CHARACTERIZATION AND OPTIMIZATION OF MICROELECTRODE ARRAYS FOR GLUTAMATE MEASUREMENTS IN THE RAT HIPPOCAMPUS

An overarching goal of the Gerhardt laboratory is the development of an implantable neural device that allows for long-term glutamate recordings in the hippocampus. Proper L-glutamate regulation is essential for hippocampal function, while glutamate dysregulation is implicated in many neurodegenerative diseases. Direct evidence for subregional glutamate regulation is lacking in previous in vivo studies because of limitations in the spatio-temporal resolution of conventional experimental techniques. We used novel enzyme-coated microelectrode arrays (MEAs) for rapid measurements (2Hz) of extracellular glutamate in urethane-anesthetized rats. Potassium-evoked glutamate release was highest in the cornu ammonis 1 (CA1) subregion and lowest in the cornu ammonis 3 (CA3). In the dentate gyrus (DG), evoked-glutamate release was diminished at a higher potassium concentration but demonstrated faster release kinetics. These studies are the first to show subregion specific regulation of glutamate release in the hippocampus. To allow for in vivo glutamate measurements in awake rats, we have adapted our MEAs for chronic use. Resting glutamate measurements were obtained up to six days post-implantation but recordings were unreliable at later time points. To determine the cause(s) for recording failure, a detailed investigation of MEA surface characteristics was conducted. Scanning electron microscopy and atomic force microscopy showed that PT sites have unique surface chemistry, a microwell geometry and nanometer-sized features, all of which appear to be favorable for high sensitivity recordings. Accordingly, studies were initiated to improve enzyme coatings using a computer-controlled microprinting system (Microfab Technologies, Plano, TX). Preliminary testing showed that microprinting allowed greater control over the coating process and produced MEAs that met our performance criteria. Our final studies investigated the effects of chronic MEA implantation. Immunohistochemical analysis showed that the MEA produced minimal damage in the hippocampus at all time points from 1 day to 6 months. Additionally, tissue attachment to the MEA surface was minimal. Taken together with previous electrophysiology data supporting that MEAs are functional up to six months, these studies established that our chronic MEAs technology is capable of maintaining a brain-device interface that is both functional and biocompatible for extended periods of time

Mechanisms of amyloid-beta cytotoxicity in hippocampal network function : rescue strategies in Alzheimer's disease

The origin and nature of cognitive processes are strongly associated with synchronous rhythmic activity in the brain. Gamma oscillations that span the frequency range of 30–80 Hz are particularly important for sensory perception, attention, learning, and memory. These oscillations occur intrinsically in brain regions, such as the hippocampus, that are directly linked to memory and disease. It has been reported that gamma and other rhythms are impaired in brain disorders such as Alzheimer’s disease, Parkinson’s disease, and schizophrenia; however, little is known about how these oscillations are affected. In the studies contained in this thesis, we investigated a possible involvement of toxic Amyloid-beta (Aβ) peptide associated with Alzheimer’s disease in degradation of gamma oscillations and the underlying cellular mechanismsin rodent hippocampi. We also aimed to prevent possible Aβ- induced effects by using specially designed molecular tools known to reduce toxicity associated with Aβ by interfering with its folding and aggregation steps. Using electrophysiological techniques to study thelocal field potentials and cellular properties in the CA3 region of the hippocampus, we found that Aβ in physiological concentrations acutely degrades pharmacologically- induced hippocampal gamma oscillations in vitro in a concentration- and time- dependent manner. The severity of degradation also increased with the amount of fibrillar Aβ present. We report that the underlying cause of degradation of gamma oscillations is Aβ-induced desynchronization of action potentials in pyramidal neurons and a shift in the equilibrium of excitatory-inhibitory synaptic transmission. Using specially designed molecular tools such as Aβ-binding ligands and molecular chaperones, we provide evidence that Aβ-induced effects on gamma oscillations, cellular firing, and synaptic dynamics can be prevented. We also show unpublished data on Aβ effects on parvalbumin-positive baskets cells or fast-spiking interneurons, in which Aβ causes an increase in firing rate during gamma oscillations. This is similar to what is observed in neighboring pyramidal neurons, suggesting a general mechanism behind the effect of Aβ. The studies in this thesis provide a correlative link between Aβ-induced effects on excitatory and inhibitory neurons in the hippocampus and extracellular gamma oscillations, and identify the Aβ aggregation state responsible for its toxicity. We demonstrate that strategies aimed at preventing peptide aggregation are able to prevent the toxic effects of Aβ on neurons and gamma oscillations. The studies have the potential to contribute to the design of future therapeutic interventions that are aimed at preserving neuronal oscillations in the brain to achieve cognitive benefits for patients

