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Paired-like homeobox gene Phox2b is predominantly expressed in pre-inspiratory neurons in the parafacial respiratory group (pFRG) in newborn rat rostral ventrolateral medulla. To analyse detailed local networks of the respiratory centre using optogenetics, the effects of selective activation of Phox2b-positive neurons in the ventral medulla on respiratory rhythm generation were examined in brainstem–spinal cord preparations isolated from transgenic newborn rats with Phox2b-positive cells expressing channelrhodopsin variant ChRFR(C167A). Photostimulation up to 43 s increased the respiratory rate > 200% of control, whereas short photostimulation (1.5 s) of the rostral pFRG reset the respiratory rhythm. At the cellular level, photostimulation depolarised Phox2b-positive pre-inspiratory, inspiratory and respiratory-modulated tonic neurons and Phox2b-negative pre-inspiratory neurons. In contrast, changes in membrane potential of Phox2b-negative inspiratory and expiratory neurons varied depending on characteristics of ongoing synaptic connections in local respiratory networks in the rostral medulla. In the presence of tetrodotoxin, photostimulation depolarised Phox2b-positive cells, but caused no significant changes in membrane potential of Phox2b-negative cells. We concluded that depolarisation of Phox2b-positive neurons was due to cell-autonomous photo-activation and summation of excitatory postsynaptic potentials, whereas membrane potential changes of Phox2b-negative neurons depended on the network configuration. Our findings shed further light on local networks among respiratory-related neurons in the rostral ventrolateral medulla and emphasise the important role of pre-inspiratory neurons in respiratory rhythm generation in the neonatal rat en bloc preparation.

This work studied the effect of age at photostimulation on reproductive performance of Beijing-You Chicken (BYC) breeders. A total of 384 fourteen-week-old BYC breeder hens were randomly allocated to 4 treatments of 96 birds each, with 2 replicates per treatment. The treatments represent photostimulation at 16, 18, 20, and 22 wk of age, respectively (PS16, PS18, PS20, and PS22) by incrementally increasing day length from 8L:16D to 14L:10D and by increasing lighting intensity from 10 to 80 lx. Egg production was recorded for each replicate until 51 wk. Four birds randomly selected from each treatment were sacrificed to characterize sexual organ development at 4 time points: 1 D before photostimulation and 2, 4, and 6 wk after photostimulation. Eggshell quality at peak laying was measured. The results showed that the changes in ovary and oviduct weight in PS22 were 8.68- and 4.27-fold higher than in PS16 at 6 wk after photostimulation. PS16 had an earlier age at 5% egg production than PS20 and PS22 (P = 0.003). The interval from photostimulation to age at 5% egg production in PS20 and PS22 was shorter than in other treatments (P = 0.005). Later peak egg production was seen in PS20 (211.0 D) and PS22 (218.0 D) than in PS16 (183.0 D) and PS18 (190.0 D, P = 0.020), but the laying rate of PS20 decreased slowly after peak laying. PS20 and PS22 had higher egg weights than PS16 and PS18 at peak laying (P = 0.018). Age at photostimulation had no effect on egg number, defective egg number, or hatchability (P > 0.05). In summary, the onset of lay can be advanced by photostimulation at early ages, but there is no difference on egg production until 51 wk. Photostimulation at 20 wk improves peak of laying and laying uniformity.

In this study, systemic neurophysiological and neuropsychological mechanisms providing processing of visual information in subjects suffering from auditory deprivation were examined. In 30 men (21 to 25 year old) with complete congenital deafness and 30 control normally hearing men of the same age (groups D and C, respectively), cortical visual evoked potentials (VEPs, photostimulation of the right and left eyes by LED flashes, recording from the O1 and O2 loci) and neurodynamic characteristics of processing of visual information within the go/nogo/go paradigm were analyzed. Under conditions of the respective tests, all indices that characterize processing of simple visual information in deaf subjects (including number of processed stimuli, minimum exposure of the signal, and number of errors) were significantly worse than in the control group. It was also found that median values of the latency of the early VEP components (P1, N1, and P2) in group D were significantly smaller than the respective values in group C. At the same time, median latencies of the late VEP waves (N2 and P3) in deaf subjects were significantly greater than the analogous C-group values. Median values of the peak-to-peak amplitudes of all, with no exceptions, VEP components in group D were significantly (two times or even more) smaller than those in control subjects. Patterns of correlations between the indices of visual information processing and time/amplitude parameters of visual VEPs in the examined groups noticeably differed from each other. Thus, specific brain mechanisms responsible for processing of visual information in persons with auditory deprivation and with normal hearing demonstrate significant dissimilarity; central mechanisms of the visual system in deaf subjects undergo considerable cross-modality modifications.

The inefficient endosomal escape of drugs or macromolecules is a major obstacle to achieving successful delivery to therapeutic targets. An efficient approach to circumvent this barrier is photochemical internalization (PCI), which uses light and photosensitizers for endosomal escape of the delivered macromolecules. The PCI mechanism is related to photogenerated singlet oxygen, but the mechanism is still unclear. In this study, we examined the relation of PCI to heat, pH and Ca2+ ions using cell penetrating peptide (CPP)-cargo-photosensitizer (Alexa546 or Alexa633) conjugates. A cell temperature changing experiment demonstrated that heat (thermal mechanism) does not significantly contribute to the photoinduced endosomal escape. Inhibition of V-ATPase proton pump activity and endosomal pH upregulation indicated that PCI-mediated endosomal escape needs endosomal acidification prior to photoirradiation. Imaging of the CPP-cargo-photosensitizer and Ca2+ ions during photostimulation showed that intracellular calcium increase is not the cause of the endosomal escape of the complex. The increment is mainly due to Ca2+ influx. These findings show the importance of extra- and intracellular milieu conditions in the PCI mechanism and enrich our understanding of PCI-related changes in cell.

Both in vivo neuropharmacology and optogenetic stimulation can be used to decode neural circuitry, and can provide therapeutic strategies for brain disorders. However, current neuronal interfaces hinder long-term studies in awake and freely behaving animals, as they are limited in their ability to provide simultaneous and prolonged delivery of multiple drugs, are often bulky and lack multifunctionality, and employ custom control systems with insufficiently versatile selectivity for output mode, animal selection and target brain circuits. Here, we describe smartphone-controlled, minimally invasive, soft optofluidic probes with replaceable plug-like drug cartridges for chronic in vivo pharmacology and optogenetics with selective manipulation of brain circuits. We demonstrate the use of the probes for the control of the locomotor activity of mice for over four weeks via programmable wireless drug delivery and photostimulation. Owing to their ability to deliver both drugs and photopharmacology into the brain repeatedly over long time periods, the probes may contribute to uncovering the basis of neuropsychiatric diseases.

