, Kruskal–Wallis test followed by Dunn's multiple comparison test.
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Related In: Results  -  Collection

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Figure 7: The inhibitory output of hilar mossy cells to granule cells is reduced in both pre-symptomatic and symptomatic Syn II−/− mice. (A) Scheme of the experimental setup. (B) Representative traces of an eEPSC (−80 mV, inward) and an eIPSC (0 mV, outward) recorded in the voltage-clamp configuration from the same pre-symptomatic Syn II−/− granule cell after the stimulation of the perforant path. (C,D) Aligned dot-plots representing the amplitude of the eEPSCs (C) or eIPSCs (D) from young WT (black), pre-symptomatic (red) and symptomatic (green) Syn II−/− mice. (E,F) Plots of the normalized mean amplitude of excitatory (E) or inhibitory (F) responses vs. time showing the multiple-pulse depression during a 2-s train at 40 Hz in young WT (black), pre-symptomatic (red) and symptomatic (green) Syn II−/− mice. (G) Mean (±s.e.m.) ratio between the amplitudes of eEPSCs and eIPSCs from the same granule neurons (young WT, black; pre-symptomatic Syn II−/−, red; symptomatic Syn II−/−, green) in the case of a single stimulus (left) or for the last 10 stimuli in a 40 Hz train (right); **p < 0.001, Kruskal–Wallis test followed by Dunn's multiple comparison test.
Mentions: The axons of hilar mossy cells project into the inner molecular layer of the DG, where they make excitatory synapses directly with granule cells, or with GABA interneurons, leading to disynaptic inhibition of granule cells (Scharfman and Myers, 2012; Jinde et al., 2013). Since Syn II is abundantly and specifically expressed in the mossy cell terminals of the inner molecular layer (Figure 3), the next step was to evaluate the net effect of the mossy cell output on granule cell activity. To this aim, we patched granule neurons from the granule cell layer and stimulated the axons of mossy cells in the region below the granule layer (Figure 7A). When granule neurons are voltage-clamped at -80 mV, close to the reversal potential of Cl−, the stimulation should result in an evoked excitatory inward response non-contaminated by inhibition. On the contrary, when the clamped voltage is shifted to 0 mV, the Cl− drive will be predominant, and the stimulation should elicit a net inhibitory, outward response representing the fast-forward inhibition resulting from the intermediate activation of GABA interneurons (Figure 7B). Single stimulation did not reveal any difference between the amplitude of both eEPSC and eIPSCs in WT and pre-symptomatic and adult Syn II−/− slices (n = 16 neurons/6 mice for WT, 10 neurons/4 mice for young Syn II−/− and 13 neurons/3 mice for adult Syn II−/−; One-Way ANOVA followed by the Bonferroni's multiple comparison test, p = 0.344 and 0.751 for eEPSCs and eIPSC, respectively) (Figures 7C,D). Instead, the application of a 40 Hz tetanic stimulation revealed that depression was significantly increased at inhibitory synapses in both young and adult Syn II−/− granule neurons (Figure 7F), while it was similar between genotypes at excitatory synapses (Figure 7E). We quantified this effect by measuring the ratio between the evoked excitatory and inhibitory responses (E/I ratio) on the same granule cell. Single pulse stimulation of the mossy cell axon produced postsynaptic inhibitory and excitatory currents whose ratio was similar between genotypes (p = 0.938; One-Way ANOVA followed by the Bonferroni's multiple comparison test) (Figure 7G, left). However, when the E/I ratio was measured for the last 10 responses in the train, it was significantly increased in both pre-symptomatic and symptomatic Syn II−/− slices, with a decrease of inhibitory responses (E/I ratio = 0.3 ± 0.04 for WT, n = 6 neurons/3 mice; 2.6 ± 0.6 for young Syn II−/−, n = 7 neurons/4 mice; 1.8 ± 0.6 for adult Syn II−/−, n = 6 neurons/3 mice; p = 0.0011; Kruskal–Wallis test followed by the Dunn's multiple comparison test) (Figure 7G, right). This change in the ratio between excitation and inhibition may be responsible for the hyperexcitability of the DG under conditions of sustained high frequency synaptic input, as seen in Figure 2.

Bottom Line: Neuronal circuit disturbances that lead to hyperexcitability in the cortico-hippocampal network are one of the landmarks of temporal lobe epilepsy.We made use of a high-resolution microelectrode array (4096 electrodes) and patch-clamp recordings, and found that in acute hippocampal slices of young pre-symptomatic (3-6 week-old) Syn II(-/-) mice excitatory synaptic output of the mossy fibers is reduced.Moreover, we showed that the main excitatory neurons present in the polymorphic layer of the DG, hilar mossy cells, display a reduced excitability.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Brain Technologies, Fondazione Istituto Italiano di Tecnologia Genoa, Italy ; International Max-Planck Research School for Neurosciences Göttingen, Germany.

ABSTRACT
Neuronal circuit disturbances that lead to hyperexcitability in the cortico-hippocampal network are one of the landmarks of temporal lobe epilepsy. The dentate gyrus (DG) network plays an important role in regulating the excitability of the entire hippocampus by filtering and integrating information received via the perforant path. Here, we investigated possible epileptogenic abnormalities in the function of the DG neuronal network in the Synapsin II (Syn II) knockout mouse (Syn II(-/-)), a genetic mouse model of epilepsy. Syn II is a presynaptic protein whose deletion in mice reproducibly leads to generalized seizures starting at the age of 2 months. We made use of a high-resolution microelectrode array (4096 electrodes) and patch-clamp recordings, and found that in acute hippocampal slices of young pre-symptomatic (3-6 week-old) Syn II(-/-) mice excitatory synaptic output of the mossy fibers is reduced. Moreover, we showed that the main excitatory neurons present in the polymorphic layer of the DG, hilar mossy cells, display a reduced excitability. We also provide evidence of a predominantly inhibitory regulatory output from mossy cells to granule cells, through feed-forward inhibition, and show that the excitatory-inhibitory ratio is increased in both pre-symptomatic and symptomatic Syn II(-/-) mice. These results support the key role of the hilar mossy neurons in maintaining the normal excitability of the hippocampal network and show that the late epileptic phenotype of the Syn II(-/-) mice is preceded by neuronal circuitry dysfunctions. Our data provide new insights into the mechanisms of epileptogenesis in the Syn II(-/-) mice and open the possibility for early diagnosis and therapeutic interventions.

No MeSH data available.


Related in: MedlinePlus