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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.

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.

Funding: Engineering and Physical Sciences Research Council (Grant Number(s): EP/R010595/1). National Science Foundation (Grant Number(s): 1706207). Defense Sciences Office, DARPA (Grant Number(s): N66001-17-C-4012). Leverhulme Trust (Grant Number(s): RPG-2017-231). Alexander von Humboldt-Stiftung (Grant Number(s): Humboldt Professur). National Research Foundation of Korea (GrantNumber(s): 2017R1A6A3A03012331). China Sponsorship Council. Optogenetic control of retinal cells transduced with light-sensitive channelrhodopsins can enable restoration of visual perception in patients with vision loss. However, a light intensity orders of magnitude higher than ambient light conditions is required to achieve robust cell activation. Relatively bulky wearable light amplifiers are currently used to deliver sufficient photon flux (>1016 photons/cm2/s in a ±10° emission cone) at a suitable wavelength (e.g., 600 nm for channelrhodopsin ChrimsonR). Here, ultrahigh brightness organic light-emitting diodes (OLEDs) with highly directional emission are developed, with the ultimate aim of providing high-resolution optogenetic control of thousands of retinal cells in parallel from a compact device. The orange-emitting phosphorescent OLEDs use doped charge transport layers, generate narrowband emission peaking at 600 nm, and achieve a luminance of 684 000 cd m–2 at 15 V forward bias. In addition, tandem-stack OLEDs with a luminance of 1 152 000 cd m–2 and doubled quantum efficiency are demonstrated, which greatly reduces electrical and thermal stress in these devices. At the photon flux required to trigger robust neuron firing in genetically modified retinal cells and when using heat sinking and realistic duty cycles (20% at 12.5 Hz), the tandem-stack OLEDs therefore show a greatly improved half-brightness lifetime of 800 h. Publisher PDF

There is compelling evidence that midbrain dopamine (DA) neurons and their projections to the ventral striatum provide a mechanism for motivating reward-seeking behavior, and for utilizing information about unexpected rewards (prediction errors, RPEs) to guide behavior based on current, rather than historical, outcomes. When this mechanism is compromised in addictions, it may produce patterns of maladaptive behavior that remain obdurate in the face of contrary information and even adverse consequences. Nonetheless, DAergic contributions to performance on behavioral tasks that rely on an ability to flexibly update stimulus-reward relationships remains incomplete understood. In the current study, we used a discrimination and reversal paradigm to monitor subsecond DA release in mouse NAc core (NAc) using in vivo fast-scan cyclic voltammetry (FSCV). We observed post-choice elevations in phasic NAc DA release; however, increased DA transients were only evident during early reversal when mice made responses at the newly-rewarded stimulus. Based on this finding, we used in vivo optogenetic (halorhodopsin (eNpHR3.0)) photosilencing and (Channelrhodopsin2 (ChR2)) photostimulation to assess the effects of manipulating VTA-DAergic fibers in the NAc on reversal performance. Photosilencing the VTA→NAc DAergic pathway during early reversal increased errors, while photostimulation did not demonstrably affect behavior. Taken together, these data provide additional evidence of the importance of NAc DA release as a neural substrate supporting adjustments in learned behavior after a switch in expected stimulus-reward contingencies. These findings have possible implications for furthering understanding the role of DA in persistent, maladaptive decision-making characterizing addictions.

Key points Causal relationships between central cardiovascular pathways and sympathetic vasomotor tone have not been evidenced. This study aimed to verify the sympathoexcitatory role of hypothalamic paraventricular nucleus neurons that project to the rostral ventrolateral medulla (PVN-RVLM neurons). By using optogenetic techniques, we demonstrated that stimulation of PVN-RVLM glutamatergic neurons increased renal sympathetic nerve activity and arterial pressure via, at least in part, stimulation of RVLM C1 neurons in rats. This monosynaptic pathway may function in acute sympathetic adjustments to stressors and/or be a component of chronic sympathetic hyperactivity in pathological conditions such as heart failure. Abstract The rostral ventrolateral medulla (RVLM), which is known to play an important role in regulating sympathetic vasomotor tone, receives axonal projections from the hypothalamic paraventricular nucleus (PVN). However, no studies have proved that excitation of the PVN neurons that send axonal projections to the RVLM (PVN-RVLM neurons) causes sympathoexcitation. This study aimed to directly examine the sympathoexcitatory role of PVN-RVLM neurons. Male rats received microinjections into the PVN with an adeno-associated virus (AAV) vector that encoded a hybrid of channelrhodopsin-2/1 with the reporter tdTomato (ChIEF-tdTomato), or into the RVLM with a retrograde AAV vector that encoded a channelrhodopsin with green fluorescent protein (ChR2-GFPretro ). Under anaesthesia with urethane and α-chloralose, photostimulation (473 nm wavelength) of PVN-RVLM neurons, achieved by laser illumination of either RVLM of ChIEF-tdTomato rats (n = 8) or PVN of ChR2-GFPretro rats (n = 4), elicited significant renal sympathoexcitation. Immunofluorescence confocal microscopy showed that RVLM adrenergic C1 neurons of ChIEF-tdTomato rats were closely associated with tdTomato-labelled, PVN-derived axons that contained vesicular glutamate transporter 2. In another subset of anaesthetized ChIEF-tdTomato rats (n = 6), the renal sympathoexcitation elicited by photostimulation of the PVN was suppressed by administering ionotropic glutamate receptor blockers into the RVLM. These results demonstrate that excitation of PVN-RVLM glutamatergic neurons leads to sympathoexcitation via, at least in part, stimulation of RVLM C1 neurons.

