Orion P. Keifer

The Physiology of Fear: Reconceptualizing the Role of the Central Amygdala in Fear Learning.

The historically understood role of the central amygdala (CeA) in fear learning is to serve as a passive output station for processing and plasticity that occurs elsewhere in the brain. However, recent research has suggested that the CeA may play a more dynamic role in fear learning. In particular, there is growing evidence that the CeA is a site of plasticity and memory formation, and that its activity is subject to tight regulation. The following review examines the evidence for these three main roles of the CeA as they relate to fear learning. The classical role of the CeA as a routing station to fear effector brain structures like the periaqueductal gray, the lateral hypothalamus, and paraventricular nucleus of the hypothalamus will be briefly reviewed, but specific emphasis is placed on recent literature suggesting that the CeA 1) has an important role in the plasticity underlying fear learning, 2) is involved in regulation of other amygdala subnuclei, and 3) is itself regulated by intra- and extra-amygdalar input. Finally, we discuss the parallels of human and mouse CeA involvement in fear disorders and fear conditioning, respectively.

Voxel-based morphometry predicts shifts in dendritic spine density and morphology with auditory fear conditioning

Neuroimaging has provided compelling data about the brain. Yet the underlying mechanisms of many neuroimaging techniques have not been elucidated. Here we report a voxel-based morphometry (VBM) study of Thy1-YFP mice following auditory fear conditioning complemented by confocal microscopy analysis of cortical thickness, neuronal morphometric features and nuclei size/density. Significant VBM results included the nuclei of the amygdala, the insula and the auditory cortex. There were no significant VBM changes in a control brain area. Focusing on the auditory cortex, confocal analysis showed that fear conditioning led to a significantly increased density of shorter and wider dendritic spines, while there were no spine differences in the control area. Of all the morphology metrics studied, the spine density was the only one to show significant correlation with the VBM signal. These data demonstrate that learning-induced structural changes detected by VBM may be partially explained by increases in dendritic spine density.

A DTI Tractography analysis of Infralimbic and Prelimbic Connectivity in the Mouse using High-throughput MRI

BACKGROUND High throughput, brain-wide analysis of neural circuit connectivity is needed to understand brain function across species. Combining such tractography techniques with small animal models will allow more rapid integration of systems neuroscience with molecular genetic, behavioral, and cellular approaches. METHODS We collected DTI and T2 scans on 3 series of 6 fixed mouse brains ex vivo in a 9.4 Tesla magnet. The DTI analysis of ten mouse brains focused on comparing prelimbic (PL) and Infralimbic (IL) probabilistic tractography. To validate the DTI results a preliminary set of 24 additional mice were injected with BDA into the IL and PL. The DTI results and preliminary BDA results were also compared to previously published rat connectivity. RESULTS We focused our analyses on the connectivity of the mouse prelimbic (PL) vs. infralimbic (IL) cortices. We demonstrated that this DTI analysis is consistent across scanned mice, with prior analyses of rat IL/PL connectivity, and with mouse PL and IL projections using the BDA tracer. CONCLUSIONS High-throughput ex vivo DTI imaging in the mouse delineated both common and differential connectivity of the IL and PL cortex. The scanning methodology provided a balance of tissue contrast, signal-to-noise ratio, resolution and throughput. Our results are largely consistent with previously published anterograde staining techniques in rats, and the preliminary tracer study of the mouse IL and PL provided here.

Gene and protein therapies utilizing VEGF for ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that is usually fatal within 2-5years. Unfortunately, the only treatment currently available is riluzole, which has a limited efficacy. As a redress, there is an expanding literature focusing on other potential treatments. One such potential treatment option utilizes the vascular endothelial growth factor (VEGF) family, which includes factors that are primarily associated with angiogenesis but are now increasingly recognized to have neurotrophic effects. Reduced expression of a member of this family, VEGF-A, in mice results in neurodegeneration similar to that of ALS, while treatment of animal models of ALS with either VEGF-A gene therapy or VEGF-A protein has yielded positive therapeutic outcomes. These basic research findings raise the potential for a VEGF therapy to be translated to the clinic for the treatment of ALS. This review covers the VEGF family, its receptors and neurotrophic effects as well as VEGF therapy in animal models of ALS and advances towards clinical trials.

