Anxiety and Anxiety Related Disorders

Introduction

Anxiety. Anxiety is an evolutionarily adaptive response to danger that helps facilitate avoidance behavior, but it becomes maladaptive when it interferes with daily life[1]. Maladaptive anxiety is characterized by excessive and enduring fear and avoidance of threats (out of proportion to the threat or to nonexistent threats) [2]. Current scientific evidence indicates that anxiety arises from disruptions in the neural circuits used to process sensory stimuli and determine threat from those stimuli, leading to a state of high arousal and negative valence [3]. There are noticeable differences between fear and anxiety; fear responses are due to clear and perceived threats, with an immediate fight or flight response, and it subsides as the threat is removed. In comparison, anxiety responses are due to unknown threats, and they last longer than fear responses [4, 5].

Epidemiology. Anxiety related disorders are the most prevalent form of psychiatric illness in the world, and one of the leading causes of disability [6, 7]. One in four individuals is likely to have or be diagnosed with an anxiety disorder in their lifetime [8], and the 12 month prevalence of anxiety disorders in Europe is 14% [6]. Disability is exacerbated by a relatively early age of onset in comparison to other psychiatric disorders [1, 9], along with a high likelihood symptom recurrence [1, 9, 10]. Anxiety is highly comorbid with other mental disorders which can complicate treatment and diagnosis [9, 11]. Anxiety disorders can disrupt cognitive development and social rules, and can reduce quality of life by causing problems including school failure, underachievement, unemployment or underemployment, and social problems [2, 12].

Diagnosis of Anxiety. The diagnostic criteria for individual anxiety disorders varies across disorder and diagnostic manual used, either the DSM-5 or ICD-10. Genetic markers, blood tests, and psychophysiological testing lack the sensitivity or specificity to be used for the diagnosis of these disorders, so a trained physician must rely on the clinical interview, screening questionnaire, and subjective judgement [2, 4]. Distinguishing anxiety from other medical conditions is a major challenge to the proper diagnosis of anxiety, as presence of other mental or physical conditions are commonly associated with anxiety[13, 14].

Treatment of anxiety. Behavioral therapies are the first line treatment, but pharmacological treatment can be used as an alternative or adjunctive therapy[2, 15, 16]. Some of the pharmacological targets in anxiety include serotonergic, adrenergic, glutamatergic, neuropeptide, and endocannabinoid systems [15, 16]. Antidepressants, such as SSRIs or SNRIs, are the first line treatment for anxiety because of their efficacy, but they are associated with some adverse effects, including common side effects similar to symptoms of anxiety, discontinuation syndrome, and risk of increased suicidal ideation in youth [17].

Brain Regions Associated with Anxiety. Many regions, including amygdala (BLA and CeA), ventral hippocampus (vHPC), ventral tegmental area (VTA), medial dorsal thalamus (MdThal), nucleus accumbens (NAc), and several areas in the prefrontal cortex (cingulate cortex CingCx, prelimbic cortex PrL, infralimbic cortex IL) are important for anxiety[18-27].

Brain Region

Actions

Amygdala (BLA) identification and interpretation of threat as aversive or dangerous [3], positive or negative valence, predicts whether something will be threatening [28-30]

Amygdala (CeA and BNST) directly invigorate anxiety response, influence threat appraisal likelihood [31]
mPFC (Cingulate Cortex) regulator of anxiogenic activity: resistance to extinction of fear memories [32]
PrL PFC fear memory acquisition and freezing response to cues [33-35]
IL PFC Fear memory extinction [34, 36-40]
NaC and VTA adjusting motivation for appetitive behaviors: cost/benefit analysis, providing reward for behaviors, suppression of anxiety behaviors when benefits are estimated to outweigh costs [41-46]

Call to Action

In the field of psychiatry, multiple challenges affect diagnosis and treatment of anxiety disorders. Diagnosis and treatment are only sought between 3 to 30 years after onset of symptoms, varying by country, with the median globally being 21 years[47]. This delay can be due to a lack of mechanistic diagnostic tests [4, 48], patients presenting with somatic symptoms that are complications of anxiety[48], underuse of systematic application diagnostic criteria [49] and that symptoms overlap considerably among different diagnoses while varying significantly among patients with the same diagnosis [50]. Previous research has found anxiety to be partially heritable, but exact replicable loci have yet to be found [2, 51, 52]. Brain based taxonomy is lacking [53], and efforts to find new therapeutic targets have languished due to the lack of a mechanistic understanding of these disorders [3].

The goal for research in the future, then, is to identify neural substrates of anxiety and develop clinically relevant predictors. Utilizing and understanding the electrophysiology of pathological anxiety will allow for better diagnosis and treatment of anxiety disorders. Rodent models in particular hold promise in understanding the conserved circuits that underlie anxiety [3]

We postulated that a neural signature predicting anxiety related behavior exists at the network level. To test this hypothesis, we tested multiple forms of anxiogenic drug (Fluoxetine, FG-7142) in C57Bl/6J mice in the elevated plus maze (EPM), injected fluoxetine in these mice while in the homecage and conducted multi-circuit in vivo recordings from a subset of anxiety related regions including the prelimbic cortex (PrL_Cx), infralimbic cortex (IL_Cx), NAc, central nucleus of the amygdala (CeA), basolateral amygdala (BLA), VTA, and VHip, then used machine learning to elucidate the anxiety related signature.