SummaryBackground The algal protein Channelrhodopsin-2 (ChR2) has been widely used in recent years in optogenetic technique to investigate the functions of complex neuronal networks through minimally invasive and temporally precise photostimulation of genetically defined neurons. However, as with any other new technique, current optogentic approaches have various limitations. In addition, how ChR2 may behave in response to complex biochemical changes associated with various physiological/pathological conditions is largely unknown. Aim In this study, we investigated whether a change in redox state of the cell affects the activity of ChR2 channels. Methods Whole-cell patch-clamp recordings were used to examine the effect of reducing and oxidizing agents on ChR2 currents activated by blue light. Results We show that the reducing agent dithiothreitol (DTT) dramatically potentiates the ChR2 currents in a reversible and concentration-dependent manner. Glutathione, an endogenous reducing agent, shows a similar effect on ChR2 currents. The oxidizing agent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) has no effect on ChR2 currents by itself; however, it completely reverses the potentiating effect of DTT. DTT also causes a shift in the current–voltage relationship by 23 ± 4.31 mV, suggesting a change in ion selectivity. Conclusion Taken together, these data suggest that redox modification of ChR2 plays an important role in its sensitivity to the light stimulation. Our findings not only help for a better understanding of how ChR2 may behave in physiological/pathological conditions where changes in redox state are common, but also provide a new direction for further optimization of this important opsin.

Photodynamic therapy (PDT) and low level laser therapy (LLLT) may mutually improve the outcomes on the healing process of chronic wounds and other skin pathologies, through processes known to stimulate the proliferation of dermal cellular structures, as well as antimicrobial application. This study proposes the use of nanoemulsion containing aluminium phthalocyanine chloride (ClAlPc) as photosensitizer (PS), to establish the most appropriate protocol for photostimulation in human skin biopsies, associated to type I collagen and elastin production. The combined effect of PS and light (diode laser at 670 nm) at three different doses is compared to the effect of light itself at doses of 70, 140 and 700 mJ ċ cm-2 , 7 and 14 days after irradiation. Histological analysis reveals the increase in collagen and elastin, higher than 20%, 14 days after treatment with PS and light at 140 mJ ċ cm-2 . Higher doses of light promote an inhibitory effect, leading to tissue degradation. In addition, the expression levels of the enzymes MMP-2 and MMP-9 (Gelatinases A and B - participant in various processes including tumoral progression and wound healing) are detected by gelatin zymography, reinforcing the efficacy of the combined treatment with PS and light at the intermediate dose.

Key points Using functional mapping assays, we conducted a quantitative assessment of both excitatory and inhibitory synaptic laminar connections to excitatory neurons in layers 2/3–6 of the mouse visual cortex (V1). Laminar-specific synaptic wiring diagrams of excitatory neurons were constructed on the basis of circuit mapping. The present study reveals that that excitatory and inhibitory synaptic connectivity is spatially balanced across excitatory neuronal networks in V1. Abstract In the mammalian neocortex, excitatory neurons provide excitation in both columnar and laminar dimensions, which is modulated further by inhibitory neurons. However, our understanding of intracortical excitatory and inhibitory synaptic inputs in relation to principal excitatory neurons remains incomplete, and it is unclear how local excitatory and inhibitory synaptic connections to excitatory neurons are spatially organized on a layer-by-layer basis. In the present study, we combined whole cell recordings with laser scanning photostimulation via glutamate uncaging to map excitatory and inhibitory synaptic inputs to single excitatory neurons throughout cortical layers 2/3–6 in the mouse primary visual cortex (V1). We find that synaptic input sources of excitatory neurons span the radial columns of laminar microcircuits, and excitatory neurons in different V1 laminae exhibit distinct patterns of layer-specific organization of excitatory inputs. Remarkably, the spatial extent of inhibitory inputs of excitatory neurons for a given layer closely mirrors that of their excitatory input sources, indicating that excitatory and inhibitory synaptic connectivity is spatially balanced across excitatory neuronal networks. Strong interlaminar inhibitory inputs are found, particularly for excitatory neurons in layers 2/3 and 5. This differs from earlier studies reporting that inhibitory cortical connections to excitatory neurons are generally localized within the same cortical layer. On the basis of the functional mapping assays, we conducted a quantitative assessment of both excitatory and inhibitory synaptic laminar connections to excitatory cells at single cell resolution, establishing precise layer-by-layer synaptic wiring diagrams of excitatory neurons in the visual cortex.