Mechanical allodynia is a cardinal feature of pathological pain. Recent work has demonstrated the necessity of Aβ-low-threshold mechanoreceptors (Aβ-LTMRs) for mechanical allodynia-like behaviors in mice, but it remains unclear whether these neurons are sufficient to produce pain under pathological conditions. We generated a transgenic mouse in which channelrhodopsin-2 (ChR2) is conditionally expressed in vesicular glutamate transporter 1 (Vglut1) sensory neurons (Vglut1-ChR2), which is a heterogeneous population of large-sized sensory neurons with features consistent with Aβ-LTMRs. In naive male Vglut1-ChR2 mice, transdermal hindpaw photostimulation evoked withdrawal behaviors in an intensity- and frequency-dependent manner, which were abolished by local anesthetic and selective A-fiber blockade. Surprisingly, male Vglut1-ChR2 mice did not show significant differences in light-evoked behaviors or real-time aversion after nerve injury despite marked hypersensitivity to punctate mechanical stimuli. We conclude that optogenetic activation of cutaneous Vglut1-ChR2 neurons alone is not sufficient to produce pain-like behaviors in neuropathic mice. SIGNIFICANCE STATEMENT Mechanical allodynia, in which innocuous touch is perceived as pain, is a common feature of pathological pain. To test the contribution of low-threshold mechanoreceptors (LTMRs) to nerve-injury-induced mechanical allodynia, we generated and characterized a new transgenic mouse (Vglut1-ChR2) to optogenetically activate cutaneous vesicular glutamate transporter 1 (Vglut1)-positive LTMRs. Using this mouse, we found that light-evoked behaviors were unchanged by nerve injury, which suggests that activation of Vglut1-positive LTMRs alone is not sufficient to produce pain. The Vglut1-ChR2 mouse will be broadly useful for the study of touch, pain, and itch.

Although molecular tools for controlling neuronal activity by light have vastly expanded, there are still unmet needs which require development and refinement. For example, light delivery into the brain is still a major practical challenge that hinders potential translation of optogenetics in human patients. In addition, it would be advantageous to manipulate neuronal activity acutely and precisely as well as chronically and non-invasively, using the same genetic construct in animal models. We have previously addressed these challenges by employing bioluminescence and have created a new line of opto-chemogenetic probes termed luminopsins by fusing light-sensing opsins with light-emitting luciferases. In this report, we incorporated Chlamydomonas channelrhodopsin 2 with step-function mutations as the opsin moiety in the new luminopsin fusion protein termed step-function luminopsin (SFLMO). Bioluminescence-induced photocurrent lasted longer than the bioluminescence signal due to very slow inactivation of the mutated channel. In addition, bioluminescence was able to activate most of the channels on the cell surface due to the extremely high light sensitivity of the channel. This efficient channel activation was partly mediated by radiationless bioluminescence resonance energy transfer due to the proximity of luciferase and opsin. To test the utility of SFLMOs in vivo, we transduced the substantia nigra unilaterally via a viral vector in male rats. Injection of the luciferase substrate as well as conventional photostimulation via fiber optics elicited circling behaviors. Thus, SFLMOs expand the current approaches for manipulation of neuronal activity in the brain and add more versatility and practicality to optogenetics in freely behaving animals.

Significance The three spectral types of cone photoreceptors underlie color perception and are largely responsible for inherited and acquired color vision anomalies. In vivo mapping of the trichromatic cone mosaic by imaging provides the most direct and quantitative means to assess the role of photoreceptors in color vision, but remains challenging because cone reflections only weakly differentiate cone types. Here, we show a noninvasive light microscopy modality that reveals the cell’s spectral type, using the optical phase change that arises within the cell when stimulated with light. Our procedure is orders of magnitude faster and more accurate than prior approaches and makes in vivo cone classification promising for a much wider range of color vision applications., Human color vision is achieved by mixing neural signals from cone photoreceptors sensitive to different wavelengths of light. The spatial arrangement and proportion of these spectral types in the retina set fundamental limits on color perception, and abnormal or missing types are responsible for color vision loss. Imaging provides the most direct and quantitative means to study these photoreceptor properties at the cellular scale in the living human retina, but remains challenging. Current methods rely on retinal densitometry to distinguish cone types, a prohibitively slow process. Here, we show that photostimulation-induced optical phase changes occur in cone cells and carry substantial information about spectral type, enabling cones to be differentiated with unprecedented accuracy and efficiency. Moreover, these phase dynamics arise from physiological activity occurring on dramatically different timescales (from milliseconds to seconds) inside the cone outer segment, thus exposing the phototransduction cascade and subsequent downstream effects. We captured these dynamics in cones of subjects with normal color vision and a deuteranope, and at different macular locations by: (i) marrying adaptive optics to phase-sensitive optical coherence tomography to avoid optical blurring of the eye, (ii) acquiring images at high speed that samples phase dynamics at up to 3 KHz, and (iii) localizing phase changes to the cone outer segment, where photoactivation occurs. Our method should have broad appeal for color vision applications in which the underlying neural processing of photoreceptors is sought and for investigations of retinal diseases that affect cone function.

As broiler breeders face increased reproductive challenges specifically related to overfeeding, a clear understanding of the physiological effects of BW and rearing photoperiod on reproductive development is needed. The objective was to use mathematical models to compare plasma estradiol-17β (E2) concentration to characterize the effect of BW and rearing photoperiod on E2 levels. A 2 × 3 factorial arrangement of treatments was used. Hens (n = 180) were fed with a precision feeding system to allocate feed individually to achieve the breeder-recommended BW curve (Standard) or to a BW curve reaching the 21 wk target at 18 wk (High). Hens were on 8L:16D, 10L:14D, or 12L:12D photoschedules during rearing and were photostimulated at 21 wk. Age at first egg (AFE) was recorded. Plasma E2 levels were determined weekly between week 20 and 28. Two modified Gompertz models described E2 level as a function of (a) chronological or (b) physiological (relative to AFE) age. Timing of E2-inflection point was compared between models and treatments. Differences were reported as significant at P ≤ 0.05. The chronological age model inferred that High BW reduced the duration between the E2-inflection point and AFE, whereas the physiological age model inferred that High BW only reduced the duration between photostimulation and the E2-inflection point. Hens on the Standard BW treatment had a longer period between photostimulation and the E2-inflection point compared to hens on the High-BW treatment (11.03 vs. 1.50 wk, respectively, based on physiological age). Hens on the 12L:12D photoschedule had a longer period between photostimulation and the E2-inflection point compared to hens on the 8L:16D or 10L:14D photoschedule, both in the Standard and High BW (28.91 vs. 1.78 and 2.40 wk, 2.65 vs. 0.93 and 0.94 wk, respectively, based on physiological age). The described methodology and results provide quantitative insight into E2 dynamics and serves as a model for future endocrinological studies in poultry reproduction.

NG2 glia, also known as oligodendrocyte precursor cells (OPCs), play an important role in proliferation and give rise to myelinating oligodendrocytes during early brain development. In contrast to other glial cell types, the most intriguing aspect of NG2 glia is their ability to directly sense synaptic inputs from neurons. However, whether this synaptic interaction is bidirectional or unidirectional, or its physiological relevance has not yet been clarified. Here, we report that NG2 glia form synaptic complexes with hippocampal interneurons and that selective photostimulation of NG2 glia (expressing channelrhodopsin-2) functionally drives GABA release and enhances inhibitory synaptic transmission onto proximal interneurons in a microcircuit. The mechanism involves GAD67 biosynthesis and VAMP-2 containing vesicular exocytosis. Further, behavioral assays demonstrate that NG2 glia photoactivation triggers anxiety-like behavior in vivo and contributes to chronic social defeat stress., Nerve/glial antigen 2 (NG2) glia can sense synaptic inputs from neurons. Here, the authors show NG2 glia form functional GABAergic synapses by regulating inhibitory synaptic transmission onto adjacent hippocampal interneurons, and activation of NG2 glia induces anxiety-like behaviour in a mouse model of chronic social defeat stress.

