The role of serotonin in depression-A historical roundup and future directions.

Depression is one of the most common psychiatric disorders worldwide, affecting approximately 280 million people, with probably much higher unrecorded cases. Depression is associated with symptoms such as anhedonia, feelings of hopelessness, sleep disturbances, and even suicidal thoughts. Tragically, more than 700 000 people commit suicide each year. Although depression has been studied for many decades, the exact mechanisms that lead to depression are still unknown, and available treatments only help a fraction of patients. In the late 1960s, the serotonin hypothesis was published, suggesting that serotonin is the key player in depressive disorders. However, this hypothesis is being increasingly doubted as there is evidence for the influence of other neurotransmitters, such as noradrenaline, glutamate, and dopamine, as well as larger systemic causes such as altered activity in the limbic network or inflammatory processes. In this narrative review, we aim to contribute to the ongoing debate on the involvement of serotonin in depression. We will review the evolution of antidepressant treatments, systemic research on depression over the years, and future research applications that will help to bridge the gap between systemic research and neurotransmitter dynamics using biosensors. These new tools in combination with systemic applications, will in the future provide a deeper understanding of the serotonergic dynamics in depression.

A Bright Future? A Perspective on Class C GPCR Based Genetically Encoded Biosensors.

One of the major challenges in molecular neuroscience today is to accurately monitor neurotransmitters, neuromodulators, peptides, and various other biomolecules in the brain with high temporal and spatial resolution. Only a comprehensive understanding of neuromodulator dynamics, their release probability, and spatial distribution will unravel their ultimate role in cognition and behavior. This Perspective offers an overview of potential design strategies for class C GPCR-based biosensors. It briefly highlights current applications of GPCR-based biosensors, with a primary focus on class C GPCRs and their unique structural characteristics compared with other GPCR subfamilies. The discussion offers insights into plausible future design approaches for biosensor development targeting members of this specific GPCR subfamily. It is important to note that, at this stage, we are contemplating possibilities rather than presenting a concrete guide, as the pipeline is still under development.

Illuminating the brain-genetically encoded single wavelength fluorescent biosensors to unravel neurotransmitter dynamics.

Understanding how neuronal networks generate complex behavior is one of the major goals of Neuroscience. Neurotransmitter and Neuromodulators are crucial for information flow between neurons and understanding their dynamics is the key to unravel their role in behavior. To understand how the brain transmits information and how brain states arise, it is essential to visualize the dynamics of neurotransmitters, neuromodulators and neurochemicals. In the last five years, an increasing number of single-wavelength biosensors either based on periplasmic binding proteins (PBPs) or on G-protein-coupled receptors (GPCR) have been published that are able to detect neurotransmitter release in vitro and in vivo with high spatial and temporal resolution. Here we review and discuss recent progress in the development of these sensors, their limitations and future directions.

Next generation genetically encoded fluorescent sensors for serotonin.

We developed a family of genetically encoded serotonin (5-HT) sensors (sDarken) on the basis of the native 5-HT1A receptor and circularly permuted GFP. sDarken 5-HT sensors are bright in the unbound state and diminish their fluorescence upon binding of 5-HT. Sensor variants with different affinities for serotonin were engineered to increase the versatility in imaging of serotonin dynamics. Experiments in vitro and in vivo showed the feasibility of imaging serotonin dynamics with high temporal and spatial resolution. As demonstrated here, the designed sensors show excellent membrane expression, have high specificity and a superior signal-to-noise ratio, detect the endogenous release of serotonin and are suitable for two-photon in vivo imaging.

Orthogonally-polarized excitation for improved two-photon and second-harmonic-generation microscopy, applied to neurotransmitter imaging with GPCR-based sensors.

Fluorescent proteins are excited by light that is polarized parallel to the dipole axis of the chromophore. In two-photon microscopy, polarized light is used for excitation. Here we reveal surprisingly strong polarization sensitivity in a class of genetically encoded, GPCR-based neurotransmitter sensors. In tubular structures such as dendrites, this effect led to a complete loss of membrane signal in dendrites running parallel to the polarization direction of the excitation beam. To reduce the sensitivity to dendritic orientation, we designed an optical device that generates interleaved pulse trains of orthogonal polarization. The passive device, which we inserted in the beam path of an existing two-photon microscope, removed the strong direction bias from fluorescence and second-harmonic (SHG) images. We conclude that for optical measurements of transmitter concentration with GPCR-based sensors, orthogonally polarized excitation is essential.

AAV1 is the optimal viral vector for optogenetic experiments in pigeons (Columba livia).