Hippocampus and Human Disease

This chapter focuses on two disorders in which the role of the hippocampus has been extensively investigated: Alzheimer's disease and temporal lobe epilepsy. Although in Alzheimer's disease the disease eventually results in widespread destruction of the cerebral cortex, the damage in the earliest stages of disease is restricted to the entorhinal cortex and the hippocampus, and the memory impairment that results from this disruption of the hippocampal formation represents one of the common characteristics of early onset Alzheimer's disease. In temporal lobe epilepsy, the pathological damage is often restricted to the hippocampus in the form of hippocampal sclerosis. However, unlike Alzheimer's disease, in which the hippocampal damage is secondary to the underlying pathological process, the hippocampus in temporal lobe epilepsy is not only sensitive to damage by seizure activity but can also act as the substrate for epileptic seizure generation

Characterization of Ambra1 heterozygous mice as genetic mouse model of female-specific autism

Autism is known as a heritable neurodevelopmental disorder, diagnosed prior to the age of three years in humans based on three major domains: (1) impairment in social interaction (2) communication deficits (3) restricted interests and repetitive behaviors. Since it is a very heterogeneous disorder with various causes and different combinations of phenotypes, it is also called autism spectrum disorder (ASD). Monogenic heritable forms of ASD enable us to develop genetic mouse models of autism in order to obtain mechanistic insight in this disorder. Ambra1 is a positive regulator of Beclin1, a major player in the formation of autophagosomes during the process of autophagy. While Ambra1 null mutation leads to embryonic lethality, we could show that Ambra1 heterozygous mice (Ambra1+/-) display autism-like behavior only in females. Purpose of this thesis was therefore to characterize this mouse line further. It turned out that communication deficits, measured by ultrasound vocalization, start in the neonatal stage of females, while physical or neurological development is normal in Ambra1+/-. Female Ambra1 mutants had a stronger reduction in Ambra1 expression than male mutants, which gives first hints of the female-specific autism-like behavior in this mouse line. Mild enlargement of whole brain and hippocampus was detected in both Ambra1+/- males and females, with no change of ventricle size. Since β-galactosidase, used as reporter expressed under the Ambra1 promoter, was found only in neuronal cells, I focused on understanding the neural mechanism of its phenotype. Short-term and long-term synaptic plasticity in the hippocampus was normal for males and females of both genotypes. However, the power of gamma oscillations (γ-power), indicative of change in the balance of excitation and inhibition, was age-dependently altered in Ambra1+/- females only. However, this difference was not detected in male. Moreover, increased susceptibility to seizures, a known comorbid condition of ASD was restricted to females, suggesting an association between autism-like behavior, gamma oscillation and seizure propensity in female Ambra1+/- mice. Next, I approached the neuronal substrate of these three phenotypes by morphological analysis of hippocampal pyramidal neurons, such as dendritic arborization and synapse number. A genotype-associated difference of dendritic arborization was detected in neither males nor females. The quantification of spines or synapses and cellular electrophysiology are still on-going. First signals point to an imbalance between excitation and inhibition as a cause of the female autism-like behavior in Ambra1+/- mice

Characterization of Ambra1 heterozygous mice as genetic mouse model of female-specific autism

Autism is known as a heritable neurodevelopmental disorder, diagnosed prior to the age of three years in humans based on three major domains: (1) impairment in social interaction (2) communication deficits (3) restricted interests and repetitive behaviors. Since it is a very heterogeneous disorder with various causes and different combinations of phenotypes, it is also called autism spectrum disorder (ASD). Monogenic heritable forms of ASD enable us to develop genetic mouse models of autism in order to obtain mechanistic insight in this disorder. Ambra1 is a positive regulator of Beclin1, a major player in the formation of autophagosomes during the process of autophagy. While Ambra1 null mutation leads to embryonic lethality, we could show that Ambra1 heterozygous mice (Ambra1+/-) display autism-like behavior only in females. Purpose of this thesis was therefore to characterize this mouse line further. It turned out that communication deficits, measured by ultrasound vocalization, start in the neonatal stage of females, while physical or neurological development is normal in Ambra1+/-. Female Ambra1 mutants had a stronger reduction in Ambra1 expression than male mutants, which gives first hints of the female-specific autism-like behavior in this mouse line. Mild enlargement of whole brain and hippocampus was detected in both Ambra1+/- males and females, with no change of ventricle size. Since β-galactosidase, used as reporter expressed under the Ambra1 promoter, was found only in neuronal cells, I focused on understanding the neural mechanism of its phenotype. Short-term and long-term synaptic plasticity in the hippocampus was normal for males and females of both genotypes. However, the power of gamma oscillations (γ-power), indicative of change in the balance of excitation and inhibition, was age-dependently altered in Ambra1+/- females only. However, this difference was not detected in male. Moreover, increased susceptibility to seizures, a known comorbid condition of ASD was restricted to females, suggesting an association between autism-like behavior, gamma oscillation and seizure propensity in female Ambra1+/- mice. Next, I approached the neuronal substrate of these three phenotypes by morphological analysis of hippocampal pyramidal neurons, such as dendritic arborization and synapse number. A genotype-associated difference of dendritic arborization was detected in neither males nor females. The quantification of spines or synapses and cellular electrophysiology are still on-going. First signals point to an imbalance between excitation and inhibition as a cause of the female autism-like behavior in Ambra1+/- mice