Experimental Background

Electrophysiology. Local field potentials (LFPs) are measurements of pooled brain activity of neurons within 1 mm of an implanted electrode, their inputs, and their outputs [54]. They are neural responses to experimental events and are analyzed by decomposing the spectrum into frequency bands[55]. LFPs have been used to study mental illnesses, including anxiety [citation needed]. [Note: will be further completed next semester when LFP work is conducted]

Elevated Plus Maze as Measure of Mouse Anxiety. The elevated plus maze is a widely used behavioral assay for anxiety, that has been well validated across several measures. The elevated plus maze is assumed to be an ethological and unconditioned model that, unlike other anxiety tests, does not rely on noxious stimuli or conditioned responses [56-58]. Previously, the EPM has been used to assess drug effects, including anxiolytics and anxiogenics [59, 60], to determine brain site or mechanisms associated with fear and anxiety [61-67], as a measure indicative of altered emotionality in animals subjected to biochemical or gene manipulation [68-71] and subtypes of anxiety disorders [61]. Specifically, it has been used to measure the anxiogenic effects of FG-7142, and fluoxetine [69, 70, 72].
The maze has four arms, two open and two closed, all with open roofs. The arms are arranged in a plus shape with the same types of arms opposite each other [57, 60]. Anxiety is traditionally assessed by using the ratio of time spent on the open arms to time spent on the closed arms, and percentage of entries [56, 57, 69]. The aversive open arms are avoided, and freezing and defecation are increased in the open arms [59]. Behavior is typically recorded and scored for the first 5 minutes of the test, with time spent and entries made on the open and closed arms are measured, as well as avoidance behaviors and risk assessment [56, 61].

Open Field Test as Measure of Mouse Anxiety. The open field test is a simple measure of anxiety, ambulatory motion, and other behaviors. In measurement of anxiety, it is similar to the EPM in that it measures the innate avoidance of open spaces, which indicates anxiety. Mice who spend less time in the center of the open field test are coded as more anxious [73].

Beta Carboline FG-7142. The beta carboline class of molecules acts as an inverse agonists on the benzodiazepine site of GABA-A receptors[74]. Inverse agonists as a class are convulsant or pro-convulsant and have shown anxiogenic properties by self report [74]. FG-7142 (N-methyl-b-carboline-3-carboxamide) is a partial inverse agonist of benzodiazepine receptors that causes anxiety in both humans[75] and mice[69], has kindling effects [74], upregulates adrenoreceptors, and decreases subsequent actions of GABA and beta carboline agonists [72]. The molecule is not selective, but does have higher affinity for the ?±1 subunit-containing GABA-A receptor than other ?± subunits [75, 76] . FG-7142 has several effects on the brain, including activating of the basal forebrain cholinergic neurons, related to attention and vigilance, and activation of mesolimbocortical dopamine output to the PFC leading to increased sensory drive output from the BLA [72, 77-79]. While FG-7142 is a convulsant as well as an anxiogenic, the dose required for anxiogenic purposes is lower than that required for convulsive purposes, allowing for usefulness for experimental induction of anxiety sans convulsions [69]. In the elevated plus maze, FG-7142 decreases the amount of time spent in open arms [69, 72], and its anxiogenic action may be mediated by the 3 subunit of the GABA-A receptor [72].

Fluoxetine. In clinical populations, chronic SSRI treatment is useful for the treatment of anxiety and depression, but has been shown to paradoxically increase anxiety and depression upon acute treatment [80, 81]. The treatment lag and anxiogenic effect of SSRIs in the acute phase may contribute to treatment noncompliance[81]. In preclinical animal research populations, namely mice and rats, fluoxetine, an SSRI that acts as a selective 5-HT reuptake inhibitor [71, 82-84], has been shown to increase anxiety related behaviors in the EPM[85-87], open field test [71], light/dark test [71], the free exploration test [85] and will also potentiate defensive behaviors[84]. In C57Bl/6J mice specifically, fluoxetine has been shown to be anxiogenic in the EPM at a concentration of 20 mg/kg [70, 88].

Methods

Subjects

Subjects were adult male C57BL/6J mice, purchased from Jackson Labs. The mice were housed in groups of 5 per cage, housed in a humidity and temperature controlled environment, with a standard 12h light/dark cycle where lights are on at 7 am. Water and food were available ad libitum. In the testing of FG-7142 and fluoxetine in the EPM, mice weighed between 20-35 g and were 9 weeks at the time of Elevated Plus Maze testing. All mice were na??ve to the EPM. In the testing of fluoxetine in the home cage, mice were implanted at 9 weeks, and were tested at 11 weeks in the home cage. Studies were conducted with approved protocols from Duke University Institutional Animal Care and Use Committee and were in accordance with the NIH guidelines for the Care and Use of Laboratory Animals.

