Development of an optogenetic toolkit for neural circuit dissection in squirrel monkeys

Optogenetic tools have opened a rich experimental landscape for understanding neural function and disease. Here, we present the first validation of eight optogenetic constructs driven by recombinant adeno-associated virus (AAV) vectors and a WGA-Cre based dual injection strategy for projection targeting in a widely-used New World primate model, the common squirrel monkey Saimiri sciureus. We observed opsin expression around the local injection site and in axonal projections to downstream regions, as well as transduction to thalamic neurons, resembling expression patterns observed in macaques. Optical stimulation drove strong, reliable excitatory responses in local neural populations for two depolarizing opsins in anesthetized monkeys. Finally, we observed continued, healthy opsin expression for at least one year. These data suggest that optogenetic tools can be readily applied in squirrel monkeys, an important first step in enabling precise, targeted manipulation of neural circuits in these highly trainable, cognitively sophisticated animals. In conjunction with similar approaches in macaques and marmosets, optogenetic manipulation of neural circuits in squirrel monkeys will provide functional, comparative insights into neural circuits which subserve dextrous motor control as well as other adaptive behaviors across the primate lineage. Additionally, development of these tools in squirrel monkeys, a well-established model system for several human neurological diseases, can aid in identifying novel treatment strategies

Decrease in coccolithophore calcification and CO2 since the middle Miocene

International audienceMarine algae are instrumental in carbon cycling and atmospheric carbon dioxide (CO2) regulation. One group, coccolithophores, uses carbon to photosynthesize and to calcify, covering their cells with chalk platelets (coccoliths). How ocean acidification influences coccolithophore calcification is strongly debated, and the effects of carbonate chemistry changes in the geological past are poorly understood. This paper relates degree of coccolith calcification to cellular calcification, and presents the first records of size-normalized coccolith thickness spanning the last 14 Myr from tropical oceans. Degree of calcification was highest in the low-pH, high-CO2 Miocene ocean, but decreased significantly between 6 and 4 Myr ago. Based on this and concurrent trends in a new alkenone εp record, we propose that decreasing CO2 partly drove the observed trend via reduced cellular bicarbonate allocation to calcification. This trend reversed in the late Pleistocene despite low CO2, suggesting an additional regulator of calcification such as alkalinity

Optogenetics: a new method for the causal analysis of neuronal networks in vivo

The causal analysis of neuronal network function requires a selective manipulation of genetically defined neuronal subpopulations in the intact living brain. Here, we highlight the method of optogenetics, meeting those needs. We cover methodological aspects, limitations and practical applications in the field of neurosciences. The basis of optogenetics are light-sensitive transmembrane channels and light-driven ion pumps, which can be genetically encoded, without requiring the application of exogenous cofactors. These opsins are expressed in neurons by means of viral gene transfer and cell-specific promoters. Light for stimulation can be non-or minimally invasively delivered by optical fibers. Illumination of opsins results in a depolarization or hyperpolarization of genetically modified neurons, depending on the type of optogenetic actuator. Strong expression levels and sufficient light densities provided, neuronal activity can be optically controlled in the intact network with millisecond precision. By applying fluorescent indicators of neuronal activity, an all-optical neurophysiological approach becomes reality

Real-time detection of neural oscillation bursts allows behaviourally relevant neurofeedback

Neural oscillations as important information carrier in the brain, are increasingly interpreted as transient bursts rather than as sustained oscillations. Short (<150 ms) bursts of beta-waves (15-30 Hz) have been documented in humans, monkeys and mice. These events were correlated with memory, movement and perception, and were even suggested as the primary ingredient of all beta-band activity. However, a method to measure these short-lived events in real-time and to investigate their impact on behaviour is missing. Here we present a real-time data analysis system, capable to detect short narrowband bursts, and demonstrate its usefulness to increase the beta-band burst-rate in rats. This neurofeedback training induced changes in overall oscillatory power, and bursts could be decoded from the movement of the rats, thus enabling future investigation of the role of oscillatory bursts. Karvat et al. describe a real-time digital signal-processing method to detect neural oscillations in the form of short and narrowband bursts. Using this real-time method, they show that neurofeedback increases burst-related behaviours and that these bursts can be decoded from the movements of the rats