Angiotensin type 1a receptors on corticotropin-releasing factor neurons contribute to the expression of conditioned fear.

Although generally associated with cardiovascular regulation, angiotensin II receptor type 1a (AT1aR) blockade in mouse models and humans has also been associated with enhanced fear extinction and decreased post‐traumatic stress disorder (PTSD) symptom severity, respectively. The mechanisms mediating these effects remain unknown, but may involve alterations in the activities of corticotropin‐releasing factor (CRF)‐expressing cells, which are known to be involved in fear regulation. To test the hypothesis that AT1aR signaling in CRFergic neurons is involved in conditioned fear expression, we generated and characterized a conditional knockout mouse strain with a deletion of the AT1aR gene from its CRF‐releasing cells (CRF‐AT1aR(−/−)). These mice exhibit normal baseline heart rate, blood pressure, anxiety and locomotion, and freeze at normal levels during acquisition of auditory fear conditioning. However, CRF‐AT1aR(−/−) mice exhibit less freezing than wild‐type mice during tests of conditioned fear expression—an effect that may be caused by a decrease in the consolidation of fear memory. These results suggest that central AT1aR activity in CRF‐expressing cells plays a role in the expression of conditioned fear, and identify CRFergic cells as a population on which AT1R antagonists may act to modulate fear extinction.

Mapping of the Mouse Olfactory System with Manganese-Enhanced Magnetic Resonance Imaging and Diffusion Tensor Imaging

As the power of studying mouse genetics and behavior advances, research tools to examine systems level connectivity in the mouse are critically needed. In this study, we compared statistical mapping of the olfactory system in adult mice using manganese-enhanced MRI (MEMRI) and diffusion tensor imaging (DTI) with probabilistic tractography. The primary goal was to determine whether these complementary techniques can determine mouse olfactory bulb (OB) connectivity consistent with known anatomical connections. For MEMRI, 3D T1-weighted images were acquired before and after bilateral nasal administration of MnCl2 solution. Concomitantly, high-resolution diffusion-tensor images were obtained ex vivo from a second group of mice and processed with a probabilistic tractography algorithm originating in the OB. Incidence maps were created by co-registering and overlaying data from the two scan modalities. The resulting maps clearly show pathways between the OB and amygdala, piriform cortex, caudate putamen, and olfactory cortex in both the DTI and MEMRI techniques that are consistent with the known anatomical connections. These data demonstrate that MEMRI and DTI are complementary, high-resolution neuroimaging tools that can be applied to mouse genetic models of olfactory and limbic system connectivity.

Deep Brain Stimulation for Chronic Pain: Intracranial Targets, Clinical Outcomes, and Trial Design Considerations

For over half a century, neurosurgeons have attempted to treat pain from a diversity of causes using acute and chronic intracranial stimulation. Targets of stimulation have included the sensory thalamus, periventricular and periaqueductal gray, the septum, the internal capsule, the motor cortex, posterior hypothalamus, and more recently, the anterior cingulate cortex. The current work focuses on presenting and evaluating the evidence for the efficacy of these targets in a historical context while also highlighting the major challenges to having a double-blind placebo-controlled clinical trial. Considerations for pain research in general and use of intracranial targets specifically are included.