The Association Between Elevated Hippocampal Glutamate Levels and Cognitive Deficits in Epilepsy

The purpose of this study was to investigate the association between extracellular basal hippocampal glutamate levels and cognitive function in epileptic patients. We used the zero-flow microdialysis method to measure the extracellular concentrations of glutamate in the epileptogenic and non-epileptogenic hippocampus of 23 awake epileptic patients during the interictal period. All patients underwent extensive neuropsychological testing to assess cognitive functioning prior to probe implantation. Basal glutamate levels in the epileptogenic hippocampus were significantly higher than the non-epileptogenic hippocampus (mean, 11.96 micromolar (µM) versus 2.92 µM, respectively). Elevated basal glutamate levels in the epileptogenic hippocampus correlated with decreased scores on the Verbal Selective Reminding Test (V-SRT) (R2 = 0.36, p = 0.0244). When controlling for MRI-detected hippocampal atrophy within epileptogenic regions, elevated basal glutamate levels within atrophic hippocampus correlated with decreased cognitive functioning measured by both the V-SRT (R2 = 0.7764, p = 0.0204) and Performance Intelligence Quotient (PIQ) (R2 = 0.7324, p = 0.0297), but not within non-atrophic hippocampus (V-SRT: R2 = 0.1013, p = 0.4424; PIQ: R2 = 0.2303, p = 0.2288). These data suggest that elevated basal glutamate levels in the epileptogenic hippocampus may be implicated in the pathogenesis of hippocampal atrophy and may contribute to impaired cognitive functioning involving verbal memory and visual-spatial skills in patients with temporal lobe epilepsy

Investigating the mechanisms of action of phytocannabinoids and a novel cognitive enhancer to target the comorbidity of temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is the most common type of epilepsy and exists with memory loss as a comorbidity. The conventional therapy available to treat these disorders achieves only modest therapeutic efficacy at best. This study investigates two potential treatments: phytocannabinoids to alleviate seizures, and a novel cognitive enhancer to restore/halt memory deficits. The anti-convulsant properties of cannabidiol (CBD) were first examined with regards to the neuropathology of two major types of hippocampal interneurons expressing parvalbumin (PV) and cholecystokinin (CCK) which are thought to dysfunction during epilepsy. Immunohistochemistry experiments using an in vivo kainic-acid induced epileptic rat model, revealed that PV- and CCK-immunopositive interneurons were significantly affected during epilepsy. This effect was greatly reduced following CBD treatment, suggesting that CBD exerts a neuroprotective function. The effects of CBD on the intrinsic membrane properties of these interneurons, together with hippocampal pyramidal cells, were further investigated in acute brain slices of rat seizure models of TLE (in vivo kainic acid-induced and in vitro Mg2+ free-induced). Whole-cell recordings revealed that bath application of CBD (10 µM) normalised the firing frequency of epileptic adapting pyramidal cells to healthy control levels. A similar effect was seen in hippocampal CCK-immunopositive Schaffer collateral associated (SCA) interneurons. In contrast, CBD resulted in an increased firing of PV-immunopositive interneurons, thus increasing their excitability and restoring the impaired membrane properties of the cells apparent in the epileptic models. The effects of cannabidivarin (CBDV), a similar cannabinoid compound, on the intrinsic membrane properties of these cell types were also evaluated. Additionally, CBDV affected excitatory postsynaptic currents by reducing excitation. In an attempt to address the memory impairment aspect associated with TLE, I investigated the neuronal effects of a5AM21, a novel potential memory enhancer. Electrophysiological experiments revealed that a5AM21 preferentially acts on 5-containing gamma (γ)-aminobutyric acid (GABA) type A (GABAA) receptors, reducing their inhibitory effects. Furthermore, data obtained using behavioural experiment paradigm, the eight-arm radial maze, suggest a significant improvement in short- and long-term memory retrieval in rats treated with a5AM21. In conclusion, the results reveal the potential mechanisms of action of two therapies to alleviate seizures and memory impairment, and the future goals would be to combine CBD/CBDV and a5AM21 as a promising novel targeted therapy for TLE