SummaryDopaminergic inputs to basal amygdala (BA) instruct learning of motivational salience. Here, we investigated the dynamics of dopamine release and downstream signaling during multiple salient events occurring within tens of seconds. We established in vitro and in vivo real-time tracking and manipulation of cAMP – a key intracellular plasticity signal downstream of dopamine receptor activation. Optogenetically-evoked release of dopamine drove proportional increases in cAMP in almost all BA glutamatergic neurons, suggesting widespread actions of dopamine across neurons preferring positive or negative valence. These cAMP responses decayed more slowly than dopamine release, potentially extending the window of plasticity. cAMP levels accumulated following direct photostimulation of cAMP but not repeated stimulation of dopamine axons, due to potent depression of dopamine release. cAMP and protein kinase A (PKA) responses to repeated appetitive or aversive stimuli also exhibited pronounced depression. Thus, history-dependent dynamics of dopamine and cAMP may regulate learning of temporally clustered, salient stimuli.

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is currently the only treatment for life-threatening ventricular fibrillation. However, defibrillation may have side-effects, including intolerable pain, tissue damage, and worsening of prognosis, indicating a significant medical need for the development of more gentle cardiac rhythm management strategies. Besides energy-reducing electrical approaches, cardiac optogenetics was introduced as a powerful tool to influence cardiac activity using light-sensitive membrane ion channels and light pulses. In the present study, a robust and valid method for successful photostimulation of Langendorff perfused intact murine hearts will be described based on multi-site pacing applying a 3 x 3 array of micro light-emitting diodes (micro-LED). Simultaneous optical mapping of epicardial membrane voltage waves allows the investigation of the effects of region-specific stimulation and evaluates the newly induced cardiac activity directly on-site. The obtained results show that the efficacy of defibrillation is strongly dependent on the parameters chosen for photostimulation during a cardiac arrhythmia. It will be demonstrated that the illuminated area of the heart plays a crucial role for termination success as well as how the targeted control of cardiac activity during illumination for modifying arrhythmia patterns can be achieved. In summary, this technique provides a possibility to optimize the on-site mechanism manipulation on the way to real-time feedback control of cardiac rhythm and, regarding the region specificity, new approaches in reducing the potential harm to the cardiac system compared to the usage of non-specific electrical shock applications.

Animals have an innate motivation to explore objects and environments with unknown values. To this end, they need to activate neural pathways that enable exploration. Here, we reveal that photostimulation of a subset of medial preoptic area (MPA) neurons expressing the vesicular-GABA transporter gene (vgat+) and sending axonal projections to the ventrolateral periaqueductal gray (vPAG) increases exploration in a chamber but causes no place preference when tested there without photostimulation. Photoinhibition of MPAvgat–vPAG projections leads to no emotional changes as measured by normal activity in an open field assay. Electrophysiological recordings revealed that most GABAergic vPAG neurons are inhibited by MPAvgat neurons. In contrast to a previous report that suggested that MPAvgat–vPAG neurons may impart positive valence to induce place preference, our results suggest that these neurons can increase innate exploration.

A normally functioning nervous system requires normal extracellular potassium ion concentration ([K]o). Throughout the nervous system, several processes, including those of an astrocytic nature, are involved in [K]o regulation. In this study we investigated the effect of astrocytic photostimulation on [K]o. We hypothesized that in vivo photostimulation of eNpHR-expressing astrocytes leads to a decreased [K]o. Using optogenetic and electrophysiological techniques we showed that stimulation of eNpHR-expressing astrocytes resulted in a significantly decreased resting [K]o and evoked K responses. The amplitude of the concomitant spreading depolarization-like events also decreased. Our results imply that astrocytic membrane potential modification could be a potential tool for adjusting the [K]o.

Purpose: The aim is to discuss the efficacy of micropulse laser treatment in a patient suspected of polypoidal choroidal vasculopathy. Case Presentation: A 61-year-old male patient presented with decreased vision in his left eye. He had a dome-shaped lesion below the macula and macular swelling in the fundus examination. Optical Coherence Tomography (OCT) showed neurosensorial serous detachment of the macula and polypoid appearance in the region consistent with the dome-shaped lesion. The patient was recommended Flourescein Angiography (FA), indocyanine green angiography (ICGA), but for economic reasons he refused to perform ICGA. After that, only FA was performed. As a result of the examinations, it was evaluated as central serous chorioretinopathy by a retina specialist. Three months later, because of subretinal fluid persistence, micropulse laser treatment including polyp-like lesion was applied. Results: At the 6th month follow-up, it was found that the fluid completely regressed and the Pigment Epithelial Detachment (PED) appearance occurred in place of the polypoidal appearance in OCT. In the 22nd month, subretinal fluid disappeared completely and PED appearance was observed. Conclusions: Although the characteristics of OCT of this case is similar to the OCT appearance in PCV, it would be more reasonable to consider this case as CSC in the light of current information, as this case responded to MPL treatment. Therefore, prospective studies with large groups are needed.

Recent advances combining two-photon calcium imaging and two-photon optogenetics with digital holography now allow us to read and write neural activity in vivo at cellular resolution with millisecond temporal precision. Such “all-optical” techniques enable experimenters to probe the impact of functionally defined neurons on neural circuit function and behavioural output with new levels of precision. This protocol describes the experimental strategy and workflow for successful completion of typical all-optical interrogation experiments in awake, behaving head-fixed mice. We describe modular procedures for the setup and calibration of an all-optical system, the preparation of an indicator and opsin-expressing and task-performing animal, the characterization of functional and photostimulation responses and the design and implementation of an all-optical experiment. We discuss optimizations for efficiently selecting and targeting neuronal ensembles for photostimulation sequences, as well as generating photostimulation response maps from the imaging data that can be used to examine the impact of photostimulation on the local circuit. We demonstrate the utility of this strategy using all-optical experiments in three different brain areas – barrel cortex, visual cortex and hippocampus – using different experimental setups. This approach can in principle be adapted to any brain area for all-optical interrogation experiments to probe functional connectivity in neural circuits and for investigating the relationship between neural circuit activity and behaviour.