Although optogenetics has revolutionized rodent neuroscience, it is still rarely used in other model organisms as the efficiencies of viral gene transfer differ between species and comprehensive viral transduction studies are rare. However, for comparative research, birds offer valuable model organisms as they have excellent visual and cognitive capabilities. Therefore, the following study establishes optogenetics in pigeons on histological, physiological, and behavioral levels. We show that AAV1 is the most efficient viral vector in various brain regions and leads to extensive anterograde and retrograde ChR2 expression when combined with the CAG promoter. Furthermore, transient optical stimulation of ChR2 expressing cells in the entopallium decreases pigeons' contrast sensitivity during a grayscale discrimination task. This finding demonstrates causal evidence for the involvement of the entopallium in contrast perception as well as a proof of principle for optogenetics in pigeons and provides the groundwork for various other methods that rely on viral gene transfer in birds.

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice.

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest. Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light. The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

Enhanced activity of pyramidal neurons in the infralimbic cortex drives anxiety behavior.

We show that in an animal model of anxiety the overall excitation, particularly in the infralimbic region of the medial prefrontal cortex (IL), is increased and that the activity ratio between excitatory pyramidal neurons and inhibitory interneurons (AR PN/IN) is shifted towards excitation. The same change in AR PN/IN is evident for wildtype mice, which have been exposed to an anxiety stimulus. We hypothesize, that an elevated activity and the imbalance of excitation (PN) and inhibition (IN) within the neuronal microcircuitry of the prefrontal cortex is responsible for anxiety behaviour and employed optogenetic methods in freely moving mice to verify our findings. Consistent with our hypothesis elevation of pyramidal neuron activity in the infralimbic region of the prefrontal cortex significantly enhanced anxiety levels in several behavioural tasks by shifting the AR PN/IN to excitation, without affecting motor behaviour, thus revealing a novel mechanism by which anxiety is facilitated.

Serotonin neurons in the dorsal raphe mediate the anticataplectic action of orexin neurons by reducing amygdala activity.

Narcolepsy is a sleep disorder caused by the loss of orexin (hypocretin)-producing neurons and marked by excessive daytime sleepiness and a sudden weakening of muscle tone, or cataplexy, often triggered by strong emotions. In a mouse model for narcolepsy, we previously demonstrated that serotonin neurons of the dorsal raphe nucleus (DRN) mediate the suppression of cataplexy-like episodes (CLEs) by orexin neurons. Using an optogenetic tool, in this paper we show that the acute activation of DRN serotonin neuron terminals in the amygdala, but not in nuclei involved in regulating rapid eye-movement sleep and atonia, suppressed CLEs. Not only did stimulating serotonin nerve terminals reduce amygdala activity, but the chemogenetic inhibition of the amygdala using designer receptors exclusively activated by designer drugs also drastically decreased CLEs, whereas chemogenetic activation increased them. Moreover, the optogenetic inhibition of serotonin nerve terminals in the amygdala blocked the anticataplectic effects of orexin signaling in DRN serotonin neurons. Taken together, the results suggest that DRN serotonin neurons, as a downstream target of orexin neurons, inhibit cataplexy by reducing the activity of amygdala as a center for emotional processing.

Optogenetic Destabilization of the Memory Trace in CA1: Insights into Reconsolidation and Retrieval Processes.

Reactivation of memory can cause instability necessitating the reconsolidation of the trace. This process can be blocked by amnestic treatments administered after memory reactivation resulting in subsequent memory deficits. While the basolateral amygdala (BLA) is known to be crucial for reconsolidation, evidence for a contribution of the hippocampal CA1 region has only started to accumulate. Moreover, the effect of a reconsolidation blockade in CA1 has only been evaluated behaviorally, and it is unknown whether this manipulation has a long-term effect on neuronal activity. We combined optogenetic and high-resolution molecular imaging techniques to inhibit cell firing in CA1 following the reactivation of a fear memory in mice, evaluated memory performance and imaged neuronal activity the next day upon reexposure to the conditioning context. Blocking memory reconsolidation led to severe memory impairments that were associated with reduced neuronal activity not only in CA1 but also in CA3 and the BLA. Thus, our results indicate that CA1 is necessary for reconsolidation and suggest the involvement of a CA3-CA1-BLA network in the retrieval of contextual fear memory. Further investigations of this network might contribute to the validation of new brain targets for the treatment of pathologies such as posttraumatic stress disorders.

Melanopsin Variants as Intrinsic Optogenetic On and Off Switches for Transient versus Sustained Activation of G Protein Pathways.