This study sought to characterize the rapid intraovarian mRNA response of key folliculogenic factors that may contribute to the restoration of folliculogenesis during 2-10 days of photostimulation in Siberian hamsters. Adult hamsters were exposed to short photoperiod (8L:16D) for 14 weeks (SD). A subset were then transferred to long photoperiod (16L:8D) for 2 (PT day-2), 4 (PT day-4), or 10 days (PT day-10). Quantitative real-time PCR was used to measure intraovarian mRNA expression of: gonadotropin releasing hormone (GnRH), follicle stimulating hormone β-subunit (FSHβ-subunit), luteinizing hormone β-subunit (LHβ-subunit), FSH and LH receptors, estrogen receptors α and β (Esr1 and Esr2), matrix metalloproteinase (MMP)-2 and -9, anti-Mullerian hormone (AMH), inhibin-α subunit, fibroblast growth factor-2 (FGF-2) and proliferating cell nuclear antigen (PCNA). Compared to SD, plasma FSH concentrations increased on PT day-4 and the number of antral follicles and corpora lutea increased on PT day-10. FSHR and inhibin-α mRNA expression also increased on PT day-4, whereas LHR and proliferation marker PCNA both increased on PT day-10 as compared to SD. Esr1 mRNA increased on PT day-2 and remained significantly increased as compared to SD, whereas Esr1 mRNA increased only on PT day-2, similar to FGF-2 and MMP-2 results. No differences were observed in mRNA expression in ovarian GnRH, FSHβ- and LHβ-subunits, AMH, and MMP-9 mRNA with 2-10 days of photostimulation. Rapid increases in intraovarian FSHR and inhibin-α mRNA and antral follicle/corpora lutea numbers suggest that the ovary is primed to react quickly to the FSH released in response to brief periods of photostimulation.

Purpose The aim of this pilot study, the first of this kind, was to evaluate the safety and efficacy of Subthreshold Yellow Micropulsed Laser (SML) to treat eleven patients with chronic residual subfoveal Retinal Detachment (RD) after surgery to repair Rhegmatogenous Retinal Detachment (RRD). Methods Eleven eyes with residual subfoveal RD after surgery, dating from eight to sixteen months before treatment have been evaluated. Evaluation included visual acuity, Amsler test, ophthalmoscopy, Autofluorescence (AF) and Spectral-Domain Optical Coherence Tomography (SD-OCT). Results After treatment we recorded improved visual acuity and Amsler test, disappearance of subfoveal detachment by ophthalmoscopy, reduced retinal pigment epithelial distress by AF and restored macular retinal profile without neuroretinal alterations by SD-OCT evaluation in nine eyes. Conclusions Photostimulation with SML, selective for the Retinal Pigment Epithelium (RPE), is proved to be painless, effective and safe. The possibility to reabsorb subretinal liquid by foveal and parafoveal RPE photostimulation, without neuroretinal damage, opens new prospects for the therapy of this pathology. If further studies confirm the results of the present pilot study, SML treatment can be considered the first and only non invasive option for chronic residual RD after retinal surgery to repair RRD.