Paraventricular thalamus (PVT) is a midline thalamic area that receives dense GABA projections from zona incerta (ZI) for the regulation of feeding behaviours. Activation of central serotonin (5-HT) signalling is known to inhibit food intake. Although previous studies have reported both 5-HT fibres and receptors in the PVT, it remains unknown how 5-HT regulates PVT neurons and whether PVT 5-HT signalling is involved in the control of food intake. Using slice patch-clamp recordings in combination with optogenetics, we found that 5-HT not only directly excited PVT neurons by activating 5-HT7 receptors to modulate hyperpolarization-activated cyclic nucleotide-gated (HCN) channels but also disinhibited these neurons by acting on presynaptic 5-HT1A receptors to reduce GABA inhibition. Specifically, 5-HT depressed photostimulation-evoked inhibitory postsynaptic currents (eIPSCs) in PVT neurons innervated by channelrhodopsin-2-positive GABA axons from ZI. Using paired-pulse photostimulation, we found 5-HT increased paired-pulse ratios of eIPSCs, suggesting 5-HT decreases ZI-PVT GABA release. Furthermore, we found that exposure to a high-fat-high-sucrose diet for 2 weeks impaired both 5-HT inhibition of ZI-PVT GABA transmission and 5-HT excitation of PVT neurons. Using retrograde tracer in combination with immunocytochemistry and slice electrophysiology, we found that PVT-projecting dorsal raphe neurons expressed 5-HT and were inhibited by food deprivation. Together, our study reveals the mechanism by which 5-HT activates PVT neurons through both direct excitation and indirect disinhibition from the ZI. The downregulation in 5-HT excitation and disinhibition of PVT neurons may contribute to the development of overeating and obesity after chronic high-fat diet. KEY POINTS: Serotonin (5-HT) depolarizes and excites paraventricular thalamus (PVT) neurons. 5-HT7 receptors are responsible for 5-HT excitation of PVT neurons and the coupling of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels to 5-HT receptors in part mediates the excitatory effect of 5-HT. 5-HT depresses the frequency of spontaneous inhibitory but not excitatory postsynaptic currents in PVT neurons. 5-HT1A receptors contribute to the depressive effect of 5-HT on inhibitory transmissions. 5-HT inhibits GABA release from zona incerta (ZI) GABA terminals in PVT. Chronic high-fat diet not only impairs 5-HT inhibition of the ZI-PVT GABA transmission but also downregulates 5-HT excitation of PVT neurons. PVT-projecting dorsal raphe neurons express 5-HT and are inhibited by food deprivation.

We investigated time course of photoperiodically driven transcriptional responses in physiologically contrasting seasonal life-history states in migratory blackheaded buntings. Birds exhibiting unstimulated winter phenotype (photosensitive state; responsive to photostimulation) under 6-h short days, and regressed summer phenotype (photorefractory state; unresponsiveness to photostimulation) under 16-h long days, were released into an extended light period up to 22 h of the day. Increased tshβ and dio2, and decreased dio3 mRNA levels in hypothalamus, and low prdx4 and high il1β mRNA levels in blood confirmed photoperiodic induction by hour 18 in photosensitive birds. Further, at hours 10, 14, 18 and 22 of light exposure, the comparison of hypothalamus RNA-Seq results revealed transcriptional differences within and between states. Particularly, we found reduced expression at hour 14 of transthyretin and proopiomelanocortin receptor, and increased expression at hour 18 of apolipoprotein A1 and carbon metabolism related genes in the photosensitive state. Similarly, valine, leucine and isoleucine degradation pathway genes and superoxide dismutase 1 were upregulated, and cocaine- and amphetamine-regulated transcript and gastrin-releasing peptide were downregulated in the photosensitive state. These results show life-history-dependent activation of hypothalamic molecular pathways involved in initiation and maintenance of key biological processes as early as on the first long day.

Understanding how specific sets of neurons fire and wire together during cognitive-relevant activity is one of the most pressing questions in neuroscience. Two-photon, single-cell resolution optogenetics based on holographic light-targeting approaches enables accurate spatio-temporal control of individual or multiple neurons. Yet, currently, the ability to drive asynchronous activity in distinct cells is critically limited to a few milliseconds and the achievable number of targets to several dozens. In order to expand the capability of single-cell optogenetics, we introduce an approach capable of ultra-fast sequential light targeting (FLiT), based on switching temporally focused beams between holograms at kHz rates. We demonstrate serial-parallel photostimulation strategies capable of multi-cell sub-millisecond temporal control and many-fold expansion of the number of activated cells. This approach will be important for experiments that require rapid and precise cell stimulation with defined spatio-temporal activity patterns and optical control of large neuronal ensembles.

In pigs, it has been reported that increased farrowing rates and litter size have been induced by photostimulating the seminal doses for artificial insemination with red LED light. As the reproductive characteristics, production system, and outcome parameters of Iberian breed pigs are different from other commercial breeds, the aim of this study was to evaluate the possible effect of illuminating seminal doses from Duroc boars with red LED light and the fertility outcomes of Iberian females. Semen samples were obtained from 38 fertile Duroc boars. Photostimulation of the artificial insemination (AI) seminal doses was carried out by illuminating the samples with a red LED for 10 min, followed by 10 min of darkness, and finally 10 additional minutes of red light. The fertility study was conducted on two commercial farms using multiparous Iberian sows (farm A, n = 824, farm B, n = 2131), that were randomly assigned to LED (L) or control (C) groups. No differences were found between L and C groups in both farms (p &gt, 0.05) for parity, pregnancy rate, duration of pregnancy, farrowing rate, and litter size (total, alive, and stillborn piglets). Farrowing rates in farm A were 88.8% (n = 383) for control and 89.6% (n = 441, p = 0.67) for the LED group. In farm B, farrowing rates were C:90.5% (n = 1030) and L: 90.1% (n = 1101, p = 0.48). In farm A, total born piglets were 8.69 ± 0.11 for C and 8.71 ± 0.11 for L (p = 0.87). In farm B, the results were 8.72 ± 0.7 for C and 8.70 ± 0.06 (p = 0.82) for L. Under the production conditions for the Iberian breed, the photostimulation with red LED light using Duroc pig seminal doses was not effective in improving the fertility of Iberian sows.

Artificial targeted illumination has a pivotal role in reproductive processes of poultry. The light-absorption mechanism in birds consists of 2 main components: the eye (retinal photoreceptors) and extraretinal photoreceptors located in the brain. Previous studies conducted on hens have shown that photostimulation of brain extraretinal photoreceptors elevates reproductive activity, whereas retinal photostimulation suppresses it. We tested the effect of targeted differential photostimulation (TDP) on reproductive activities of broiler breeder males. Fifty broiler breeder roosters (Ross), 21 wk of age, were divided into 5 environmentally controlled light-treatment rooms (n = 10) equipped with individual cages. Rooms 1 and 2 had 2 parallel lighting systems consisting of red light (630 nm) and green light (514 nm), and rooms 3 and 4 had parallel red and blue (456 nm) lighting systems. Room 5, illuminated with white light, served as the control. Birds of all groups were kept under short day (6L:18D) for 2 wk with both lighting systems in each treatment room turned on. At 23 wk of age, birds were photostimulated by gradually increasing one of the lighting systems to 14 h of light in each room, while the other lighting system was left on short day (6L:18D). Weekly semen samples were collected until 65 wk of age and analyzed for volume, motility, concentration and vitality. Monthly blood samples were drawn for plasma hormone assays. At 65 wk of age, roosters were euthanized and hypothalamus, pituitary gland, retina and testes samples were taken for mRNA expression analysis. TDP using long-day red light and short-day green light significantly increased reproductive performance, manifested by higher semen volume, motility and concentration, and testis weight; furthermore, this group had higher plasma testosterone levels, higher GnRH mRNA expression in the hypothalamus, lower levels of aromatase in the testes, and lower mRNA expression of hypothalamic serotonin transporter, and of pituitary prolactin and its receptors in the testes. This is the first study showing a positive effect of TDP on reproduction of broiler breeder roosters.

Intraventricular hemorrhage (IVH) is the most fatal form of brain injury in adults and infants. However, therapy of IVH is very limited, and new strategies are needed to reduce hematoma expansion. For the first time, in our experiments on mice with IVH, we clearly demonstrate that transcranial low-level infra-red photostimulation (PS) accelerates the red blood cells (RBCs) evacuation from the ventricles that is associated with reducing the mortality in 1.57 times, improving emotional status and recovery of intracranial pressure. We revealed the mechanisms of PS-modulation of lymphatic contractility and tone of the lymphatic vessels underlying the lymphatic clearing functions. Our findings strongly suggest that PS-mediated stimulation of lymphatic clearance of RBCs can be a novel non-invasive, bedside, readily applicable, and commercially viable technology for the treatment of IVH.

