Matthew E. Carter

Tuning arousal with optogenetic modulation of locus coeruleus neurons

Neural activity in the noradrenergic locus coeruleus correlates with periods of wakefulness and arousal. However, it is unclear whether tonic or phasic activity in these neurons is necessary or sufficient to induce transitions between behavioral states and to promote long-term arousal. Using optogenetic tools in mice, we found that there is a frequency-dependent, causal relationship among locus coeruleus firing, cortical activity, sleep-to-wake transitions and general locomotor arousal. We also found that sustained, high-frequency stimulation of the locus coeruleus at frequencies of 5 Hz and above caused reversible behavioral arrests. These results suggest that the locus coeruleus is finely tuned to regulate organismal arousal and that bursts of noradrenergic overexcitation cause behavioral attacks that resemble those seen in people with neuropsychiatric disorders.

FOXO transcription factors

What are they? FOXO proteins are a subgroup of the Forkhead family of transcription factors. This family is characterized by a conserved DNA-binding domain (the ‘Forkhead box’, or FOX) and comprises more than 100 members in humans, classified from FOXA to FOXR on the basis of sequence similarity. These proteins participate in very diverse functions: for example, FOXE3 is necessary for proper eye development, while FOXP2 plays a role in language acquisition. Members of class ‘O’ share the characteristic of being regulated by the insulin/PI3K/Akt signaling pathway.How did this family get named ‘Forkhead’? Forkhead, the founding member of the entire family (now classified as FOXA), was originally identified in Drosophila as a gene whose mutation resulted in ectopic head structures that looked like a fork. Forkhead proteins are also sometimes referred to as ‘winged helix’ proteins because X-ray crystallography revealed that the DNA-binding domain features a 3D structure with three α-helices flanked by two characteristic loops that resemble butterfly wings.How many FOXOs are there? In invertebrates, there is only one FOXO gene, termed daf-16 in the worm and dFOXO in the fly. In mammals, there are four FOXO genes, FOXO1, 3, 4, and 6.Hey, what about FOXO2 and FOXO5?FOXO2 is identical to FOXO3 (a.k.a. FOXO3a, as opposed to FOXO3b, a pseudogene). FOXO5 is the fish ortholog of FOXO3.FOX hunting…FOXO genes were first identified in humans because three family members (1, 3, and 4) were found at chromosomal translocations in rhabdomyosarcomas and acute myeloid leukemias. Just after FOXO factors were identified in human tumor cells, the crucial role of DAF-16 in organismal longevity was discovered in worms. DAF-16 activity was shown to be negatively regulated by the insulin/PI3K/Akt signaling pathway. Subsequent experiments in mammalian cells showed that mammalian FOXO proteins were directly phosphorylated and inhibited by Akt in response to insulin/growth factor stimulation. Thus, FOXO factors are evolutionarily conserved mediators of insulin and growth factor signaling.Why are they important? FOXO transcription factors are at the interface of crucial cellular processes, orchestrating programs of gene expression that regulate apoptosis, cell-cycle progression, and oxidative-stress resistance (Figure 1Figure 1). For example, FOXO factors can initiate apoptosis by activating transcription of FasL, the ligand for the Fas-dependent cell-death pathway, and by activating the pro-apoptotic Bcl-2 family member Bim. Alternatively, FOXO factors can promote cell-cycle arrest; for example, FOXO factors upregulate the cell-cycle inhibitor p27kip1 to induce G1 arrest or GADD45 to induce G2 arrest. FOXO factors are also involved in stress resistance via upregulation of catalase and MnSOD, two enzymes involved in the detoxification of reactive oxygen species. Additionally, FOXO factors facilitate the repair of damaged DNA by upregulating genes, such as GADD45 and DDB1. Other FOXO target genes have been shown to play a role in glucose metabolism, cellular differentiation, muscle atrophy, and even energy homeostasis.Figure 1In the absence of insulin or growth factors, FOXO transcription factors are located in the nucleus, where they specify target gene expression (see text for details).View Large Image | View Hi-Res Image | Download PowerPoint SlideHow are they regulated? FOXO proteins are tightly regulated to ensure that transcription of specific target genes is responsive to environmental conditions. A major form of regulation is Akt-mediated phosphorylation of FOXO in response to insulin or growth factors (Figure 1Figure 1). Phosphorylation at three conserved residues results in the export of FOXO factors from the nucleus to the cytoplasm, thereby inhibiting FOXO-dependent transcription. FOXO proteins are also