Catecholaminergic neurons of the rostral ventrolateral medulla (RVLM-CA neurons; C1 neurons) contribute to the sympathetic, parasympathetic and neuroendocrine responses elicited by physical stressors such as hypotension, hypoxia, hypoglycemia, and infection. Most RVLM-CA neurons express vesicular glutamate transporter (VGLUT)2, and may use glutamate as a ionotropic transmitter, but the importance of this mode of transmission in vivo is uncertain. To address this question, we genetically deleted VGLUT2 from dopamine-β-hydroxylase-expressing neurons in mice [DβH(Cre/0) ;VGLUT2(flox/flox) mice (cKO mice)]. We compared the in vivo effects of selectively stimulating RVLM-CA neurons in cKO vs. control mice (DβH(Cre/0) ), using channelrhodopsin-2 (ChR2-mCherry) optogenetics. ChR2-mCherry was expressed by similar numbers of rostral ventrolateral medulla (RVLM) neurons in each strain (~400 neurons), with identical selectivity for catecholaminergic neurons (90-99% colocalisation with tyrosine hydroxylase). RVLM-CA neurons had similar morphology and axonal projections in DβH(Cre/0) and cKO mice. Under urethane anesthesia, photostimulation produced a similar pattern of activation of presumptive ChR2-positive RVLM-CA neurons in DβH(Cre/0) and cKO mice. Photostimulation in conscious mice produced frequency-dependent respiratory activation in DβH(Cre/0) mice but no effect in cKO mice. Similarly, photostimulation under urethane anesthesia strongly activated efferent vagal nerve activity in DβH(Cre/0) mice only. Vagal responses were unaffected by α1 -adrenoreceptor blockade. In conclusion, two responses evoked by RVLM-CA neuron stimulation in vivo require the expression of VGLUT2 by these neurons, suggesting that the acute autonomic responses driven by RVLM-CA neurons are mediated by glutamate.

The primary goal of this study was to map the transverse distribution of local excitatory and inhibitory synaptic inputs to mouse lamina I spinal dorsal horn neurons, using laser scanning photostimulation. A sample of lamina II neurons was also studied for comparison. Lamina I neurons received excitatory synaptic input from both laminae I-II and the outer part of III-IV, especially the II/III border region, while the inhibitory input zones were mostly confined within I-II. The excitatory synaptic input zones showed a pronounced medial asymmetry, which was correlated with a matching asymmetry in the dendritic fields of the neurons. Inhibitory input from laminae III-IV was found in a subpopulation of neurons occupying a highly restricted zone, essentially one cell layer thick, immediately below the lamina I/II border, with morphological and physiological properties that were distinct from other laminar populations in the superficial dorsal horn, and that suggest a critical role in interlaminar communication. This subpopulation also received excitatory input from laminae III-IV. Within this subpopulation, inhibitory III-IV input was correlated with the presence of long ventral dendrites. Correlations between the distribution of synaptic input zones and dendritic fields support the concept that interlaminar communication is mediated in part via contacts made onto ventrally extending dendrites of superficial laminae neurons. The results point to the presence of cell type specificity in dorsal horn circuitry, and show how the study of connectivity can itself help identify previously unrecognized neuronal populations.

Controlling neural activity with high spatio-temporal resolution is desired for studying how neural circuit dynamics control animal behavior. Conventional methods for manipulating neural activity, such as electrical microstimulation or pharmacological blockade, have poor spatial and/or temporal resolution. Algal protein channelrhodopsin-2 (ChR2) enables millisecond-precision control of neural activity. However, a photostimulation method for high spatial resolution mapping in vivo is yet to be established. Here, we report a novel optical/electrical probe, consisting of optical fiber bundles and metal electrodes. Optical fiber bundles were used as a brain-insertable endoscope for image transfer and stimulating light delivery. Light-induced activity from ChR2-expressing neurons was detected with electrodes bundled to the endoscope, enabling verification of light-evoked action potentials. Photostimulation through optical fiber bundles of transgenic mice expressing ChR2 in layer 5 cortical neurons resulted in single-whisker movement, indicating spatially restricted activation of neurons in vivo. The probe system described here and a combination of various photoactive molecules will facilitate studies on the causal link between specific neural activity patterns and behavior.

