Faculty Bibliography

Cannabidiol (CBD), a non-euphoric component of cannabis, reduces seizures in multiple forms of pediatric epilepsies, but the mechanism(s) of anti-seizure action remain unclear. In one leading model, CBD acts at glutamatergic axon terminals, blocking the pro-excitatory actions of an endogenous membrane phospholipid, lysophosphatidylinositol (LPI), at the G-protein-coupled receptor GPR55. However, the impact of LPI-GPR55 signaling at inhibitory synapses and in epileptogenesis remains underexplored. We found that LPI transiently increased hippocampal CA3-CA1 excitatory presynaptic release probability and evoked synaptic strength in WT mice, while attenuating inhibitory postsynaptic strength by decreasing GABA_ARγ₂ and gephyrin puncta. LPI effects at excitatory and inhibitory synapses were eliminated by CBD pre-treatment and absent after GPR55 deletion. Acute pentylenetrazole-induced seizures elevated GPR55 and LPI levels, and chronic lithium-pilocarpine-induced epileptogenesis potentiated LPI's pro-excitatory effects. We propose that CBD exerts potential anti-seizure effects by blocking LPI's synaptic effects and dampening hyperexcitability.

OBJECTIVE:To test the hypothesis that high frequency oscillations (HFOs) between 250 and 500Hz occur in mouse models of Alzheimer's disease (AD) and thus are not unique to epilepsy. METHODS:Experiments were conducted in three mouse models of AD: Tg2576 mice that simulate a form of familial AD, presenilin 2 knock-out (PS2KO) mice, and the Ts65Dn model of Down's syndrome. We recorded HFOs using wideband (0.1-500Hz, 2kHz) intra-hippocampal and cortical surface EEG at 1month until 24months-old during wakefulness, slow wave sleep (SWS) and rapid eye movement (REM) sleep. Interictal spikes (IIS) and seizures were also analyzed for the possible presence of HFOs. Comparisons were made to the intra-hippocampal kainic acid and pilocarpine models of epilepsy. RESULTS:We describe for the first time that hippocampal and cortical HFOs are a new EEG abnormality in AD mouse models. HFOs occurred in all transgenic mice but no controls. They were also detectable as early as 1month of age and prior to amyloid-β plaque neuropathology. HFOs were most frequent during SWS (vs. REM or wakefulness). Notably, HFOs in the AD and epilepsy models were indistinguishable in both spectral frequency and duration. HFOs also occurred during IIS and seizures in the AD models, although with altered spectral properties compared to isolated HFOs. SIGNIFICANCE/CONCLUSIONS:Our data demonstrate that HFOs, an epilepsy biomarker with high translational value, are not unique to epilepsy and thus not disease specific. Our findings also strengthen the idea of hyperexcitability in AD and its significant overlap with epilepsy. HFOs in AD mouse models may serve as an EEG biomarker which is detectable from the scalp and thus amenable to non-invasive detection in people at risk for AD.

Background: Alzheimer"™s disease (AD) is a neurodegenerative illness characterized by progressive accumulation of amyloid beta (Aβ) and neurofibrillary tangles, with cognitive impairment and altered neural activity. Hyperexcitability in the early stages of AD contribute to Aβ accumulation and cognitive impairment, aggravating the progression of AD. However, the hyperexcitability origin is not clear. This study aimed to test whether mossy cells (MCs), an excitatory cell of the hippocampal dentate gyrus, show increased excitability at early stages of AD and contribute to the increased network excitability generation. Indeed, alterations of MCs contribute to hyperexcitability and cognitive impairment in epilepsy. However, the role of MCs in AD has not been substantially explored. Methods: Intrinsic and synaptic properties of MCs and granule cells (GCs) from WT and Tg2576 mice at early ages (1-2 m.o.) were characterized by whole-cell patch-clamp recordings. Synaptic properties included the frequency and amplitude of spontaneous excitatory postsynaptic potentials (EPSPs) and excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs). Deterioration in MCs morphology was evaluated using Nissl staining and GluR2/3 labeling by light- and confocal microscopy. Aβ deposition was evaluated using the McSA1 antibody. Results: Tg2576 GCs did not have any significant difference in their intrinsic properties, as we shown previously in mice ∼3 m.o. However, an enhanced excitatory and inhibitory input to GCs, depicted by augmented IPSC (7.16 vs 14.04 events/s) and NMDA-mediated EPSC frequencies (0.81 vs 1.41 events/s) were found. Interestingly, Tg2576 MCs had an augmented EPSP frequency (5.75 vs 9.44 events/s), and their intrinsic properties showed a depolarized RMP (-72.88 vs -58.36 mV), and reduced rheobase (145.56 vs 47.14 pA), AP amplitude (98.14 vs 76.66 mV), time-to-peak (552.75 vs 266.16 ms) and maximum rise (171.44 vs 88.68 mV/ms) and decay slopes (-61.17 vs -42.38 mV/ms). The correlation between #APs and current injected showed Tg2576 MCs fired significantly more APs (SEZD = 0.34; z = 2.48). Tg2576 MCs showed robust intracellular Aβ aggregation without any significant morphological change. Conclusions: MCs changes in excitability and early accumulation of Aβ suggest that MCs could be the cause of increased excitability occurring later in GCs. In this manner, MCs could be an important contributor to AD.