G-protein-coupled receptors (GPCRs) represent the major protein family for cellular modulation in mammals. Therefore, various strategies have been developed to analyze the function of GPCRs involving pharmaco- and optogenetic approaches [1, 2]. However, a tool that combines precise control of the activation and deactivation of GPCR pathways and/or neuronal firing with limited phototoxicity is still missing. We compared the biophysical properties and optogenetic application of a human and a mouse melanopsin variant (hOpn4L and mOpn4L) on the control of Gi/o and Gq pathways in heterologous expression systems and mouse brain. We found that GPCR pathways can be switched on/off by blue/yellow light. The proteins differ in their kinetics and wavelength dependence to activate and deactivate G protein pathways. Whereas mOpn4L is maximally activated by very short light pulses, leading to sustained G protein activation, G protein responses of hOpn4L need longer light pulses to be activated and decline in amplitude. Based on the different biophysical properties, brief light activation of mOpn4L is sufficient to induce sustained neuronal firing in cerebellar Purkinje cells (PC), whereas brief light activation of hOpn4L induces AP firing, which declines in frequency over time. Most importantly, mOpn4L-induced sustained firing can be switched off by yellow light. Based on the biophysical properties, hOpn4L and mOpn4L represent the first GPCR optogenetic tools, which can be used to switch GPCR pathways/neuronal firing on an off with temporal precision and limited phototoxicity. We suggest to name these tools moMo and huMo for future optogenetic applications.

Gq/5-HT2c receptor signals activate a local GABAergic inhibitory feedback circuit to modulate serotonergic firing and anxiety in mice.

Serotonin 2c receptors (5-HT2c-Rs) are drug targets for certain mental disorders, including schizophrenia, depression, and anxiety. 5-HT2c-Rs are expressed throughout the brain, making it difficult to link behavioral changes to circuit specific receptor expression. Various 5-HT-Rs, including 5-HT2c-Rs, are found in the dorsal raphe nucleus (DRN); however, the function of 5-HT2c-Rs and their influence on the serotonergic signals mediating mood disorders remain unclear. To investigate the role of 5-HT2c-Rs in the DRN in mice, we developed a melanopsin-based optogenetic probe for activation of Gq signals in cellular domains, where 5-HT2c-Rs are localized. Our results demonstrate that precise temporal control of Gq signals in 5-HT2c-R domains in GABAergic neurons upstream of 5-HT neurons provides negative feedback regulation of serotonergic firing to modulate anxiety-like behavior in mice.

Vertebrate cone opsins enable sustained and highly sensitive rapid control of Gi/o signaling in anxiety circuitry.

G protein-coupled receptors (GPCRs) coupling to Gi/o signaling pathways are involved in the control of important physiological functions, which are difficult to investigate because of the limitation of tools to control the signaling pathway with precise kinetics and specificity. We established two vertebrate cone opsins, short- and long-wavelength opsin, for long-lasting and repetitive activation of Gi/o signaling pathways in vitro and in vivo. We demonstrate for both opsins the repetitive fast, membrane-delimited, ultra light-sensitive, and wavelength-dependent activation of the Gi/o pathway in HEK cells. We also show repetitive control of Gi/o pathway activation in 5-HT1A receptor domains in the dorsal raphe nucleus (DRN) in brain slices and in vivo, which is sufficient to modulate anxiety behavior in mice. Thus, vertebrate cone opsins represent a class of tools for understanding the role of Gi/o-coupled GPCRs in health and disease.

Modulation of firing and synaptic transmission of serotonergic neurons by intrinsic G protein-coupled receptors and ion channels.

Serotonergic neurons project to virtually all regions of the central nervous system and are consequently involved in many critical physiological functions such as mood, sexual behavior, feeding, sleep/wake cycle, memory, cognition, blood pressure regulation, breathing, and reproductive success. Therefore, serotonin release and serotonergic neuronal activity have to be precisely controlled and modulated by interacting brain circuits to adapt to specific emotional and environmental states. We will review the current knowledge about G protein-coupled receptors and ion channels involved in the regulation of serotonergic system, how their regulation is modulating the intrinsic activity of serotonergic neurons and its transmitter release and will discuss the latest methods for controlling the modulation of serotonin release and intracellular signaling in serotonergic neurons in vitro and in vivo.

Optogenetic control of motor coordination by Gi/o protein-coupled vertebrate rhodopsin in cerebellar Purkinje cells.

G protein-coupled receptors are involved in the modulation of complex neuronal networks in the brain. To investigate the impact of a cell-specific G(i/o) protein-mediated signaling pathway on brain function, we created a new optogenetic mouse model in which the G(i/o) protein-coupled receptor vertebrate rhodopsin can be cell-specifically expressed with the aid of Cre recombinase. Here we use this mouse model to study the functional impact of G(i/o) modulation in cerebellar Purkinje cells (PCs). We show that in vivo light activation of vertebrate rhodopsin specifically expressed in PCs reduces simple spike firing that is comparable with the reduction in firing observed for the activation of cerebellar G(i/o)-coupled GABA(B) receptors. Notably, the light exposure of the cerebellar vermis in freely moving mice changes the motor behavior. Thus, our studies directly demonstrate that spike modulation via G(i/o)-mediated signaling in cerebellar PCs affects motor coordination and show a new promising approach for studying the physiological function of G protein-coupled receptor-mediated signaling in a cell type-specific manner.