The challenges of developing a gene therapy for amyotrophic lateral sclerosis

Since Charcot’s description of the neuropathological characteristics of Amyotrophic Lateral Sclerosis (ALS) in 1869, our understanding of the disease has developed substantially. However, therapy development has lagged behind. Since ALS is linked to over 40 different mutations, gene therapy is of particular interest as an approach to attenuate disease progression [1]. ALS is broadly divided into familial cases (fALS) and sporadic cases (sALS) with respect to inheritance patterns and association with known mutations. Generally, the gene therapy strategy for sALS focuses on introducing neuroprotective agents like neurotrophic factors. The strategy for fALS focuses on addressing specific gene mutations [2,3]. On both fronts, preliminary research has shown progress, but successes are tempered by subsequent failures to scale up to translation. Here, we will discuss the challenges involved with developing a gene therapy for ALS and the concurrent potential solutions. Broadly, we will review the following areas: gene mutations, underlying pathogenesis, research models, vector selection, and surgical delivery paradigms. A major challenge in the development of a gene therapy for amyotrophic lateral sclerosis (ALS) is the continued idiopathic nature of the majority of cases. It is estimated that 10% of cases are familial ALS (fALS), of which 70% are linked to a known genetic lesion [4,5]. However, 90% of cases are sporadic ALS (sALS), of which 90% have an unidentified etiology [5]. In other words, of every 100 patients with ALS, only about 16 patients will have known genetic mutations. There are efforts to address this deficiency in knowledge of genetic etiology. For example, recent ALS associated mutations in the C9orf72 and NEK1 genes were discovered through the recruitment of large patient cohorts and advanced genetic analyses [6,7]. Given the rarity of ALS, discovery of genetic lesions will require large-scale collaborations across institutions and nations. One promising international initiative is Project MinE, which is collecting and analyzing 15,000 ALS patient samples to identify novel genetic lesions [8]. Projects like this will be the foundation of understanding the genetic component of ALS and therefore potential gene therapies, particularly for fALS. Downstream of the genetic lesions, our understanding of the underlying pathogenesis is an ongoing challenge to gene therapy development. In this domain, current knowledge is surprisingly limited. For example, we do not understand the function of the native protein that is impacted by the C9orf72 mutation [9]. Even the well characterized SOD1 mutation does not have a definitive mechanism beyond a toxic gain of function [1]. To cloud the field even further, there are as many proposed pathological mechanisms as there are mutations. These include, but are not limited to, glutamate excitotoxicity, glial cell dysfunction, protein/RNA toxicity, and mitochondrial/ cytoskeletal dysfunction [1]. While the reality of the disease pathogenesis may be multifactorial, the paucity of a clear mechanism limits therapeutic development progress. This knowledge gap is only addressable through experimental work with in vitro and animal models. One historical success has been SOD1 small-animal models. These models have provided a space to draw the crucial understanding of a toxic gain of function in SOD1-associated ALS. The result is that many strategies have focused on mitigating effects related to the SOD1 gain of function. Indeed, there have been successes in SOD1 gene-silencing approaches (e.g. various adenoassociated virus (AAV), siRNA, and shRNA constructs, and antisense oligonucleotides), showing delayed disease onset and extension of life-span [5]. For example, the use of viral vectors (particularly AAV9) with silencing constructs have shown delayed disease progression and improved survival in SOD1 mouse models [10,11]. Further, to address the proposed excito-oxidative stress mechanism, other groups have added a combination of neuroprotective and neurotrophic transgenes (e.g. EAAT2, GDH2, and NRF2) leading to improvements in survival, body weight and neurological findings [12]. While promising, it is not clear if these strategies will succeed outside of SOD1 mutants, meaning, at best, they would treat only 1% of ALS patients. In order to further address this challenge of underlying pathophysiology and the other 99% of patients, additional research models must be generated. Directly addressing these concerns, there have been strides in both the use of in vitro models and efforts to develop other genespecific murine models (e.g. C9orf72 and NEK1) [3,13]. However, one great difficulty is that the symptomology and histology of ALS are not necessarily recapitulated in these models, meaning refinements are necessary [3]. A critical translational gap is that small-animal and in vitro models do provide a space for proof-of-principle, but have not adequately paralleled human disease phenotypes [3,14]. The