Cortical gamma-band resonance preferentially transmits coherent input

Synchronization has been implicated in neuronal communication, but causal evidence remains indirect. We use optogenetics to generate depolarizing currents in pyramidal neurons of the cat visual cortex, emulating excitatory synaptic inputs under precise temporal control, while measuring spike output. The cortex transforms constant excitation into strong gamma-band synchronization, revealing the well-known cortical resonance. Increasing excitation with ramps increases the strength and frequency of synchronization. Slow, symmetric excitation profiles reveal hysteresis of power and frequency. White-noise input sequences enable causal analysis of network transmission, establishing that the cortical gamma-band resonance preferentially transmits coherent input components. Models composed of recurrently coupled excitatory and inhibitory units uncover a crucial role of feedback inhibition and suggest that hysteresis can arise through spike-frequency adaptation. The presented approach provides a powerful means to investigate the resonance properties of local circuits and probe how these properties transform input and shape transmission

Current Biology

The ability to plan and execute appropriately timed responses to external stimuli is based on a well-orchestrated balance between movement initiation and inhibition. In impulse control disorders involving the prefrontal cortex (PFC) [1], this balance is disturbed, emphasizing the critical role that PFC plays in appropriately timing actions [2-4]. Here, we employed optogenetic and electrophysiological techniques to systematically analyze the functional role of five key subareas of the rat medial PFC (mPFC) and orbitofrontal cortex (OFC) in action control [5-9]. Inactivation of mPFC subareas induced drastic changes in performance, namely an increase (prelimbic cortex, PL) or decrease (infralimbic cortex, IL) of premature responses. Additionally, electrophysiology revealed a significant decrease in neuronal activity of a PL subpopulation prior to premature responses. In contrast, inhibition of OFC subareas (mainly the ventral OFC, i.e., VO) significantly impaired the ability to respond rapidly after external cues. Consistent with these findings, mPFC activity during response preparation predicted trial outcomes and reaction times significantly better than OFC activity. These data support the concept of opposing roles of IL and PL in directing proactive behavior and argue for an involvement of OFC in predominantly reactive movement control. By attributing defined roles to rodent PFC sections, this study contributes to a deeper understanding of the functional heterogeneity of this brain area and thus may guide medically relevant studies of PFC-associated impulse control disorders in this animal model for neural disorders [10-12]

Translational Neuroscience: Toward New Therapies

Classically, research into human disease tends to be done in a top- down or bottom- up manner, starting from symptoms or genes, respectively. While bottom-up approaches may work well in oncology, and might advance understanding of monogenic neuropsy- chiatric diseases, successful application for complex, multifactorial disorders is more difficult and has resulted in many translational failures. This chapter investigates the existing obstacles and explores options to overcome them. Complex diseases need to be dissected into measureable, manageable factors and investigated in a comparable, compatible assembly of model systems to test hypotheses, concepts, and ultimately drug candidates or other therapeutic interventions. While some of these factors might best be investigated top down, a bottom-up approach might be more effective for oth- ers. Both approaches may only be successful up to a specific point. Thus, the two must be linked and a bidirectional approach pursued. Inclusion of patients is essential as are behavioral readouts, since disease-associated dysfunctions or symptoms are often behavioral in nature. To connect models and humans, behavioral readouts need ide- ally to be linked to evolutionary conserved neural substrates. Some anchor points al- ready exist and new promising ones, such as induced pluripotent stem cells (iPSCs), are emerging. Recent developments may speed up translation of research into clinical applications (e.g., faster drug screens in a patient-specific manner). When positioning different models, it is important to characterize their predictive power diligently, to em- phasize their scientific rigor, and to not overstate their application potential. Finally, to effect faster transition from research to clinical applications, organizational structures are needed to foster interdisciplinary research and collaborations between academia and industry. A “ third space” concept is proposed to conduct early proof of principle studies (Phase 0 and I). To increase the success rate in clinical development so as to provide actual benefit for patients, proactive interaction is needed between all organizational entities involved in drug development and therapeutic discovery (e.g., academia, guid- ance agencies, biotech, device and pharmaceutical companies, regulatory agencies, and funding agencies)

Pathophysiological Toolkit; Genes to Circuits

Understanding the etiology and pathophysiology of neuropsychiatric disease requires the development of new tools (ranging from evolving diagnostic strategies to biomarkers) that can address the unique challenges of neuropsychiatric disease, including the current lack of tractable interfaces between what we can learn in the clinic and the tools available using model systems in the laboratory. This chapter outlines some of these tools, addressing pitfalls and opportunities, while acknowledging the iterative nature of bridging the gaps between different levels of inquiry