Light- and drug-activated G-protein-coupled receptors to control intracellular signalling.

G-protein-coupled receptors (GPCRs) integrate extracellular cues into intracellular signals to modulate the cellular state. Owing to their diverse modulatory functions, GPCRs represent one of the major drug targets of the pharmaceutical industry. Until now, the characterization and control of GPCRs and their intracellular signalling cascades have mainly relied on chemical compounds, which either activate or inhibit GPCR pathways, albeit with limited receptor and cell-type specificity. Recently, new approaches have been developed to control signalling cascades in cell- and receptor-type-specific ways. The chemical approach focuses on GPCR design and activation by an inert chemical compound, whereas the physical approach uses designer GPCRs and activation by physical stimuli, such as light.

Sensitivity of the goldfish motion detection system revealed by incoherent random dot stimuli: comparison of behavioural and neuronal data.

BACKGROUND: Global motion detection is one of the most important abilities in the animal kingdom to navigate through a 3-dimensional environment. In the visual system of teleost fish direction-selective neurons in the pretectal area (APT) are most important for global motion detection. As in all other vertebrates these neurons are involved in the control of slow phase eye movements during gaze stabilization. In contrast to mammals cortical pathways that might influence motion detection abilities of the optokinetic system are missing in teleost fish. RESULTS: To test global motion detection in goldfish we first measured the coherence threshold of random dot patterns to elicit horizontal slow phase eye movements. In addition, the coherence threshold of the optomotor response was determined by the same random dot patterns. In a second approach the coherence threshold to elicit a direction selective response in neurons of the APT was assessed from a neurometric function. Behavioural thresholds and neuronal thresholds to elicit slow phase eye movements were very similar, and ranged between 10% and 20% coherence. In contrast to these low thresholds for the optokinetic reaction and APT neurons the optomotor response could only be elicited by random dot patterns with coherences above 40%. CONCLUSION: Our findings suggest a high sensitivity for global motion in the goldfish optokinetic system. Comparison of neuronal and behavioural thresholds implies a nearly one-to-one transformation of visual neuron performance to the visuo-motor output. In addition, we assume that the optomotor response is not mediated by the optokinetic system, but instead by other motion detection systems with higher coherence thresholds.

Comparative neurobiology of the optokinetic reflex.

A comprehensive overview of the optokinetic reflex (OKR) in vertebrates is given. The main objective is to compare the asymmetry in optokinetic reactions when patterns presented to one eye move horizontally in temporo-nasal or naso-temporal directions. Different hypotheses concerning the evolution of this asymmetry or symmetry in monocular horizontal OKR are discussed.

Question of reference frames: visual direction-selective neurons in the accessory optic system of goldfish.

We investigated if visual direction-selective neurons in the pretectal area (APT) of goldfish (Carassius auratus auratus) preferred visual stimuli resulting from rotations around axes corresponding to the best responsive axes of the semicircular canals [optic flow that is consistent to a maximal activation of the horizontal canal pair (yaw), to a maximal activation of the right anterior/left posterior semicircular canal pair (RALP), and to a maximal activation of the left anterior/right posterior semicircular canal pair (LARP)]. Our sample of neurons recorded in the left pretectum had two preferred axes of rotation: first, rotation around the yaw axis and second, rotation around the RALP axis. Both axes of rotation correspond to best responsive axes of the semicircular canals. For this reason, coding in a reference frame defined by the vestibular system or the pulling direction of the eye muscles is suggested. In our population of recorded APT neurons, we did not find segregation of different preferred axes of rotation into different anatomical structures. Furthermore in all axes no bias for clockwise or counterclockwise rotations was obvious. This is particularly noteworthy for the yaw axis because preference for temporo-nasal and naso-temporal rotations was found at the same recording side. Hence we conclude that in fish the accessory optic system may consist of one nucleus on each side of the midbrain only, the APT. Segregation into different nuclei coding for different axes and different senses of rotation probably first developed in amphibians.

The optokinetic reaction in foveate and afoveate geckos.

To investigate determinants of symmetry of the monocular horizontal optokinetic reaction (hOKR) in vertebrates, we performed behavioural studies in diurnal foveate, as well as nocturnal afoveate geckos. During binocular viewing hOKR gain was equal for movement to the left or right, during monocular stimulation, all afoveate geckos (Lepidodactylus lugubris, Gekko gecko, Eublepharis macularius) and the foveate Lygodactylus spp. exclusively reacted to temporo-nasal stimulation with stabilising head movements whereas in Phelsuma madagascariensis a naso-temporal component of hOKR could be elicited albeit much weaker. Thus, neither the presence of a fovea nor lifestyle seems to be decisive for a symmetrical monocular hOKR.