Faculty Bibliography

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.

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.

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

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.

It has been reported that hyperexcitability occurs in a subset of patients with Alzheimer's disease (AD) and hyperexcitability could contribute to the disease. Several studies have suggested that the hippocampal dentate gyrus (DG) may be an important area where hyperexcitability occurs. Therefore, we tested the hypothesis that the principal DG cell type, granule cells (GCs), would exhibit changes at the single-cell level which would be consistent with hyperexcitability and might help explain it. We used the Tg2576 mouse, where it has been shown that hyperexcitability is robust at 2-3 months of age. GCs from 2 to 3-month-old Tg2576 mice were compared to age-matched wild type (WT) mice. Effects of muscarinic cholinergic antagonism were tested because previously we found that Tg2576 mice exhibited hyperexcitability in vivo that was reduced by the muscarinic cholinergic antagonist atropine, counter to the dogma that in AD one needs to boost cholinergic function. The results showed that GCs from Tg2576 mice exhibited increased frequency of spontaneous excitatory postsynaptic potentials/currents (sEPSP/Cs) and reduced frequency of spontaneous inhibitory synaptic events (sIPSCs) relative to WT, increasing the excitation:inhibition (E:I) ratio. There was an inward NMDA receptor-dependent current that we defined here as a novel synaptic current (nsC) in Tg2576 mice because it was very weak in WT mice. Intrinsic properties were distinct in Tg2576 GCs relative to WT. In summary, GCs of the Tg2576 mouse exhibit early electrophysiological alterations that are consistent with increased synaptic excitation, reduced inhibition, and muscarinic cholinergic dysregulation. The data support previous suggestions that the DG contributes to hyperexcitability and there is cholinergic dysfunction early in life in AD mouse models.

Angelman syndrome (AS), a genetic disorder that primarily affects the nervous system, is characterized by delayed development, intellectual disability, severe speech impairment, and problems with movement and balance (ataxia). Most affected children also have recurrent seizures (epilepsy). No existing therapies are capable of comprehensively treating the deficits in AS; hence, there is an urgent need to identify new treatments. Here we show that insulin-like growth factor 2 (IGF-2) and mannose-6-phosphate (M6P), ligands of two independent binding sites of the cation-independent M6P/IGF-2 receptor (CIM6P/IGF-2R), reverse most major deficits of AS modeled in mice. Subcutaneous injection of IGF-2 or M6P in mice modeling AS restored cognitive impairments as assessed by measurements of contextual and recognition memories, motor deficits assessed by rotarod and hindlimb clasping, and working memory/flexibility measured by Y-maze. IGF-2 also corrected deficits in marble burying and significantly attenuated acoustically induced seizures. An observational battery of tests confirmed that neither ligand changed basic functions including physical characteristics, general behavioral responses, and sensory reflexes, indicating that they are relatively safe. Our data provide strong preclinical evidence that targeting CIM6P/IGF-2R is a promising approach for developing novel therapeutics for AS. LAY SUMMARY: There is no effective treatment for the neurodevelopmental disorder Angelman syndrome (AS). Using a validated AS mouse model, the Ube3a^m-/p+ , in this study we show that systemic administration of ligands of the cation independent mannose-6-phosphate receptor, also known as insulin-like growth factor 2 receptor (CIM6P/IGF-2R) reverses cognitive impairment, motor deficits, as well as seizures associated with AS. Thus, ligands that activate the CIM6P/IGF-2R may represent novel, potential therapeutic targets for AS.

Spreading depolarization (SD) is a sudden, large, and synchronous depolarization of principal cells which also involves interneurons and astrocytes. It is followed by depression of neuronal activity, and it slowly propagates across brain regions like cortex or hippocampus. SD is considered to be mechanistically relevant to migraine, epilepsy, and traumatic brain injury (TBI), but there are many questions about its basic neurophysiology and spread. Research into SD in hippocampus using slices is often used to gain insight and SD is usually triggered by a focal stimulus with or without an altered extracellular buffer. Here, we optimize an in vitro experimental model allowing us to record SD without focal stimulation, which we call spontaneous. This method uses only an altered extracellular buffer containing 0 mM Mg²⁺ and 5 mM K⁺ and makes it possible for simultaneous patch and extracellular recording in a submerged chamber plus intrinsic optical imaging in slices of either sex. We also add methods for quantification and show the quantified optical signal is much more complex than imaging alone would suggest. In brief, acute hippocampal slices were prepared with a chamber holding a submerged slice but with flow of artificial cerebrospinal fluid (aCSF) above and below, which we call interface-like. As soon as slices were placed in the chamber, aCSF with 0 Mg²⁺/5 K⁺ was used. Most mouse slices developed SD and did so in the first hour of 0 Mg²⁺/5 K⁺ aCSF exposure. In addition, prolonged bursts we call seizure-like events (SLEs) occurred, and the interactions between SD and SLEs suggest potentially important relationships. Differences between rats and mice in different chambers are described. Regarding optical imaging, SD originated in CA3 and the pattern of spread to CA1 and the dentate gyrus was similar in some ways to prior studies but also showed interesting differences. In summary, the methods are easy to use, provide new opportunities to study SD, new insights, and are inexpensive. They support previous suggestions that SD is diverse, and also suggest that participation by the dentate gyrus merits greater attention.