ApoE4 is the main genetic risk factor for Alzheimer’s disease (AD) and causes dysfunction and death in inhibitory interneurons in humans and AD mouse models. In apoE4 knock-in mice (apoE4-KI), a model of late-onset AD, replacing lost inhibitory interneurons with GABAergic progenitors restores inhibition and rescues learning and memory behavior. Postsynaptic inhibitory transmission depends on receptors for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and on the equilibrium potential for chloride (ECl). To determine whether increasing postsynaptic inhibitory transmission – rather than replacing lost interneurons themselves – rescues learning and memory in apoE4-KI mice, we evaluated two strategies to increase postsynaptic inhibition. The diuretic drug bumetanide hyperpolarizes ECl by inhibiting the chloride importer NKCC1 in neurons. We show that aged apoE4-KI mice have increased expression of NKCC1 and that chronic treatment with bumetanide normalizes learning and memory behavior. We also show that increasing expression of GABAA receptor subunit δ in the hippocampus of aged apoE4-KI mice rescues cognitive flexibility and anxiety-like behavior, and ameliorates inhibitory interneuron losses. These results suggest that postsynaptic inhibition is an effective, druggable target for apoE4-related AD with potential disease-modifying effects.

The endocannabinoid (eCB) system is a complex network of proteins and ligands primarily found in the central nervous system. Though the eCB system is most notorious for producing psychotropic effects following consumption of the Cannabis sativa plant, it also modulates a wide range of physiological processes including neuronal development, neuroinflammation, anxiety, memory, appetite, lipid homeostasis, pain, and immunity. Harnessing the eCB system has been a successful therapeutic strategy for treating diseases, as in the cases of cannabidiol (CBD) for epilepsy treatment, or Cannabis as an anti-emetic for patients undergoing chemotherapy. Though there have been great strides in our understanding of the eCB system, unanswered questions remain in regards to its potential for additional therapeutic uses, toxicological considerations for Cannabis consumers, and more fundamentally, the mechanistic workings of eCB system signaling in the context of neural circuitry.

One approach to gain insights on the eCB system is through utilization of zebrafish (Danio rerio), a powerful model organism used in biological research. Proteins of interest can be easily targeted by both pharmacological agents and genetic alterations within zebrafish, allowing for examination of complex phenotypes resulting from desired perturbations. A diverse panel of zebrafish behavioral assays are available, allowing for examination of memory, addiction, sociability, aggression, and anxiety-like behaviors. Unlike mammalian models, development is relatively fast and breeding produces a large amount of progeny, allowing for quick generation progression and large sample sizes. Also, unlike mammals, development occurs externally and the brain is transparent, allowing for accessible and clear visualization of neurodevelopment, neuronal activity, and fluorescent markers of interest throughout the whole brain. These attributes make zebrafish an excellent model for studying the eCB system in vivo. In this dissertation, we explore the roles of several eCB proteins through utilization of genetic and pharmacological manipulation of the zebrafish model.

We start off in Chapter 1 with an in-depth examination of techniques in zebrafish that have paved the way for gaining insights on neurobiological mechanisms. The techniques we cover include forward and reverse genetic screens, and chemical screens, which have shed light on mechanisms of neuronal subtype differentiation and maintenance, habituation, candidate genes implicated in autism spectrum disorders, genes involved in electrical synapse formation, and regulators of sleep/wake states, to name a few examples.

Next, in Chapter 2, we introduce the known roles of the eCB system in vertebrates, with an emphasis on the zebrafish model. We describe each component of the eCB system, how each eCB protein and ligand work together to facilitate signaling cascades, and the involvement of the eCB system in addiction, development, anxiety, lipid homeostasis, appetite, immunity, and neuroinflammation. We examine spatial expression of two eCB genes, cb1 and cnrip1a, in developing zebrafish larvae, and quantitative expression of 18 eCB genes across 10 developmental stages and 11 organs in adult fish.

In the next two chapters, we use techniques described in Chapter 1 to target eCB genes described in Chapter 2. In Chapter 3, we utilize CRISPR-Cas9 gene editing to produce knockout zebrafish lines of cb1, dagla, daglb, abhd4, mgll, and faah. Following homology analysis, we determined that all 6 of these genes are conserved in zebrafish with amino acid sequence identity ranging from 45-70%. After sequencing each eCB knockout line, we found that all mutant alleles contained a frameshift mutation, suggesting a deleterious effect. We proceeded to phenotype dagla knockout fish and observed in increase in locomotor activity, as well as alterations in mRNA transcript of dagla, gpr55a, and fas. We next examine the effects of pharmacological agents that target CB1 (WIN55212-2, 2-arachadonoylglycerol [2-AG], anandamide [AEA]), MGLL (MJN110, JZL-184) and FAAH (PF-3845) in Chapter 4. Through the light-dark preference assay, we observed changes in dark avoidance (an anxiety-like behavior) and locomotor activity following eCB protein-targeting drug treatment. These genetic and pharmacological studies provide an excellent opportunity for gaining insight on individual gene involvement in behavior and physiological processes.