The connection between early brain injury and subsequent development of disorders is unknown. Neonatal hypoxia-ischemia (HI) alters circuits associated with subplate neurons (SPNs). SPNs are among the first maturing cortical neurons, project to thalamorecipient layer 4 (L4), and are required for the development of thalamocortical connections. Thus, early HI might influence L4 and such influence might persist. We investigated functional circuits to L4 neurons in neonatal rat HI models of different severities (mild and moderate) shortly after injury and at adolescence. We used laser-scanning photostimulation in slices of auditory cortex during P5–10 and P18–23. Mild injuries did not initially (P6/P7) alter the convergence of excitatory inputs from L2/3, but hyperconnectivity emerged by P8–10. Inputs from L4 showed initial hypoconnectivity which resolved by P8–10. Moderate injuries resulted in initial hypoconnectivity from both layers which resolved by P8–10 and led to persistent strengthening of connections. Inhibitory inputs to L4 cells showed similar changes. Functional changes were mirrored by reduced dendritic complexity. We also observed a persistent increase in similarity of L4 circuits, suggesting that HI interferes with developmental circuit refinement and diversification. Altogether, our results show that neonatal HI injuries lead to persistent changes in intracortical connections.

Stimulation of the visual cortex is a promising method for the restoration of vision, covering a broad range of patients with visual impairments. Optogenetic stimulation has the potential to advance visual restoration. To design better optogenetic implants, an appropriate combination of specific cell types and optogenetic proteins should be identified. Here, we developed a photo-stimulator system and characterized two combinations in the primary visual cortex: targeting excitatory neurons with the red-light-sensitive opto-stimulator chrimsonR and targeting parvalbumin-positive inhibitory neurons with the red-light-sensitive opto-silencer eNpHR-3.0. A custom-developed system consisting of a mini-LED device emitting red-shift light and two convex lenses was installed above the V1 for light focus. We recorded and compared the electrophysiological responses to both visual stimuli and photostimulation. The optogenetic stimulations resulted in a slightly larger number of silence-evoked neurons than excitation-evoked neurons, with a similar number of neurons that responded to visual stimuli.

Hypersomnolence disorder (HD) is characterized by excessive sleep, which is a common sequela following stroke, infection, or tumorigenesis. HD is traditionally thought to be associated with lesions of wake-promoting nuclei. However, lesions of a single wake-promoting nucleus, or even two simultaneously, did not exert serious HD. Therefore, the specific nucleus and neural circuitry for HD remain unknown. Here, we observed that the paraventricular nucleus of the hypothalamus (PVH) exhibited higher c-fos expression during the active period (23:00) than during the inactive period (11:00) in mice. Therefore, we speculated that the PVH, in which most neurons are glutamatergic, may represent one of the key arousal-controlling centers. By using vesicular glutamate transporter 2 (vglut2Cre) mice together with fiber photometry, multichannel electrophysiological recordings, and genetic approaches, we found that PVHvglut2 neurons were most active during wakefulness. Chemogenetic activation of PVHvglut2 neurons induced wakefulness for 9 hr, and photostimulation of PVHvglut2→parabrachial complex/ventral lateral septum circuits immediately drove transitions from sleep to wakefulness. Moreover, lesioning or chemogenetic inhibition of PVHvglut2 neurons dramatically decreased wakefulness. These results indicate that the PVH is critical for arousal promotion and maintenance.

Interneurons are critical for information processing in the cortex. In vitro optogenetic studies in mouse primary visual cortex (V1) have sketched the connectivity of a local neural circuit comprising excitatory pyramidal neurons and distinct interneuron subtypes that express parvalbumin (Pvalb+), somatostatin (SOM+), or vasoactive intestinal peptide (VIP+). However, in vivo studies focusing on V1 orientation tuning have ascribed discrepant computational roles to specific interneuron subtypes. Here, we sought to clarify the differences between interneuron subtypes by examining the effects of optogenetic activation of Pvalb+, SOM+, or VIP+ interneurons on contrast tuning of V1 neurons while also accounting for cortical depth and photostimulation intensity. We found that illumination of the cortical surface produced a similar spectrum of saturating additive photostimulation effects in all 3 interneuron subtypes, which varied with cortical depth rather than light intensity in Pvalb+ and SOM+ cells. Pyramidal cell modulation was well explained by a conductance-based model that incorporated these interneuron photostimulation effects.

As neuronal subtypes are increasingly categorized, delineating their functional role is paramount. The preBötzinger Complex (preBötC) subpopulation expressing the neuropeptide somatostatin (SST) is classified as mostly excitatory, inspiratory-modulated and not rhythmogenic. We further characterized their phenotypic identity; 87% were glutamatergic and the balance were glycinergic and/or GABAergic. We then used optogenetics to investigate their modulatory role in both anesthetized and freely moving mice. In anesthetized mice, short photostimulation (100 ms) of preBötC SST+ neurons modulated breathing-related variables in a combinatory phase- and state-dependent manner; changes in inspiratory duration, inspiratory peak amplitude (Amp), and phase were different at higher (≥2.5 Hz) vs. lower (f in normoxia, hypoxia, or hypercapnia, and increased Amp and produced a phase advance, which was similar to the results in anesthetized mice when f≥2.5 Hz. We conclude that preBötC SST+ neurons are a key mediator of the extraordinary and essential lability of breathing pattern.Key points summaryWe transfected preBötzinger Complex (preBötC) somatostatin-expressing (SST+) neurons, which modulate respiratory pattern, but are not rhythmogenic, with channelrhodopsin to investigate phase- and state-dependent modulation of breathing pattern in anesthetized and freely behaving mice in normoxia, hypoxia, and hypercapnia.In anesthetized mice, photostimulation of preBötC SST+ neurons during inspiration increased inspiratory duration and amplitude regardless of baseline breathing frequency, f.In anesthetized mice with low f (+ neurons during expiration evoked either phase advance or phase delay, whereas in anesthetized mice with high f (≥2.5 Hz) and in freely behaving mice in normoxia, hypoxia, or hypercapnia, photostimulation always evoked phase advance.Phase- and state-dependency is a function of overall breathing network excitability.The f-dependent probabilistic modulation of breathing pattern by preBötC SST+ neurons was unexpected, requiring reconsideration of current models of preBötC function, which neither predict nor can readily account for such responses.

The topographic organization of sensory cortices is a prominent feature, but its functional role remains unclear. Particularly, how activity is integrated within a cortical area depending on its topography is unknown. Here, we trained mice expressing channelrhodopsin in cortical excitatory neurons to track a bar photostimulation that rotated smoothly over the primary somatosensory cortex (S1). When photostimulation was aimed at vS1, the area which contains a contiguous representation of the whisker array at the periphery, mice could learn to discriminate angular positions of the bar to obtain a reward. In contrast, they could not learn the task when the photostimulation was aimed at the representation of the trunk and legs in S1, where neighboring zones represent distant peripheral body parts, introducing discontinuities. Mice demonstrated anticipation of reward availability, specifically when cortical topography enabled to predict future sensory activation. These results are particularly helpful for designing efficient cortical sensory neuroprostheses.TeaserOptogenetic stimulation sweeping the cortical surface: A way to provide precise sensory information and guide behaviour.

