Amanda A. Freeman

Effects of Interferon-Alpha on Rhesus Monkeys: A Nonhuman Primate Model of Cytokine-Induced Depression

BACKGROUND Interferon (IFN)-alpha is an innate immune cytokine that causes high rates of depression in humans and therefore has been used to study the impact of cytokines on the brain and behavior. To establish a nonhuman primate model of cytokine-induced depression, we examined the effects of IFN-alpha on rhesus monkeys. METHODS Eight rhesus monkeys were administered recombinant human (rHu)-IFN-alpha (20 MIU/m(2)) or saline for 4 weeks in counterbalanced fashion, and videotaped behavior, as well as plasma and cerebrospinal fluid (CSF), were obtained at regular intervals to assess behavioral, neuroendocrine, immune, and neurotransmitter parameters. Additionally, expression and activity of IFN-alpha/beta receptors in monkey peripheral blood mononuclear cells (PBMCs) were assessed. RESULTS Compared with saline treatment, IFN-alpha administration was associated with persistent increases in anxiety-like behaviors and decreases in environmental exploration. In addition, IFN-alpha induced significant increases in plasma concentrations of corticotropin (ACTH), cortisol, and interleukin-6 that tended to diminish after chronic administration, especially in dominant animals. Interestingly, in three animals, depressive-like, huddling behavior was observed. Monkeys that displayed huddling behavior exhibited significantly higher plasma concentrations of ACTH and lower CSF concentrations of the dopamine metabolite homovanillic acid. Rhesus monkey PBMCs were found to express mRNA and protein for the IFN-alpha/beta receptor. Moreover, treatment of PBMCs with rHu-IFN-alpha led to induction of STAT1, one of the primary IFN-alpha-induced signaling molecules. CONCLUSIONS IFN-alpha evoked behavioral, neuroendocrine, and immune responses in rhesus monkeys that are similar to humans. Moreover, alterations in hypothalamic-pituitary-adrenal axis responses and dopamine metabolism may contribute to IFN-alpha-induced depressive-like huddling behavior.

Nigrostriatal collaterals to thalamus degenerate in parkinsonian animal models.

Movement, cognition, emotion, and positive reinforcement are influenced by mesostriatal, mesocortical, and mesolimbic dopamine systems. Here, we describe a fourth major pathway originating from mesencephalic dopamine neurons: a mesothalamic system. The dopamine transporter, specific to dopamine containing axons, was histochemically visualized in thalamic motor and limbic‐related nuclei and regions that modulate behavioral state as opposed to sensory nuclei in rats, nonhuman primates, and humans. Anatomical tracing established this innervations origin via axon collaterals from the mesostriatal pathway. These findings implicate the thalamus as a novel site for disease specific alterations in dopamine neurotransmission, such as exist with nigral degeneration attending Parkinsons disease. This was confirmed in hemiparkinsonian animals where reduction of thalamic dopamine innervation occurred coincident with signs of active axonal degeneration. Individual mesencephalic dopamine neurons therefore have the potential to modulate normal and pathologic behavior not only through traditional nigrostriatal pathways but also by way of axon collaterals that innervate the thalamus.

Modulation of Vigilance in the Primary Hypersomnias by Endogenous Enhancement of GABAA Receptors