phosphorylated by other protein kinases, including JNK or Mst1, which phosphorylate FOXO under conditions of oxidative stress. This phosphorylation causes the translocation of FOXO from the cytoplasm to the nucleus, thus opposing Akts action. In addition to being post-translationally modified by phosphorylation, FOXO proteins also bind to co-activator or co-repressor complexes and become acetylated or deacetylated. For example, the deacetylase SIRT1 increases FOXO DNA-binding ability by deacetylating FOXO in response to oxidative stress. FOXO proteins are also monoubiquitinated under conditions of oxidative stress and this increases transcriptional activity. Finally, FOXO proteins can also be polyubiquitinated and targeted for protein degradation. The unique phosphorylation, acetylation, and ubiquitination status of FOXO under specific environmental conditions may provide specificity in the regulation of subsets of FOXO target genes.What is the role of FOXO in longevity? FOXO factors have been shown to prolong lifespan in invertebrates. The worm orthologue, DAF-16, activates a program of genes that extend longevity by promoting resistance to oxidative stress, pathogens, and damage to protein structure. Mutations in the insulin receptor or PI3K extend longevity up to threefold, and this extension is reverted when daf-16 is mutated. In flies, overexpression of dFOXO is sufficient to increase longevity. The role of FOXO factors in mammalian longevity is currently being explored. Mice that are deficient for either the insulin receptor or the insulin-like growth factor receptor-1 can live up to 30% longer than wild-type mice, suggesting that FOXO factors could be involved in mammalian longevity. Furthermore, FOXO target genes involved in stress resistance are conserved between invertebrates and mammals, suggesting that the function of FOXO in organismal stress resistance and longevity is evolutionarily conserved.Isnt it strange that FOXO could induce both stress resistance and cell death? The regulation of stress-resistance genes and pro-apoptotic genes by FOXO is not necessarily a paradox. FOXO factors may orchestrate different patterns of gene expression based on the intensity of the stimulus, perhaps activating stress-resistance genes under mild conditions but pro-apoptotic genes when the intensity of stress stimuli increases beyond a certain threshold. It is also possible that FOXO factors regulate different genes in different cell types, causing apoptosis in some cells (e.g. neurons, lymphocytes) while promoting survival in others. Importantly, the induction of apoptosis by FOXO may cause the death of damaged or abnormal cells, therefore benefiting the longevity of the entire organism.Is there a connection between FOXO and cancer? Because FOXO proteins were originally identified in human tumors, and because they play an important role in cell-cycle arrest, DNA repair, and apoptosis — cell functions that go awry in cancer — the FOXO family is thought to coordinate the balance between longevity and tumor suppression. Consistent with this idea, in certain breast cancers, FOXO3 is sequestered in the cytoplasm and inactivated. Expression of active forms of FOXO in tumor cells prevents tumor growth in vivo. Additionally, protein partners of FOXO, such as p53 and SMAD transcription factors, are tumor suppressors. Investigating the ensemble of FOXO protein partners will provide insight into the connection between aging and cancer.Can you live without FOXO? It depends if you are a worm, a fly, or a mammal. Worms lacking daf-16 or flies lacking dFOXO are viable but do not show an increase in lifespan following mutations in the insulin/PI3K/Akt pathway. FoxO1-null mice die at embryonic day 10.5 from defects in angiogenesis. FoxO3- and FoxO4-null mice have also been produced and are viable: FoxO3-null mice exhibit an age-dependent infertility in females, while FoxO4-null mice have no apparent phenotype. FoxO6-null mice are currently being generated. The four mammalian isoforms may have both distinct and overlapping functions, and compensation of one member by another may mask the function of individual FOXOs. Investigating the role of FOXO factors in longevity and tumor suppression will require more complex mouse models in which multiple FoxO genes are deleted.What remains to be explored? More FOXO target genes remain to be discovered, as do regulators of FOXO function. An exciting area of future exploration will be to determine how FOXO factors mediate cell non-autonomous processes in the entire organism. The recent discovery that FOXO can upregulate neuropeptides in the hypothalamus suggests that FOXO can regulate animal behavior, and future studies will elucidate how hormones and neuronal signaling cause FOXO-dependent transcription of target genes that affect the entire organism.