Genetic selection for earlier sexual maturation and extended production cycles in laying hens has significantly improved reproductive efficiency. While limited emphasis has been placed on the underlying physiological changes, we hypothesize that modifications in the control of the hypothalamic-pituitary gonadal (HPG) axis have occurred. Thus, three strains of White leghorn derivatives were followed from hatch to 100 weeks of age (woa), including Lohmann LSL-lite (n = 120) as current commercial hens, heritage Shaver White leghorns (n = 100) as 2000s commercial equivalents, and Smoky Joe hens (n = 68) as 1960s commercial equivalents. Body weight (BW) and egg production were monitored, and blood samples were collected throughout to monitor estradiol (E2) concentrations. Tissue samples were collected at 12, 17, 20, 25, 45, 60, 75, and 100 woa to capture changes in mRNA levels of key genes involved in the HPG axis and monitor ovarian follicular pools. All hens, regardless of strain, age or photoperiod laid their first egg within a 64-gram BW window and, as E2 levels increased prior to photostimulation (PS) in Lohmann and Shaver hens, a metabolic trigger likely induced sexual maturation. However, increased levels of Opsin 5 (OPN5) were observed during the maturation period. Although an elevation in gonadotrophin-releasing hormone I (GnRH-I) mRNA levels was associated with early maturation, no changes in gonadotrophin-inhibitory hormone (GnIH) mRNA levels were observed. Nonetheless, a significant shift in pituitary sensitivity to GnRH was associated with maturation. Throughout the trial, Lohmann, Shaver, and Smoky Joe hens laid 515, 417, and 257 eggs, respectively (p < 0.0001). Results show that the extended laying persistency in Lohmann hens was supported by sustained pituitary sensitivity to GnRH-I, recurrent elevations in follicle-stimulating hormone (FSH) mRNA levels, and five cyclical elevations in E2 levels. This was also associated with a consistently higher pool of small white ovarian follicles. In summary, our results demonstrate first that, regardless of photoperiodic cues, meeting a specific narrow body weight threshold is sufficient to initiate sexual maturation in Leghorn chicken derivatives. Furthermore, recurrent increases in E2 and FSH may be the key to sustain extended laying period, allowing modern layers to double their reproductive capacity compared to their 1960s-counterparts.

The development of GABAergic interneurons is important for the functional maturation of cortical circuits. After migrating into the cortex, GABAergic interneurons start to receive glutamatergic connections from cortical excitatory neurons and thus gradually become integrated into cortical circuits. These glutamatergic connections are mediated by glutamate receptors including AMPA and NMDA receptors and the ratio of AMPA to NMDA receptors decreases during development. Since previous studies have shown that retinal input can regulate the early development of connections along the visual pathway, we investigated if the maturation of glutamatergic inputs to GABAergic interneurons in the visual cortex requires retinal input. We mapped the spatial pattern of glutamatergic connections to layer 4 (L4) GABAergic interneurons in mouse visual cortex at around postnatal day (P) 16 by laser-scanning photostimulation and investigated the effect of binocular enucleations at P1/P2 on these patterns. Gad2-positive interneurons in enucleated animals showed an increased fraction of AMPAR-mediated input from L2/3 and a decreased fraction of input from L5/6. Parvalbumin-expressing (PV) interneurons showed similar changes in relative connectivity. NMDAR-only input was largely unchanged by enucleation. Our results show that retinal input sculpts the integration of interneurons into V1 circuits and suggest that the development of AMPAR- and NMDAR-only connections might be regulated differently.

Iodothyronine deiodinases are enzymes capable of activating and inactivating thyroid hormones (THs) and have an important role in regulating TH action in tissues throughout the body. Three types of deiodinases (D1, D2, and D3) were originally defined based on their biochemical characteristics. Cloning of the first complementary DNAs in the 1990s (Dio1 in rat and dio2 and dio3 in frog) allowed to confirm the existence of 3 distinct enzymes. Over the years, increasing genomic information revealed that deiodinases are present in all chordates, vertebrates, and nonvertebrates and that they can even be found in some mollusks and annelids, pointing to an ancient origin. Research in nonmammalian models has substantially broadened our understanding of deiodinases. In relation to their structure, we discovered for instance that biochemical properties such as inhibition by 6-propyl-2-thiouracil, stimulation by dithiothreitol, and temperature optimum are subject to variation. Data from fish, amphibians, and birds were key in shifting our view on the relative importance of activating and inactivating deiodination pathways and in showing the impact of D2 and D3 not only in local but also whole body T3 availability. They also led to the discovery of new local functions such as the acute reciprocal changes in D2 and D3 in hypothalamic tanycytes upon photostimulation, involved in seasonal rhythmicity. With the present possibilities for rapid and precise gene silencing in any species of interest, comparative research will certainly further contribute to a better understanding of the importance of deiodinases for adequate TH action, also in humans. ispartof: ENDOCRINOLOGY vol:162 issue:6 ispartof: location:United States status: published

Vagus nerve stimulation has shown many benefits for disease therapies but current approaches involve imprecise electrical stimulation that gives rise to off-target effects, while the functionally relevant pathways remain poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate targeted neural communication with light-sensitive actuators (opsins) and can be targeted to organs of interest based on the location of viral delivery. Here, we tested whether retrograde adeno-associated virus (rAAV2-retro) injected in the heart can be used to selectively express opsins in vagus nerve fibers controlling cardiac function. Furthermore, we investigated whether perturbations in cardiac function could be achieved with photostimulation at the cervical vagus nerve. Viral injection in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Photostimulation using both one-photon stimulation and two-photon holography with a GRIN-lens incorporated nerve cuff, was tested on the pilot-cohort of injected mice. Changes in heart rate, surface electrocardiogram, and respiratory responses were observed in response to both one- and two-photon photostimulation. The results demonstrate feasibility of retrograde labeling for organ targeted optical neuromodulation.

The response of plants to the spaceflight environment and microgravity is still not well understood, although research has increased in this area. Even less is known about plants’ response to partial or reduced gravity levels. In the absence of the directional cues provided by the gravity vector, the plant is especially perceptive to other cues such as light. Here, we investigate the response of Arabidopsis thaliana 6-day-old seedlings to microgravity and the Mars partial gravity level during spaceflight, as well as the effects of red-light photostimulation by determining meristematic cell growth and proliferation. These experiments involve microscopic techniques together with transcriptomic studies. We demonstrate that microgravity and partial gravity trigger differential responses. The microgravity environment activates hormonal routes responsible for proliferation/growth and upregulates plastid/mitochondrial-encoded transcripts, even in the dark. In contrast, the Mars gravity level inhibits these routes and activates responses to stress factors to restore cell growth parameters only when red photostimulation is provided. This response is accompanied by upregulation of numerous transcription factors such as the environmental acclimation-related WRKY-domain family. In the long term, these discoveries can be applied in the design of bioregenerative life support systems and space farming., This research was funded by the Agencia Estatal de Investigación of the Spanish Ministry of Science an Innovation, Grants #ESP2015-64323-R and #RTI2018-099309-B-I00 (co-funded by EU-ERDF) to F.J.M., by pre-doctoral fellowships to A.M. and A.V. from the Spanish National Program for Young Researchers Training (MINECO, Ref. #BES-2013-063933, #BES-2016-077976) and the Seedling Growth Project for ISS experimentation #LSRA2009-0932/ 1177, a shared project of ESA-ELIPS Program and NASA, to F.J.M. and J.Z.K. In addition, J.Z.K. is funded by Grants NNX12A065G and 80NSSC17K0546. This research is related to the Space Omics TT funded by the ESA contract 4000131202/20/NL/PG to R.H.