A component of cerebrospinal fluid in excessively sleepy people activates an inhibitory signaling pathway and mimics the actions of sedative-hypnotics. Awake and Refreshed A spindle prick on the finger, and Princess Aurora couldn’t keep her eyes open; one hundred years later, Sleeping Beauty was awakened with a kiss. But persistent daytime sleepiness—hypersomnolence—is no fairy tale, and neither the cause nor a cure is apparent. Now, Rye et al. begin to illuminate, in patients with primary hypersomnias, the neurobiology that underlies sleepiness of unknown etiology. A disabling condition, primary hypersomnia is characterized by lethargy, trance-like states, and “sleep drunkenness” even after prolonged, deep, nonrestorative sleep. The authors showed that cerebrospinal fluid (CSF) from these hypersomnolent subjects contains a small (500 to 3000 daltons) trypsin-sensitive substance that stimulates the in vitro function of selected γ-aminobutyric acid (GABA) receptors only in the presence of GABA—an inhibitory neurotransmitter that stimulates GABA receptors, quells consciousness, and induces sleep. GABA receptors are known to bind a class of psychoactive sedating drugs called benzodiazepines (BZDs). Hypersomnolent CSF samples mimicked the effects of BZD on GABA receptors but did not compete with BZD binding in human brain tissue, suggesting that the newly identified substance functions by a distinct mechanism. Furthermore, the BZD receptor antagonist flumazenil reversed hypersomnolent-CSF activation of GABA signaling, even though the drug is known to be a competitive antagonist of BZD and blocks BZD action by binding to the classical BZD-binding domain of GABA receptors. Most importantly, flumazenil restored vigilance in some hypersomnolent subjects. Together, these mechanistic studies pinpoint a potential new neuropharmacological pathway for a 25-year-old drug. The current study suggests that one of the “spindle pricks” that puts hypersomnolent subjects to sleep is a substance in CSF that augments inhibitory GABA signaling. A deeper understanding of the neurobiology of primary hypersomnia should help scientists discover new “kisses” that restore wakefulness—in fewer than 100 years. The biology underlying excessive daytime sleepiness (hypersomnolence) is incompletely understood. After excluding known causes of sleepiness in 32 hypersomnolent patients, we showed that, in the presence of 10 μM γ-aminobutyric acid (GABA), cerebrospinal fluid (CSF) from these subjects stimulated GABAA receptor function in vitro by 84.0 ± 40.7% (SD) relative to the 35.8 ± 7.5% (SD) stimulation obtained with CSF from control subjects (Student’s t test, t = 6.47, P < 0.0001); CSF alone had no effect on GABAA signaling. The bioactive CSF component had a mass of 500 to 3000 daltons and was neutralized by trypsin. Enhancement was greater for α2 subunit– versus α1 subunit–containing GABAA receptors and negligible for α4 subunit–containing ones. CSF samples from hypersomnolent patients also modestly enhanced benzodiazepine (BZD)–insensitive GABAA receptors and did not competitively displace BZDs from human brain tissue. Flumazenil—a drug that is generally believed to antagonize the sedative-hypnotic actions of BZDs only at the classical BZD-binding domain in GABAA receptors and to lack intrinsic activity—nevertheless reversed enhancement of GABAA signaling by hypersomnolent CSF in vitro. Furthermore, flumazenil normalized vigilance in seven hypersomnolent patients. We conclude that a naturally occurring substance in CSF augments inhibitory GABA signaling, thus revealing a new pathophysiology associated with excessive daytime sleepiness.

Chronic Interferon- α Decreases Dopamine 2 Receptor Binding and Striatal Dopamine Release in Association with Anhedonia-Like Behavior in Nonhuman Primates

Neuroimaging studies in humans have demonstrated that inflammatory cytokines target basal ganglia function and presynaptic dopamine (DA), leading to symptoms of depression. Cytokine-treated nonhuman primates also exhibit evidence of altered DA metabolism in association with depressive-like behaviors. To further examine cytokine effects on striatal DA function, eight rhesus monkeys (four male, four female) were administered interferon (IFN)-α (20 MIU/m2 s.c.) or saline for 4 weeks. In vivo microdialysis was used to investigate IFN-α effects on DA release in the striatum. In addition, positron emission tomography (PET) with [11C]raclopride was used to examine IFN-α-induced changes in DA2 receptor (D2R) binding potential before and after intravenous amphetamine administration. DA transporter binding was measured by PET using [18F]2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane. Anhedonia-like behavior (sucrose consumption) was assessed during saline and IFN-α administration. In vivo microdialysis demonstrated decreased release of DA after 4 weeks of IFN-α administration compared with saline. PET neuroimaging also revealed decreased DA release after 4 weeks of IFN-α as evidenced by reduced displacement of [11C]raclopride following amphetamine administration. In addition, 4 weeks of IFN-α was associated with decreased D2R binding but no change in the DA transporter. Sucrose consumption was reduced during IFN-α administration and was correlated with decreased DA release at 4 weeks as measured by in vivo microdialysis. Taken together, these findings indicate that chronic peripheral IFN-α exposure reduces striatal DA release in association with anhedonia-like behavior in nonhuman primates. Future studies examining the mechanisms of cytokine effects on DA release and potential therapeutic strategies to reverse these changes are warranted.

Improvement in daytime sleepiness with clarithromycin in patients with GABA-related hypersomnia: Clinical experience

The macrolide antibiotic clarithromycin can enhance central nervous system excitability, possibly by antagonism of GABA-A receptors. Enhancement of GABA signaling has recently been demonstrated in a significant proportion of patients with central nervous system hypersomnias, so we sought to determine whether clarithromycin might provide symptomatic benefit in these patients. We performed a retrospective review of all patients treated with clarithromycin for hypersomnia, in whom cerebrospinal fluid enhanced GABA-A receptor activity in vitro in excess of controls, excluding those with hypocretin deficiency or definite cataplexy. Subjective reports of benefit and objective measures of psychomotor vigilance were collected to assess clarithromycin’s effects. Clinical and demographic characteristics were compared in responders and non-responders. In total, 53 patients (38 women, mean age 35.2 (SD 12.8 years)) were prescribed clarithromycin. Of these, 34 (64%) reported improvement in daytime sleepiness, while 10 (19%) did not tolerate its side effects, and nine (17%) found it tolerable but without symptomatic benefit. In those who reported subjective benefit, objective corroboration of improved vigilance was evident on the psychomotor vigilance task. Twenty patients (38%) elected to continue clarithromycin therapy. Clarithromycin responders were significantly younger than non-responders. Clarithromycin may be useful in the treatment of hypersomnia associated with enhancement of GABA-A receptor function. Further evaluation of this novel therapy is needed.

