Slide Session II: Animal Models

Optogenetic Stimulation of Cholinergic Brain Networks for Preventing Cortical Dysfunction during Seizures

Presenting Author: Moran Furman

Collaborating Authors: Joshua Motelow, Benjamin Lerner, Ilana Witten, Karl Deisseroth, Fahmeed Hyder, Jessica Cardin, Hal Blumenfeld

Ictal and post-ictal loss of consciousness is a major cause of disability, and currently there are no effective treatments for this debilitating side effect of epilepsy. Studies in humans and animal models have shown that during complex partial seizures, the cortex transitions into slow-wave oscillations (1-2Hz), resembling neocortical activity during deep sleep, anesthesia, and coma. Furthermore, this transition is mediated by suppression of subcortical arousal structures, including the brainstem pedunculopontine tegmental nucleus (PPT) and thalamic centro-lateral nucleus (CL), structures that are essential for maintaining the cortex in an alert and awake state. Thus, stimulating these subcortical arousal regions is a promising avenue for improving the level of consciousness during seizures.

We aim to prevent neocortical slow-wave activity in a rat model of complex partial seizures using optogenetic stimulation of subcortical arousal structures. Recent evidence from our lab shows that cholinergic PPT neurons, but not other cell types, are inhibited during limbic seizures. To selectively stimulate cholinergic neurons in the PPT, we performed stereotaxic viral injections into the PPT and confirmed expression of the light sensitive protein channelrhodopsin-2 (ChR2) specifically in PPT cholinergic neurons. Using acute electrophysiological recordings with optogenetic stimulation and in the future also fMRI imaging and behavioral testing, we will examine the effect of stimulating and inhibiting cholinergic subcortical pathways on neocortical function during limbic seizures. These experiments will advance our mechanistic understanding of impaired consciousness in epilepsy and hopefully lead to novel treatments for preventing ictal unconsciousness, particularly in cases when medications or surgical therapies are ineffective or unfavorable.

Optical Approaches to Unraveling the Function of Dendrite-Targeting GABAergic Interneurons

Presenting Author: Michael Higley

Collaborating Authors: Chiayu Chiu, Gyorgy Lur

GABAergic synaptic inhibition plays a critical but poorly understood role in shaping neuronal activity in the neocortex.  The disruption of GABAergic signaling is implicated in several neuropsychiatric disorders, including schizophrenia and autism. Hypotheses of inhibitory function have largely focused on perisomatic synapses, which control the magnitude and timing of action potential output from excitatory pyramidal neurons. However, the majority of GABAergic synapses in the neocortex are formed onto pyramidal cell dendrites, where their actions are less clear.  Here, using cell type-specific optical stimulation in combination with 2-photon Ca(2+) imaging, we show that dendritic inhibition mediated by somatostatin-expressing interneurons exerts highly compartmentalized control over postsynaptic calcium (Ca(2+)) signals within individual dendritic spines.  Anatomical and computational analyses indicate that this highly focal inhibitory action is mediated by a subset of GABAergic synapses that directly target spine heads.  Our results demonstrate that GABAergic inhibition participates in localized control of dendritic biochemical signaling. 

Neural Hyperactivity Disrupts Cerebral Microvascular Formation during a Postnatal Critical Period

Presenting Author: Christina Whiteus

Collaborating Authors: Catarina Freitas, Jaime Grutzendler

During the first postnatal month extensive synaptic and vascular remodeling occur in the mouse brain, however it is not known how these processes are coordinated. We investigated whether neural activity in the cortex influences the patterning of the vascular bed during this period. We found that increases in neuronal activity through audio and motor stimulation lead to reduced vascular branching in audio and motor cortices respectively, but had no effect in control areas. Pathological high neuronal activity induced by pilocarpine- and tetanus seizures caused an even more dramatic reduction in blood vessel branching. These branching deficits corresponded with a drop endothelial proliferation, though non-endothelial proliferation was unaffected. In vivo two-photon imaging revealed fewer vessel formations in animals experiencing seizures, however, we observed no increased elimination of either vessels or growing sprouts. The observed vascular deficits appear to be long-lasting as mice exposed to a 15 day auditory stimulation exhibit reduced vessel density up to three months later. Our results suggest the existence of a postnatal critical period during which inhibition of vascular growth in the brain might be detrimental later in life. Exposure of stimulated mice to low atmospheric oxygen reveals an increased sensitivity to hypoxia in brains with fewer vessels. This may have important clinical implications, especially during aging when vascular compensatory mechanisms are compromised by arteriosclerosis and stroke.