A detailed theoretical analysis of low-power, high-frequency and temporally precise optogenetic inhibition of neuronal spiking, with red-shifted opsins namely, NpHR, eNpHR3.0 and Jaws, has been presented. An accurate model for inhibition of spiking in these opsins expressed hippocampal neurons that includes the important rebound activity of chloride ions across the membrane has been formulated. The effect of various parameters including irradiance, pulse width, frequency, opsin-expression density and chloride concentration has been studied in detail. Theoretical simulations are in very good agreement with reported experimental results. The chloride concentration gradient directly affects the photocurrent and inhibition capacity in all three variants. eNpHR3.0 shows smallest inhibitory post-synaptic potential plateau at higher frequencies. The time delay between light stimulus and target spike is crucial to minimize irradiance and expression density thresholds for suppressing individual spike. Good practical values of photostimulation parameters have been obtained empirically for peak photocurrent, time delay and 100% spiking inhibition, at continuous and pulsed illumination. Under continuous illumination, complete inhibition of neural activity in Jaws-expressing neurons takes place at minimum irradiance of 0.2 mW mm-2 and expression density of 0.2 mS cm-2, whereas for pulsed stimulation, it is at minimum irradiance of 0.6 mW mm-2 and 5 ms pulse width, at 10 Hz. It is shown that Jaws and eNpHR3.0 are able to invoke single spike precise inhibition up to 160 and 200 Hz, respectively. The study is useful in designing new experiments, understanding temporal spike coding and bidirectional control, and curing neurological disorders.

Targeted in ovo green light (GL) photostimulation during the last days of broiler egg incubation increases embryonic expression of the somatotropic axis, similar to in ovo green light photostimulation from embryonic day (ED) 0 to the end of incubation. The aim of this study was to examine the effect of selected in ovo GL photostimulation periods on post-hatch broiler growth. Four hundred twenty fertile broiler eggs were divided into 7 treatment groups: the first incubated in the dark (standard conditions) as a negative control; the second incubated under monochromatic GL from ED0-ED20 (positive control); the third group incubated under monochromatic GL light from ED15-ED20; the fourth, fifth and sixth groups were incubated under monochromatic GL on ED16, ED17, and ED18, respectively; and the seventh group was incubated under monochromatic GL from ED18-ED20. All illumination was provided intermittently using LED lamps. After hatch, all chicks were transferred to a controlled room under standard rearing conditions. The group incubated under green light from ED18 until hatch showed similar results to the positive control group in body weights, as well as breast muscle weights (as % of body weights), and an elevation in the somatotropic axis activity during the experiment. We suggest that broiler embryos can be exposed to in ovo GL photostimulation from ED18 until hatch (hatching period), and still exhibit the same performance as obtained by photostimulation from d 0 of incubation.

Summary Two-photon (2-P) all-optical approaches combine in vivo 2-P calcium imaging and 2-P optogenetic modulations. Here, firstly, we combined in vivo juxtacellular recordings and GCaMP6f-based 2-P calcium imaging in mouse visual cortex to tune our detection algorithm towards a 100% specific identification of action potential-related calcium transients. Secondly, we minimized photostimulation artifacts by using extended-wavelength-spectrum laser sources for optogenetic stimulation. We achieved artifact-free all-optical experiments performing optogenetic stimulation from 1100 nm to 1300 nm. Thirdly, we determined the spectral range for maximizing efficacy until 1300 nm. The rate of evoked transients in GCaMP6f/C1V1-co-expressing cortical neurons peaked already at 1100 nm. By refining spike detection and defining 1100 nm as the optimal wavelength for artifact-free and effective GCaMP6f/C1V1-based all-optical physiology, we increased the translational value of these approaches, e.g., for the development of network-based therapies., Graphical abstract, Highlights • We developed an algorithm for 100 %-specific identification of AP-related calcium transients • Artifact-free all-optical experiments can be achieved from 1100 nm to 1300 nm • Efficacy of C1V1 excitation does not increase beyond 1100 nm until 1300 nm, Optical Imaging; Molecular Physiology; Cellular Neuroscience

Cell membrane perturbation is a common way to stimulate cells by using external actuators. Recently, nanotechnology has added a number of new strategies for doing this, enlarging the scope and the range of mechanisms involved. Here, we describe a number of possible perturbation actions that are driven by light, and we try to capture the underlying phenomena. The discussion is based on the simple equivalent circuit model for the cell membrane.