Underlying Mechanisms of Epilepsy

This book is a very provocative and interesting addition to the literature on Epilepsy. It offers a lot of appealing and stimulating work to offer food of thought to the readers from different disciplines. Around 5% of the total world population have seizures but only 0.9% is diagnosed with epilepsy, so it is very important to understand the differences between seizures and epilepsy, and also to identify the factors responsible for its etiology so as to have more effective therapeutic regime. In this book we have twenty chapters ranging from causes and underlying mechanisms to the treatment and side effects of epilepsy. This book contains a variety of chapters which will stimulate the readers to think about the complex interplay of epigenetics and epilepsy

Oscillatory and epileptiform activity in human and rodent cortical regions in vitro

Epilepsy is a chronic neurological disorder in which patients have spontaneous recurrent seizures. Approximately 50 million people worldwide live with epilepsy and of those ~30% fail to adequately respond to anti-epileptic drugs (AEDs), indicating a need for further research. In this study oscillatory and epileptiform activity was explored in the rodent piriform cortex (PC) in vitro, an underexplored brain region implicated in the development of epilepsy. PC gamma oscillations have been studied in both anaesthetised and awake rodents in vivo; however, to date they have not been reported in vitro. Extracellular field potential recordings were made in rodent PC brain slices prepared from 70-100g male Wistar rats in vitro. Application of kainic acid and carbachol reliably induced persistent gamma oscillations (30 – 40 Hz) in layer II of the PC. These oscillations were found to be pharmacologically similar to gamma oscillations previously found in other rodent brain regions in vitro, as they were dependent on GABAA receptors, AMPA receptors and gap junctions. Persistent oscillations were also induced and characterised for the first time in human neuronal tissue in vitro. Human brain slices were prepared from excised tissue from various brain regions (primarily temporal) from paediatric patients undergoing surgery to alleviate the symptoms of drug resistant epilepsy. As in the rodent PC, oscillations were induced by application of kainic acid and carbachol, however, these oscillations were found to be within the beta frequency range (12 – 30 Hz). Despite this difference in frequency band, these beta oscillations were pharmacologically similar to gamma oscillations found in the rodent PC. Seizure-like events (SLEs) were induced in brain slices prepared from 70-100g male Wistar rats via application of zero Mg2+ artificial cerebral spinal fluid (0[Mg]2+ aCSF). The properties of these SLEs were found to be similar between brain regions when recordings were performed in layer II of the anterior and posterior PC and lateral entorhinal cortex (LEC) and the stratum pyramidale of CA1. In the majority of recordings SLEs occurred in the PC before the LEC or CA1 and SLEs were displayed in the PC in a higher proportion of slices than the LEC. The sensitivity of these PC slices to 0[Mg]2+ aCSF was assessed at several stages (24 hours and 1 week (early latent), 4 weeks (mid latent) and 3 months+ (chronic period)) following the reduced intensity status epilepticus (SE) protocol for epilepsy induction compared to age-matched controls (AMCs). A decrease in excitability of the slices was observed in slices prepared from AMC animals with age, as the inter-event interval and latency to first SLE was observed to be longer in slices prepared from aged compared to young AMC animals. Slices prepared from SE animals maintained their youthful hyperexcitability with no difference in IEI or latency to first SLE observed in the early latent period compared to the chronic period. The pharmacoresistance (or sensitivity) of these SLEs to single and double AED challenge was evaluated. Differences in efficacy of the AEDs were found between SE and AMC in the mid-latent period; increased efficacy of Na+ channel modulating AEDs were found in slices prepared from SE compared to AMC animals. The proportion of slices that displayed pharmacoresistance of these SLEs to AEDs was found to be higher in slices prepared from young animals (early latent period and AMCs), and was similar to that found clinically in human patients. The pharmacoresistance of the SLEs to AEDs was lower in slices prepared from older animals (mid latent, chronic and AMCs) compared to young animals (early latent and AMCs). This age-dependent reduction in resistance likely reflects normal alterations in neuronal networks with ageing. SLEs induced in young control PC slices could be exploited as a new in vitro model of drug resistant epilepsy. Overall, oscillatory and epileptiform activity in the PC and human cortex in vitro could be further explored as tools to evaluate the efficacy and mechanism of action of newly developed AEDs, as well as to explore the networks involved in drug resistant epilepsy