The hippocampal CA2 region, an area important for social memory, has been suspected to play a role in temporal lobe epilepsy (TLE) because of its resistance to degeneration observed in neighboring CA1 and CA3 regions in both humans and rodent models of TLE. However, little is known about whether alterations in CA2 properties promote seizure generation or propagation. Here, we addressed the role of CA2 using the pilocarpine-induced status epilepticus model of TLE. Ex vivo electrophysiological recordings from acute hippocampal slices revealed a set of coordinated changes that enhance CA2 PC intrinsic excitability, reduce CA2 inhibitory input, and increase CA2 excitatory output to its major CA1 synaptic target. Moreover, selective chemogenetic silencing of CA2 pyramidal cells caused a significant decrease in the frequency of spontaneous seizures measured in vivo. These findings provide the first evidence that CA2 actively contributes to TLE seizure activity and may thus be a promising therapeutic target.

Intrahippocampal kainic acid (IHKA) has been widely implemented to simulate temporal lobe epilepsy (TLE), but evidence of robust seizures is usually limited. To resolve this problem, we slightly modified previous methods and show robust seizures are common and frequent in both male and female mice. We employed continuous wideband video-EEG monitoring from 4 recording sites to best demonstrate the seizures. We found many more convulsive seizures than most studies have reported. Mortality was low. Analysis of convulsive seizures at 2-4 and 10-12 wks post-IHKA showed a robust frequency (2-4 per day on average) and duration (typically 20-30 s) at each time. Comparison of the two timepoints showed that seizure burden became more severe in approximately 50% of the animals. We show that almost all convulsive seizures could be characterized as either low-voltage fast or hypersynchronous onset seizures, which has not been reported in a mouse model of epilepsy and is important because these seizure types are found in humans. In addition, we report that high frequency oscillations (>250 Hz) occur, resembling findings from IHKA in rats and TLE patients. Pathology in the hippocampus at the site of IHKA injection was similar to mesial temporal lobe sclerosis and reduced contralaterally. In summary, our methods produce a model of TLE in mice with robust convulsive seizures, and there is variable progression. HFOs are robust also, and seizures have onset patterns and pathology like human TLE. SIGNIFICANCE: Although the IHKA model has been widely used in mice for epilepsy research, there is variation in outcomes, with many studies showing few robust seizures long-term, especially convulsive seizures. We present an implementation of the IHKA model with frequent convulsive seizures that are robust, meaning they are >10 s and associated with complex high frequency rhythmic activity recorded from 2 hippocampal and 2 cortical sites. Seizure onset patterns usually matched the low-voltage fast and hypersynchronous seizures in TLE. Importantly, there is low mortality, and both sexes can be used. We believe our results will advance the ability to use the IHKA model of TLE in mice. The results also have important implications for our understanding of HFOs, progression, and other topics of broad interest to the epilepsy research community. Finally, the results have implications for preclinical drug screening because seizure frequency increased in approximately half of the mice after a 6 wk. interval, suggesting that the typical 2 wk. period for monitoring seizure frequency is insufficient.

Purpose: Although the intrahippocampal kainic acid (IHKA) model has been widely used to simulate temporal lobe epilepsy (TLE) in mice, there is variation in outcomes, with many studies showing few robust seizures long-term, especially convulsive seizures. We present an implementation of the IHKA model with frequent chronic convulsive seizures that are robust in frequency, duration and both sexes can be used.
Method(s): Our methods varied slightly from prior studies. We employed continuous wideband video-EEG from 2 cortical and 2 hippocampal sites to characterize chronic epilepsy outcomes in both sexes and 2 timepoints (2-4 and 10-12wks post-IHKA).
Result(s): Analysis of convulsive seizures at 2-4 and 10-12wks post-IHKA showed a robust frequency (2-4/day on average) and duration (typically 20-30 sec) at each time. Comparison of the 2 timepoints showed that seizure burden became more severe in approximately 50% of the animals. We show that almost all convulsive seizures could be characterized as either low-voltage fast or hypersynchronous onset seizures, which has not been reported in a mouse model of epilepsy and is important because these seizure types are found in humans. In addition, we report that high-frequency oscillations (HFOs, >250Hz) occur, resembling findings from IHKA in rats and TLE patients. Pathology in the hippocampus at the site of IHKA injection was similar to mesial temporal lobe sclerosis and reduced contralaterally.
Conclusion(s): In summary, our methods produce a model of TLE in mice with robust convulsive seizures, show variable progression, that HFOs are robust also, and that the model has seizures with onset patterns and pathology like human TLE. We believe our results will advance the ability to use the IHKA model of TLE in mice. The results also have important implications for our understanding of HFOs, progression and other topics of broad interest to the epilepsy research community including preclinical drug screening