In Chapter 5, we focus on one gene, cb1, and further examine its role in anxiety; in particular, we aim to gain a greater understanding of the neuronal populations that are involved in facilitating eCB-mediated changes in anxiety behavior. Corroborating human and rodent studies, we determine that zebrafish have increased anxiety-like behavior following either pharmacological or genetic inhibition of CB1. By leveraging high resolution in situ techniques, we discovered region-specific colocalization of cb1 mRNA with pallial and hypothalamic glutamatergic and subpallial GABAergic neuronal markers. We produced a transgenic CB1 zebrafish line, allowing access to CB1-expressing cells and visualization of anatomical connectivity throughout the entire brain.

In conclusion, this dissertation reveals the behavioral effects of perturbing eCB genes, furthering our understanding of the roles of each eCB protein, and provides insight on the mechanism by which CB1 modulates anxiety-like behavior. From this work, 7 new eCB zebrafish lines were produced, allowing for further studies to uncover mechanistic roles of cb1, dagla, daglb, abhd4, mgll, and faah. Uncovering the roles of each eCB gene on biological processes not only demystifies this broadly expressed and highly conserved pathway, but also paves the way for insight on potential therapeutic uses and toxicological effects of eCB-targeting pharmacological agents.

The thalamus is usually regarded as the gateway sensory information must cross to reach the cortex, the higher cognitive processing plant of the brain. This gateway is guarded by the nucleus Reticularis Thalami (nRT). The nRT is a group of inhibitory neurons that through synaptic connections to the thalamic relay nuclei, temper and control the information stream from thalamus to cortex. The nRT is known to play important roles in sickness and in health. In Chapter 2, we deconstruct the nRT to its various cell types and study their role in health. Traditionally, the nRT was described as a relatively homogenous structure: containing mostly Parvalbumin (PV) expressing neurons that have Transient-type (T-type) Calcium currents that confer onto them a burst firing mode. Recent work has suggested that not all nRT neurons are created equal. There have been studies suggesting that nRT cells have different molecular, electrophysiological, and circuit properties. In other areas of the brain, many of these differences between inhibitory neurons can be correlated with expression of markers such as PV and Somatostatin (SOM). We found this to hold true in the nRT as well. PV-expressing neurons are the bursting cells that are preferentially connected to somatosensory circuits and primed to be involved in generating the physiological sleep-spindle and pathological seizure activities. SOM-expressing neurons are non-bursting cells preferentially connected to limbic circuits, and seem to be less involved in sleep-spindles and seizures. In Chapter 3, we explore the role of the nRT neurons in an animal model of a human chronic neuropsychiatric disorder. Dravet syndrome (DS) is an incurable form of childhood epilepsy associated with severe seizures, autism, sleep disorders, and sudden death. DS is brought about by a loss-of-function mutation in the sodium channel gene SCN1A. SCN1A is highly expressed in nRT neurons. We found that the bursting nRT neurons had prolonged post-hyperpolarization rebound bursting activity, caused by a decrease in the small conductance calcium-activated potassium (SK) current. This increased bursting in nRT neurons caused intra-thalamic circuit hyperexcitability, which may cause the epileptic events observed in these mice. Increasing SK current through the SK agonist EBIO allowed us to normalize the bursting activity in nRT cells, reduce the intra-thalamic circuit hyperexcitability, and consequentially, reduce the seizure activity. Understanding the guardians of the gateway in sickness and in health ultimately help us understand how the brain transfers and processes information.

Bioelectric signaling is evolutionarily conserved and maintained in all living cells through the selective expression of ion channels. In excitable cells like neurons, ion channels play an essential and well characterized role in defining the properties of action potentials. Less is known about their role in non-excitable cells such as neural stem cells and neural progenitor cells. Recent studies in mice have found that the loss of a voltage-gated potassium channel, Kv1.1, leads to a dramatic increase in hippocampal neurons. The hippocampus is critical for learning and memory, and it harbors one of two well-established neurogenic niches in the adult mouse brain. Here, we investigated the role of Kv1.1 in early postnatal and adult hippocampal neurogenesis. In Chapter 2, we first examined the role of Kv1.1 in early postnatal neurogenesis. We found that loss of Kv1.1 depolarizes the membrane potential of neural progenitor cells and increases the proliferation of neural progenitor cells through the TrkB signaling pathway. Next, we assessed the role of Kv1.1 in adult hippocampal neurogenesis in Chapter 3. We observed that conditional deletion of Kv1.1 in adult neural stem cells causes an initial increase in proliferation that eventually depletes the neural stem cell pool. Furthermore, neurons produced from adult neural stem cells lacking Kv1.1 had impaired maturation and positioning. These mice also displayed hippocampal-dependent learning and memory deficits. Together, these findings support an important role for this voltage-gated ion channel in adult neurogenesis. As aberrant neurogenesis has been implicated in cognitive decline associated with aging and neurological diseases, understanding the role of Kv1.1 in adult neurogenesis provides a framework for identifying new therapeutic targets to promote healthy neurogenesis and cognition.