Drugs

FG-7142 was suspended in physiological (0.9%) saline containing 3% Tween 80 and injected i.p. at 10 mg/kg [69]. Fluoxetine HCl (Sigma-Aldrich, USA) was dissolved in physiological saline and injected i.p. at 20mg/kg [70].

Apparatus

The elevated plus maze apparatus has black plexiglass walls and flooring, with white tape on the floor of the arms. There are no walls on the open arms, but ?? cm of tape is on the edges to prevent mice from falling off the arms. The maze is mounted in a plexiglass pedestal on the floor and is 40 cm tall with 30x5.08 cm arms; walls on closed arms are 15.25 cm high.

General Procedure of the EPM

All experiments were conducted during the mouse light phase. Mice were habituated to handling and injection for 7 days prior to testing, with either saline for 5 days and vehicle for 2 days (FG-7142) or saline for 7 days (Fluoxetine). Treatment conditions were randomly assigned, and the experimenter was blind to treatment. Mice were tested in order of cage and mouse number. Light intensity was low light (50 lux) for all experiments. Mice were brought up to the testing room at least one hour before testing to allow for acclimation to the testing environment. After injection, mice were either placed in a new cage (FG-7142) or in the home cage (Fluoxetine) and allowed to acclimate for 30 minutes. In the elevated plus maze, mice were individually placed on the central square, facing a closed arm, and freely allowed to explore for 10 minutes (FG-7142) or 5 minutes (Fluoxetine). Between each mouse, the apparatus was cleaned with Rescue wipes (Virox, Oakville ON).

Fluoxetine Injections in Home Cage

Mice were handled, injected with saline, and habituated to single housing for 30 minutes after injection for 7 days prior to testing. On the day of testing, mice were habituated to the room for 60 minutes prior to testing, connected to a headstage without anesthesia (Blackrock Microsystems, UT, USA), recorded 5 minutes of baseline LFP activity, injected with fluoxetine, then placed in a separate cage and singly housed. Recordings were initiated immediately, data was used after 30 minutes. Experiment was conducted under low light (50 lux) conditions. LFP data was collected using Cerebus system (Blackrock Microsystems, Inc., Salt Lake City, UT), animals were video recorded using Neuromotive software (Blackrock Microsystems, Inc., Salt Lake City, UT) and analyzed for time spent standing still using Ethovision XT 7.1 software (Noldus Information Technology, Wageningen, Netherlands).

Open Field Test

Open field testing was conducted immediately after completion of home cage fluoxetine recordings 1 hour after fluoxetine injection. Mice were placed in a 17.5 in long x 17.5 inch wide x 11.75 in high chamber for 5 minutes. LFP data was collected using Cerebus system (Blackrock Microsystems, Inc., Salt Lake City, UT), location of animals was recorded using Neuromotive software (Blackrock Microsystems, Inc., Salt Lake City, UT) and analyzed for time spent in the center using Ethovision XT 7.1 software (Noldus Information Technology, Wageningen, Netherlands).

Neurophysiological data acquisition

Neurophysiological data was acquired at 30kHz using the Cerebus acquisition system (Blackrock Microsystems, UT, USA). LFPs were low pass filtered at 250 Hz, and stored at 1000 Hz. Voltage was measured by comparing data from each wire against a wire within the same brain area that had a SNR > 3:1.

Electrode implantation surgery

Mice were anesthetized with 1.5% isoflurane, placed in a stereotaxic device, and had screws implanted above the cerebellum and anterior cranium. Bundles were stereotaxically centered based on coordinates measured from bregma (AMY: -1.4mm and 40.86 AP, -2.9mm and 22.91 ML, -3.85 and 8.57 mm DV from the dura; NAc: 1.3 and 45.56 mm AP, -2.25 and 20.91 mm ML, -4.1 and 8.72 mm DV from the dura; PrL: 1.5 and 45.76 mm AP, 0 and 23.16mm ML, -2.25 and 10.57 mm DV from the dura; VTA: -3.4 and 40.86 mm AP, -.25 and 22.91 mm ML, -4.25 and 8.57 mm DV from the dura; VHip: -3.3 and 40.96 mm AP, -3 and 20.16 mm ML, -3.75 and 9.07 mm DV from the dura. Histology was performed on implantation sites after testing was complete to confirm recording sites used for analysis. Behavioral testing was performed when all animals had stable weights, at approximately 1.5 weeks after surgery.

Statistical Analyses

We ran the Shapiro Wilkes test for normality, the F test for equal variances, and two sample t tests for equal and unequal variances depending on the F test for equal variances.

LFPs

We used in vivo recording to quantify local field potentials (LFPs). LFP data was analyzed using non-negative matrix factorization, which is predictive of behavior and extrapolate across different subjects. It quantifies spectral power and coherence in certain frequency ranges to identify network level signatures.

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