The dentate gyrus not only gates the flow of information into the hippocampus, it also integrates and processes this information. Mossy cells (MCs) are a major type of excitatory neuron strategically located in the hilus of the dentate gyrus where they can contribute to this processing through networks of synapses with inhibitory neurons and dentate granule cells. Some prior work has suggested that MCs can form excitatory synapses with other MCs, but the role of these synapses in the network activity of the dentate gyrus has received little attention. Here, we investigated synaptic inputs to MCs in mouse hippocampal slices using a genetically encoded hybrid voltage sensor (hVOS) targeted to MCs by Cre-lox technology. This enabled optical recording of voltage changes from multiple MCs simultaneously. Stimulating granule cells and CA3 pyramidal cells activated well-established inputs to MCs and elicited synaptic responses as expected. However, the weak blockade of MC responses to granule cell layer stimulation by DCG-IV raised the possibility of another source of excitation. To evaluate synapses between MCs as this source, single MCs were stimulated focally. Stimulation of one MC above its action potential threshold evoked depolarizing responses in neighboring MCs that depended on glutamate receptors. Short latency responses of MCs to other MCs did not depend on release from granule cell axons. However, granule cells did contribute to the longer latency responses of MCs to stimulation of other MCs. Thus, MCs transmit their activity to other MCs both through direct synaptic coupling and through polysynaptic coupling with dentate granule cells. MC-MC synapses can redistribute information entering the dentate gyrus and thus shape and modulate the electrical activity underlying hippocampal functions such as navigation and memory, as well as excessive excitation during seizures.

Glutamatergic hilar mossy cells (MCs) have axons that terminate both near and far from their cell body but stay within the DG, making synapses primarily in the molecular layer. The long-range axons are considered the primary projection, and extend throughout the DG ipsilateral to the soma, and project to the contralateral DG. The specificity of MC axons for the inner molecular layer (IML) has been considered to be a key characteristic of the DG. In the present study, we made the surprising finding that dorsal MC axons are an exception to this rule. We used two mouse lines that allow for Cre-dependent viral labeling of MCs and their axons: dopamine receptor D2 (Drd2-Cre) and calcitonin receptor-like receptor (Crlr-Cre). A single viral injection into the dorsal DG to label dorsal MCs resulted in labeling of MC axons in both the IML and middle molecular layer (MML). Interestingly, this broad termination of dorsal MC axons occurred throughout the septotemporal DG. In contrast, long-range axons of ventral MCs terminated in the IML, consistent with the literature. Taken together, these results suggest that dorsal and ventral MCs differ significantly in their axonal projections. Since MC projections in the ML are thought to terminate primarily on GCs, the results suggest a dorsal-ventral difference in MC activation of GCs. The surprising difference in dorsal and ventral MC projections should therefore be considered when evaluating dorsal-ventral differences in DG function.

The dentate gyrus (DG) of the hippocampus is important for cognition and behavior. However, the circuits underlying these functions are unclear. DG mossy cells (MCs) are potentially important because of their excitatory synapses on the primary cell type, granule cells (GCs). However, MCs also activate GABAergic neurons which inhibit GCs. We used viral delivery of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in mice to implement a gain- and loss of function study of MCs in diverse behaviors. Using this approach, manipulations of MCs could bidirectionally regulate behavior. The results suggest that inhibiting MCs can reduce anxiety-like behavior and improve cognitive performance. However, not all cognitive or anxiety-related behaviors were influenced, suggesting specific roles of MCs in some but not all types of cognition and anxiety. Notably, several behaviors showed sex-specific effects, with females often showing more pronounced effects than the males. We also used the immediate early gene c-Fos to address whether DREADDs bidirectionally regulated MC or GC activity. We confirmed excitatory DREADDs increased MC c-Fos. However, there was no change in GC c-Fos, consistent with MC activation leading to GABAergic inhibition of GCs. In contrast, inhibitory DREADDs led to a large increase in GC c-Fos, consistent with a reduction in MC excitation of GABAergic neurons, and reduced inhibition of GCs. Taken together, these results suggest that MCs regulate anxiety and cognition in specific ways. We also raise the possibility that cognitive performance may be improved by reducing anxiety.SIGNIFICANCE STATEMENT: The dentate gyrus (DG) has many important cognitive roles as well as being associated with affective behavior. This study addressed how a glutamatergic DG cell type called mossy cells (MCs) contributes to diverse behaviors, which is timely because it is known that MCs regulate the activity of the primary DG cell type, granule cells (GCs), but how MC activity influences behavior is unclear. We show, surprisingly, that activating MCs can lead to adverse behavioral outcomes, and inhibiting MCs have an opposite effect. Importantly, the results appeared to be task-dependent and showed that testing both sexes was important. Additional experiments indicated what MC and GC circuitry was involved. Taken together, the results suggest how MCs influence behaviors that involve the DG.