Estudis previs realitzats en altres espècies, han demostrat que la fotoestimulació amb llum vermella augmenta la motilitat espermàtica. A més, altres estudis indiquen que la capacitat fecundant dels espermatozoides pot incrementar després de la irradiació. Tot i que els mecanismes a través dels quals la llum exerceix els seus efectes sobre els espermatozoides es desconeixen en gran mesura, l'evidència creixent atorga als fotosensibilitzadors cel·lulars endògens, especialment els presents en les mitocòndries, un paper fonamental. Per tot això, l'objectiu d'aquesta tesi doctoral va ser avaluar els efectes de diferents protocols de fotoestimulació amb un llum LED vermella a una longitud d'ona de 620-630 nm sobre els paràmetres de qualitat espermàtica, en semen de ruc i cavall; fresc, refrigerat i congelat/descongelat, a més d'avaluar la influència del medi i del color de la palleta utilitzada en els efectes observats, i determinar si els efectes produïts per l'estimulació amb llum vermella en els espermatozoides estan provocats per canvis en la funció mitocondrial induïts per l'acció de la llum sobre els citocroms mitocondrials com el citocrom C. Amb aquest propòsit es van dissenyar cinc estudis: en el primer es van avaluar els efectes de diferents patrons d'estimulació amb llum LED vermella sobre els paràmetres de motilitat espermàtica, la integritat de la membrana, el potencial de membrana mitocondrial i els nivells intracel·lulars de ROS en semen de ruc fresc i refrigerat. En el segon estudi es van analitzar els efectes de diferents patrons d'irradiació amb llum vermella sobre la motilitat espermàtica, les subpoblacions d'espermatozoides mòbils, el potencial de membrana mitocondrial, la taxa de consum d'O2, els nivells intracel·lulars d'ATP, la integritat de la membrana i la integritat de l'ADN espermàtic en semen fresc de cavall. El tercer estudi tenia com objectiu determinar si la irradiació amb llum vermella de l’esperma de cavall congelada-descongelada podria millorar la qualitat espermàtica i augmentar la seva capacitat de resistir la incubació a 38 °C per 120 minuts. En el quart estudi, es va examinar si el color de la palleta i / o la terbolesa del medi utilitzat per a la refrigeració del semen afectaven a la resposta induïda per l'estimulació amb llum vermella en el semen de cavall refrigerat. Finalment, en el cinquè treball es va estudiar si els efectes produïts per la fotoestimulació estaven relacionats amb canvis en la funció mitocondrial induïts sobre els citocroms mitocondrials, com el citocrom C. Els resultats obtinguts en aquesta Tesi Doctoral indiquen que la irradiació dels espermatozoides de ruc i cavall amb llum LED vermella modifica alguns paràmetres de motilitat espermàtica, l'estructura de les subpoblacions d'espermatozoides mòbils, la taxa de consum d'O2, l'activitat de la citocrom C oxidasa i els nivells intracel·lulars de ROS, sense afectar la integritat de la membrana plasmàtica, l'acrosoma o l'ADN espermàtic, millorant a més la seva resiliència a l'estrès tèrmic. Aquests efectes varien segons el temps i el patró d'exposició, l'espècie, el tipus de semen, el color de la palleta i la terbolesa del medi utilitzat. En conclusió, aquesta Tesi Doctoral mostra que la irradiació dels espermatozoides de ruc i cavall amb llum LED vermella (620-630 nm) és un mètode segur que produeix una millora sobre alguns paràmetres de qualitat espermàtica en el semen de burro i cavall, efectes que estan relacionats amb l'acció de la llum sobre els fotosensibilitzadors mitocondrials, com el citocrom C que modifica l'activitat de la cadena d'electrons mitocondrial. Així, la fotoestimualació podria tenir un impacte positiu en el rendiment reproductiu i en les tecnologies de reproducció assistida en aquestes espècies, així com també en la conservació del semen. Estudios previos realizados en otras especies, han demostrado que la estimulación con luz roja aumenta la motilidad espermática. Además, otros estudios han sugerido que la capacidad fecundante de los espermatozoides puede incrementarse después de la irradiación. Aunque los mecanismos a través de los cuales la luz ejerce sus efectos sobre los espermatozoides se desconocen en gran medida, la evidencia creciente otorga a los fotosensibilizadores celulares endógenos, especialmente los presentes en las mitocondrias, un papel crucial. Por todo ello, el objetivo de esta Tesis Doctoral fue evaluar los efectos de diferentes protocolos de fotoestimulación con un luz LED roja a una longitud de onda de 620-630 nm sobre los parámetros de calidad espermática, en semen de burro y caballo; fresco, refrigerado y congelado/descongelado, además de evaluar la influencia del medio/diluyente y del color de la pajuela utilizada en los efectos observados, así como estudiar si los efectos producidos por la estimulación con luz roja en los espermatozoides están provocados por cambios en la función mitocondrial inducidos por la acción de la luz sobre los citocromos mitocondriales como el citocromo C. Con este propósito, se diseñaron cinco estudios: en el primero se evaluaron los efectos de diferentes patrones de estimulación con luz LED roja sobre los parámetros de motilidad espermática, la integridad de la membrana plasmática, el potencial de membrana mitocondrial y los niveles intracelulares de ROS en semen de burro fresco y refrigerado. En el segundo estudio, se analizaron los efectos de diferentes patrones de irradiación con luz roja sobre la motilidad espermática, las subpoblaciones de espermatozoides móviles, el potencial de membrana mitocondrial, la tasa de consumo de O2, los niveles intracelulares de ATP, la integridad de la membrana plasmática y la integridad del ADN espermático en semen fresco de caballo. En el tercer estudio se propuso determinar si la irradiación con luz roja del semen de caballo congelado-descongelado podría mejorar la calidad espermática y aumentar su capacidad de resistir al estrés térmico. En el cuarto estudio se examinó si el color de la pajuela y/o la turbidez del diluyente utilizado para la refrigeración del semen afectaba a la respuesta inducida por la estimulación con luz roja en semen de caballo refrigerado. Finalmente, en el quinto trabajo se estudió si los efectos producidos por la fotoestimulación estaban relacionados con cambios en la función mitocondrial inducidos sobre los citocromos mitocondriales, como el citocromo C. Los resultados presentados en esta Tesis Doctoral indican que la irradiación de los espermatozoides de burro y caballo con luz LED roja modifica algunos parámetros de motilidad espermática, la estructura de las subpoblaciones de espermatozoides móviles, la tasa de consumo de O2, la actividad de la citocromo C oxidasa y los niveles intracelulares de ROS, sin afectar a la integridad de la membrana plasmática, el acrosoma o el ADN espermático, mejorando además su resiliencia al estrés térmico. Estos efectos varían según el tiempo y patrón de exposición, la especie, el tipo de semen, el color de la pajuela y la turbidez del diluyente utilizado. En conclusión, esta Tesis Doctoral muestra que la irradiación de los espermatozoides de burro y caballo con luz LED roja (620-630 nm) es un método seguro que produce una mejora sobre algunos parámetros de calidad espermática en el semen de burro y caballo, efectos que están relacionados con la acción de la luz sobre los fotosensibilizadores mitocondriales, como el citocromo C que modifica la actividad de la cadena de electrones mitocondrial. Así, la fotoestimulación podría tener un impacto positivo en el rendimiento reproductivo y en las tecnologías de reproducción asistida en estas especies, así como también en la conservación de semen. Previous studies carried out in other species, showed that stimulating sperm with red light increases their motility. In addition, other studies indicate sperm fertilizing ability can be increased after irradiation. While the mechanisms through which light exerts its effects on sperm are largely unknown, mounting evidence gives endogenous cellular photosensitizers, especially those present in mitochondria, a crucial role. Therefore, the objective of this Dissertation was to evaluate the effects of different irradiation protocols using a red LED light at a wavelength of 620-630 nm on freshly ejaculated, liquid-stored and frozen-thawed sperm from stallions and jackasses. The potential influence of the medium/extender and the colour of the straw was also investigated, as well as whether the effects produced by red light resulted from changes in mitochondrial function via modulation of cytochromes, such as cytochrome C. For this purpose, five studies were designed. In the first one, the effects of different red LED light patterns on sperm motility, integrity of plasma membrane, mitochondrial membrane potential, and intracellular ROS levels were investigated in fresh and stored donkey semen. The second study analysed the effects of separate red light irradiation patterns on motility, membrane integrity, motile sperm subpopulations, mitochondrial membrane potential, O2 consumption rate, intracellular ATP levels and the integrity of DNA of fresh horse sperm. The third study sought to determine whether red light irradiation of frozen-thawed horse semen could improve sperm quality and increase their ability to withstand incubation. In the fourth study, whether the colour of the straw and / or the turbidity of the medium affected the response induced by red light stimulation was examined in liquid-stored horse semen. Finally, the fifth work studied whether the effects produced by red light on sperm were related to changes in mitochondrial function induced by mitochondrial cytochromes, such as cytochrome C. The results shown in this Dissertation indicate that irradiation of donkey and horse sperm with red LED light modifies their sperm motility, the structure of motile sperm subpopulations, the rate of O2 consumption, the activity of cytochrome C oxidase and intracellular levels of ROS, without affecting the integrity of plasma membrane and acrosome or sperm DNA. In addition, stimulation with red light also improves the sperm resilience to thermal stress. These effects vary according to the time and pattern of exposure, species, type of semen, colour of the straw and the turbidity of the medium. In conclusion, this Dissertation supports that irradiation of donkey and horse sperm with red LED light (620-630 nm) is a safe method that may improve some sperm quality parameters in donkey and horse semen. These effects are related to the action of light on mitochondrial photosensitizers, such as cytochrome C, which modifies the activity of the mitochondrial electron chain. Therefore, irradiation of sperm with red light could have a positive impact on reproductive performance and assisted reproductive technologies in these species, as well as on semen preservation. Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina i Sanitat Animals