State-Dependent Inhibitory Control of Local Networks

Presenting Author: Jess Cardin

Collaborating Authors: Ulf Knoblich, Renata Batista-Brito, Mitra Miri

Brain activity is regulated by the interplay between two major types of neural cells: excitatory neurons that use the neurotransmitter glutamate and inhibitory interneurons that use the neurotransmitter GABA. Acute disruption of GABAergic inhibition profoundly alters brain activity patterns and leads to seizure, while constitutive changes in interneuron number and function are strongly linked to psychiatric disorders, such as schizophrenia and autism. In the cerebral cortex, which controls perception, cognition, and action, excitatory neurons receive a continuous barrage of synaptic inputs that must be integrated to produce output to target cells. Synaptic inhibition is thought to shape this integration and to provide a 'brake' on excitatory activity. Inhibition is thus hypothesized to be a critical regulator of healthy brain function and a key mediator of dysfunction in disease. However, little is known about the activity of inhibitory interneurons in the intact brain. Recent work has highlighted two major classes of hippocampal interneurons whose dysfunction is associated with seizure: 1) parvalbumin-expressing, fast-spiking interneurons that target the soma of excitatory neurons (PV) and 2) somatostatin-expressing interneurons that target the dendrites (SOM). These two populations are hypothesized to function differently in maintaining neural network stability. Until recently, it was not possible to target specific interneuron populations for either electrophysiological recording or perturbation of their activity in vivo. However, the recent development of transgenic optical tools for neural stimulation and suppression provides a novel approach for identification and direct manipulation of specific neural classes in vivo. In our current work, we are using a combined electrophysiological and optogenetic approach to record and manipulate the activity of genetically targeted PV and SOM interneurons under healthy conditions and in mouse models of disease.

Glutamine Synthetase and Astrocytes in the Pathophysiology of Localization-Related Epilepsy

Presenting Author: Tore Eid

Collaborating Authors: Ronnie Dhaher, Helen Wang, Nathan Tu, Hitten Zaveri, Benjamin Albright, Caroline Ong, Argyle Bumanglag, Eyiyemisi Damisah

Epilepsy is a common neurological disorder with a prevalence of approximately 1% in the general population. Up to one-third of individuals with epilepsy cannot control their seizures with current antiepileptic drugs, and the available drugs have side effects that limit their use. Uncontrolled seizures are often very disabling due to their unpredictable appearance and frequently associated features such as loss of consciousness, physical injury, and social stigmatization. Epilepsy is also associated with significant comorbidities including depression/suicide, cognitive impairment, and sudden unexpected death. Thus, our long-term goal is to facilitate the development of more efficacious and better tolerated treatments for epilepsy and its comorbidities. To attain this goal, the main scientific objective of the laboratory is to understand the cellular, molecular and metabolic mechanisms of temporal lobe epilepsy (TLE), which is one of the most common forms of medication refractory, localization related epilepsies. Our central hypothesis is that an initial brain insult (seizures, head trauma, intracranial infection) leads to proliferation of phenotypically abnormal ("reactive") astrocytes in the medial temporal lobe. The reactive astrocytes facilitate the development of recurrent seizures by perturbing the brain glutamine-glutamate-GABA homeostasis. We postulate that loss of glutamine synthetase in reactive astrocytes is a critical event in the pathophysiological process that leads to TLE.

Epileptiform Discharges in Transgenic Alzheimer's Mice Correlate with Impairments in Spatial Memory and are Reduced by Ethosuximide

Presenting Author: Haakon B. Nygaard

Collaborating Authors: Adam Kaufman, Linda L. Huh, Stephen M. Strittmatter

Background: Hyperexcitability and seizures have emerged as possible mechanisms underlying the neuronal dysfunction in Alzheimer's Disease (AD). Despite recent advances, it is not known to what extent seizures affect memory function or disease progression in AD, or whether reducing seizures could be an effective therapy in this disease.Here, we address these questions in detail utilizing rodent models of Alzheimer's disease.

Methods: APP-PSEN and 3xTg-AD mice were used, both of which harbor APPswe and PSEN1 transgenes, with the latter model also harboring the P301L tau mutation. All underwent continuous EEG monitoring for 72 hours. The APP-PSEN mice were tested in the Morris Water Maze, and their memory function correlated to the presence and severity of seizures. Subgroups with frequentseizures then underwent anticonvulsant therapy with Phenytoin, Ethosuximide, and Levetiracetam to assess effectiveseizure reduction. In addition, APP-PSEN mice lacking expression of cellular prion protein (PrPC) were also assessed for seizure reduction. The therapeutic effect of anticonvulsant therapy on the memory impairments in AD mice is now being tested.

Results: 40% ofaged APP-PSEN mice had at least 1 convulsive seizure over a 72 hour recording period, and approximately half had frequent non-convulsive spike-wave discharges (SWDs), lasting 1-2 seconds. Many SWDs were accompanied by a 1-3 second behavioral arrest.The presence of SWDs correlated with both learning and memory, with SWD-positive mice performing significantly worse in both the acquisition phase and the subsequent probe trial of the Morris Water Maze.Ethosuximide was most effective in reducing SWD frequency, with a complete resolution lasting 4 hours after dosing, with returnto baseline over 12 hours. Levetiracetam produced more than 50% reduction of SWD frequency, while phenytoin caused a slight increase in SWDs. The lack of PrPC expression completely reversed the SWD phenotype in APP-PSEN mice.

Conclusions: Our study is the firstto detail the correlation betweennon-convulsive SWDs and the behavioral impairments of AD mice. Our data suggest that the use ofanticonvulsants in the treatment of AD has therapeutic potential, either as an alternative or complimentary to current anti-amyloid interventions.