Modeling the genetic basis for human sleep disorders in Drosophila

Sleep research in Drosophila is not only here to stay, but is making impressive strides towards helping us understand the biological basis for and the purpose of sleep—perhaps one of the most complex and enigmatic of behaviors. Thanks to over a decade of sleep-related studies in flies, more molecular methods are being applied than ever before towards understanding the genetic basis of sleep disorders. The advent of high-throughput technologies that can rapidly interrogate whole genomes, epigenomes and proteomes, has also revolutionized our ability to detect genetic variants that might be causal for a number of sleep disorders. In the coming years, mutational studies in model organisms such as Drosophila will need to be functionally connected to information being generated from these whole-genome approaches in humans. This will necessitate the development of appropriate methods for interpolating data and increased analytical power to synthesize useful network(s) of sleep regulatory pathways—including appropriate discriminatory and predictive capabilities. Ultimately, such networks will also need to be interpreted in the context of fundamental neurobiological substrates for sleep in any given species. In this review, we highlight some emerging approaches, such as network analysis and mathematical modeling of sleep distributions, which can be applied to contemporary sleep research as a first step to achieving these aims. These methodologies should favorably impact not only a mechanistic understanding of sleep, but also future pharmacological intervention strategies to manage and treat sleep disorders in humans.

Clarithromycin in γ-aminobutyric acid-Related hypersomnolence: A randomized, crossover trial.

Some central hypersomnolence syndromes are associated with a positive allosteric modulator of γ‐aminobutyric acid (GABA)‐A receptors in cerebrospinal fluid. Negative allosteric modulators of GABA‐A receptors, including clarithromycin, have been reported to reduce sleepiness in these patients. We sought to systematically assess the effects of clarithromycin on objective vigilance and subjective sleepiness.

Overexpression Screen in Drosophila Identifies Neuronal Roles of GSK-3β/shaggy as a Regulator of AP-1-Dependent Developmental Plasticity

AP-1, an immediate-early transcription factor comprising heterodimers of the Fos and Jun proteins, has been shown in several animal models, including Drosophila, to control neuronal development and plasticity. In spite of this important role, very little is known about additional proteins that regulate, cooperate with, or are downstream targets of AP-1 in neurons. Here, we outline results from an overexpression/misexpression screen in Drosophila to identify potential regulators of AP-1 function at third instar larval neuromuscular junction (NMJ) synapses. First, we utilize >4000 enhancer and promoter (EP) and EPgy2 lines to screen a large subset of Drosophila genes for their ability to modify an AP-1-dependent eye-growth phenotype. Of 303 initially identified genes, we use a set of selection criteria to arrive at 25 prioritized genes from the resulting collection of putative interactors. Of these, perturbations in 13 genes result in synaptic phenotypes. Finally, we show that one candidate, the GSK-3β-kinase homolog, shaggy, negatively influences AP-1-dependent synaptic growth, by modulating the Jun-N-terminal kinase pathway, and also regulates presynaptic neurotransmitter release at the larval neuromuscular junction. Other candidates identified in this screen provide a useful starting point to investigate genes that interact with AP-1 in vivo to regulate neuronal development and plasticity.

NFAT regulates pre-synaptic development and activity-dependent plasticity in Drosophila.

The calcium-regulated transcription factor NFAT is emerging as a key regulator of neuronal development and plasticity but precise cellular consequences of NFAT function remain poorly understood. Here, we report that the single Drosophila NFAT homolog is widely expressed in the nervous system including motor neurons and unexpectedly controls neural excitability. Likely due to this effect on excitability, NFAT regulates overall larval locomotion and both chronic and acute forms of activity-dependent plasticity at the larval glutamatergic neuro-muscular synapse. Specifically, NFAT-dependent synaptic phenotypes include changes in the number of pre-synaptic boutons, stable modifications in synaptic microtubule architecture and pre-synaptic transmitter release, while no evidence is found for synaptic retraction or alterations in the level of the synaptic cell adhesion molecule FasII. We propose that NFAT regulates pre-synaptic development and constrains long-term plasticity by dampening neuronal excitability.

The molecular basis of restless legs syndrome.

Restless legs syndrome (RLS) disrupts sleep in a substantial proportion of the population and is associated with higher cross-sectional rates of affective illness and cardiovascular disease. While dopamine and iron availability in the brain modulate emergence of symptoms, and dopamine agonists and iron alleviate the sensory symptoms and motor signs of RLS, the biology of the disorder is incompletely understood. Genetic factors, as opposed to environmental ones, account for most of the disease variance. The at-risk allelic variants exist in non-coding regions of at least six genes rendering it a complex genetic disease. Nonetheless, these provide the first hypothesis independent clues that advance a better understanding of RLS pathophysiology.