Sleep homeostasis modulates hypocretin-mediated sleep-to-wake transitions.

The hypocretins (Hcrts) (also called orexins) are two neuropeptides expressed in the lateral hypothalamus that play a crucial role in the stability of wakefulness. Previously, our laboratory demonstrated that in vivo photostimulation of Hcrt neurons genetically targeted with ChR2, a light-activated cation channel, was sufficient to increase the probability of an awakening event during both slow-wave sleep and rapid eye movement sleep. In the current study, we ask whether Hcrt-mediated sleep-to-wake transitions are affected by light/dark period and sleep pressure. We found that stimulation of Hcrt neurons increased the probability of an awakening event throughout the entire light/dark period but that this effect was diminished with sleep pressure induced by 2 or 4 h of sleep deprivation. Interestingly, photostimulation of Hcrt neurons was still sufficient to increase activity assessed by c-Fos expression in Hcrt neurons after sleep deprivation, although this stimulation did not cause an increase in transitions to wakefulness. In addition, we found that photostimulation of Hcrt neurons increases neural activity assessed by c-Fos expression in the downstream arousal-promoting locus ceruleus and tuberomammilary nucleus but not after 2 h of sleep deprivation. Finally, stimulation of Hcrt neurons was still sufficient to increase the probability of an awakening event in histidine decarboxylase-deficient knock-out animals. Collectively, these results suggest that the Hcrt system promotes wakefulness throughout the light/dark period by activating multiple downstream targets, which themselves are inhibited with increased sleep pressure.

Mechanism for Hypocretin-mediated sleep-to-wake transitions

Current models of sleep/wake regulation posit that Hypocretin (Hcrt)-expressing neurons in the lateral hypothalamus promote and stabilize wakefulness by projecting to subcortical arousal centers. However, the critical downstream effectors of Hcrt neurons are unknown. Here we use optogenetic, pharmacological, and computational tools to investigate the functional connectivity between Hcrt neurons and downstream noradrenergic neurons in the locus coeruleus (LC) during nonrapid eye movement (NREM) sleep. We found that photoinhibiting LC neurons during Hcrt stimulation blocked Hcrt-mediated sleep-to-wake transitions. In contrast, when LC neurons were optically stimulated to increase membrane excitability, concomitant photostimulation of Hcrt neurons significantly increased the probability of sleep-to-wake transitions compared with Hcrt stimulation alone. We also built a conductance-based computational model of Hcrt-LC circuitry that recapitulates our behavioral results using LC neurons as the main effectors of Hcrt signaling. These results establish the Hcrt-LC connection as a critical integrator-effector circuit that regulates NREM sleep/wake behavior during the inactive period. This coupling of distinct neuronal systems can be generalized to other hypothalamic integrator nuclei with downstream effector/output populations in the brain.

The brain hypocretins and their receptors: mediators of allostatic arousal

The hypocretins (abbreviated Hcrts - also called orexins) are two neuropeptides secreted exclusively by a small population of neurons in the lateral hypothalamus. These peptides bind to two receptors located throughout the brain in nuclei associated with diverse cognitive and physiological functions. Initially, the brain Hcrt system was found to have a major role in the regulation of sleep/wake transitions. More recent studies indicate Hcrts may play a role in other physiological functions, including food intake, addiction, and stress. Taken together, these studies suggest a general role for Hcrts in mediating arousal, especially when an organism must respond to unexpected stressors and challenges in the environment.

Shining Light on Wakefulness and Arousal

Alterations in arousal states are associated with multiple neuropsychiatric disorders, including generalized anxiety disorders, addiction, schizophrenia, and depression. Therefore, elucidating the neurobiological mechanisms controlling the boundaries between arousal, hyperarousal, and hypoarousal is a crucial endeavor in biological psychiatry. Substantial research over several decades has identified distinct arousal-promoting neural populations in the brain; however, how these nuclei act individually and collectively to promote and maintain wakefulness and various arousal states is unknown. We have recently applied optogenetic technology to the repertoire of techniques used to study arousal. Here, we discuss the recent results of these experiments and propose future use of this approach as a way to understand the complex dynamics of neural circuits controlling arousal and arousal-related behaviors.

Parabrachial Calcitonin Gene-Related Peptide Neurons Mediate Conditioned Taste Aversion

Conditioned taste aversion (CTA) is a phenomenon in which an individual forms an association between a novel tastant and toxin-induced gastrointestinal malaise. Previous studies showed that the parabrachial nucleus (PBN) contains neurons that are necessary for the acquisition of CTA, but the specific neuronal populations involved are unknown. Previously, we identified calcitonin gene-related peptide (CGRP)-expressing neurons in the external lateral subdivision of the PBN (PBel) as being sufficient to suppress appetite and necessary for the anorexigenic effects of appetite-suppressing substances including lithium chloride (LiCl), a compound often used to induce CTA. Here, we test the hypothesis that PBel CGRP neurons are sufficient and necessary for CTA acquisition in mice. We show that optogenetic activation of these neurons is sufficient to induce CTA in the absence of anorexigenic substances, whereas genetically induced silencing of these neurons attenuates acquisition of CTA upon exposure to LiCl. Together, these results demonstrate that PBel CGRP neurons mediate a gastrointestinal distress signal required to establish CTA.

Optogenetic investigation of neural circuits in vivo

The recent development of light-activated optogenetic probes allows for the identification and manipulation of specific neural populations and their connections in awake animals with unprecedented spatial and temporal precision. This review describes the use of optogenetic tools to investigate neurons and neural circuits in vivo. We describe the current panel of optogenetic probes, methods of targeting these probes to specific cell types in the nervous system, and strategies of photostimulating cells in awake, behaving animals. Finally, we survey the application of optogenetic tools to studying functional neuroanatomy, behavior and the etiology and treatment of various neurological disorders.

Functional wiring of hypocretin and LC-NE neurons: implications for arousal

To survive in a rapidly changing environment, animals must sense their external world and internal physiological state and properly regulate levels of arousal. Levels of arousal that are abnormally high may result in inefficient use of internal energy stores and unfocused attention to salient environmental stimuli. Alternatively, levels of arousal that are abnormally low may result in the inability to properly seek food, water, sexual partners, and other factors necessary for life. In the brain, neurons that express hypocretin neuropeptides may be uniquely posed to sense the external and internal state of the animal and tune arousal state according to behavioral needs. In recent years, we have applied temporally precise optogenetic techniques to study the role of these neurons and their downstream connections in regulating arousal. In particular, we have found that noradrenergic neurons in the brainstem locus coeruleus (LC) are particularly important for mediating the effects of hypocretin neurons on arousal. Here, we discuss our recent results and consider the implications of the anatomical connectivity of these neurons in regulating the arousal state of an organism across various states of sleep and wakefulness.

Development of echolocation and communication vocalizations in the big brown bat, Eptesicus fuscus

Big brown bats form large maternity colonies of up to 200 mothers and their pups. If pups are separated from their mothers, they can locate each other using vocalizations. The goal of this study was to systematically characterize the development of echolocation and communication calls from birth through adulthood to determine whether they develop from a common precursor at the same or different rates, or whether both types are present initially. Three females and their six pups were isolated from our captive breeding colony. We recorded vocal activity from postnatal day 1 to 35, both when the pups were isolated and when they were reunited with their mothers. At birth, pups exclusively emitted isolation calls, with a fundamental frequency range <20xa0kHz, and duration >30xa0ms. By the middle of week 1, different types of vocalizations began to emerge. Starting in week 2, pups in the presence of their mothers emitted sounds that resembled adult communication vocalizations, with a lower frequency range and longer durations than isolation calls or echolocation signals. During weeks 2 and 3, these vocalizations were extremely heterogeneous, suggesting that the pups went through a babbling stage before establishing a repertoire of stereotyped adult vocalizations around week 4. By week 4, vocalizations emitted when pups were alone were identical to adult echolocation signals. Echolocation and communication signals both appear to develop from the isolation call, diverging during week 2 and continuing to develop at different rates for several weeks until the adult vocal repertoire is established.