C1 neurons activate sympathetic tone and stimulate the hypothalamic–pituitary–adrenal axis in circumstances such as pain, hypoxia or hypotension. They also innervate pontine noradrenergic cell groups, including the locus coeruleus (LC) and A5. Activation of C1 neurons reportedly inhibits LC neurons; however, because these neurons are glutamatergic and have excitatory effects elsewhere, we re-examined the effect of C1 activation on pontine noradrenergic neurons (LC and A5) using a more selective method. Using a lentivirus that expresses channelrhodopsin2 (ChR2) under the control of the artificial promoter PRSx8, we restricted ChR2 expression to C1 neurons (67%), retrotrapezoid nucleus neurons (20%) and cholinergic neurons (13%). The LC contained ChR2-positive terminals that formed asymmetric synapses and were immunoreactive for vesicular glutamate transporter type 2. Low-frequency photostimulation of ChR2-expressing neurons activated LC (38 of 65; 58%) and A5 neurons (11 of 16; 69%) and sympathetic nerve discharge. Locus coeruleus and A5 inhibition was not seen unless preceded by excitation. Locus coeruleus activation was eliminated by intracerebroventricular kynurenic acid. Stimulation of ChR2-expressing neurons at 20 Hz produced modest increases in LC and A5 neuronal discharge. In additional rats, the retrotrapezoid nucleus region was destroyed with substance P–saporin prior to lentivirus injection into the rostral ventrolateral medulla, increasing the proportion of C1 ChR2-expressing neurons (83%). Photostimulation in these rats activated the same proportion of LC and A5 neurons as in control rats but produced no effect on sympathetic nerve discharge owing to the destruction of bulbospinal C1 neurons. In conclusion, low-frequency stimulation of C1 neurons activates pontine noradrenergic neurons and sympathetic nerve discharge, possibly via the release of glutamate from monosynaptic C1 inputs.

In many seasonally reproducing animals, the experience of prolonged exposure to constant photoperiods results in the induction of a state of photorefractoriness, which is defined as a lack of responsiveness to a previously stimulatory photoperiod. The physiological and genetic processes that control photorefractoriness are not well understood; however, the hallmark of photorefractoriness is an endogenous change in the physiological response to a constant photoperiod. It is already known that preoptic area (POA) gnrh1 gene expression declines during the development of refractoriness to long-day stimulation in European starlings. We employed in situ hybridisation histochemistry to characterise changes in POA gnrh1 mRNA expression during the reinstatement of photosensitivity in female starlings. Photorefractory starlings moved to short days (8L:16D) increased optical density of gnrh1 expressing cells within 10 days. Exposure to 30 short days resulted in greater visible gnrh1 cell numbers, with no detectable change in measures of ovarian follicular volume and oviduct mass. We subsequently examined the extent of gnrh1 expression in response to photostimulation after incremental periods on short day lengths. A significant long-day-induced increase in both gnrh1 expression and ovarian and oviduct mass occurred only after at least 30 short days. These findings demonstrate that the recovery of photorefractoriness involves an increase in gnrh1 mRNA expression and expands upon our previous knowledge that the development of photosensitivity is associated with an increase in both the precursor proGnRH1-GAP and GnRH1 peptides in the POA. Importantly, the change in the brain sensitivity occurs well before such changes can be detected via variation in ovarian activity.

Background and Objectives We have used a 660-nm laser diode in genetic diabetic mice to stimulate the healing of wounds covered with a Tegaderm HP dressing that causes a retardation of contraction (splinted wounds). It is possible that the stimulation of healing could be due to possible diabetes-modifying properties of laser light. This has been examined by using the 660 nm laser to irradiate non-wounded diabetic mice with the same dose and at same location as for wounded diabetic mice. Materials and Methods Twenty-two diabetic mice were used and divided into two equal groups. Body weight and water intake of mice were measured daily for 7 days prior to the start of treatment (Day 0). The left flank of mice in the experimental group was irradiated with 660 nm laser, 100 mW, 20 seconds/day, 7 days; for mice in the control group, the left flank was sham-treated with the laser power supply not switched on. Body weight and water intake of mice were measured to Day 14. On Day 14, the mice were fasted for 4 hours, anaesthetized with sodium pentobarbitone (i.p.) and blood collected by cardiac puncture into heparinized tubes. The plasma was assayed for glucose and glycated hemoglobin A1c. Results There were no significant differences in body weight and water intake over 22 days between mice in the experimental group and control group. On day 14, the mean blood plasma glucose level was not significantly different between the two groups; glycated hemoglobin A1c was not detected in the samples. Conclusion Irradiation of the left flank in diabetic mice with 660 nm laser system does not have a significant hypoglycemic effect, and the laser-stimulated healing of wounds in diabetic mice is due to cellular and biochemical changes in the immediate wound environment. Lasers Surg. Med. 44:26–29, 2012. © 2011 Wiley Periodicals, Inc.

The present results show that the semicircular canal crista ampullaris of the toadfish, Opsanus tau, is sensitive to infrared radiation (IR) applied in vivo. IR pulse trains (∼1862 nm, ∼200 μs pulse⁻¹) delivered to the sensory epithelium by an optical fibre evoked profound changes in phasic and tonic discharge rates of postsynaptic afferent neurons. Phasic afferent responses to pulsed IR occurred with a latency of

Manipulation of defined neurons using excitatory opsins, including channelrhodopsin, enables studies of connectivity and the functional role of these circuit components in the brain. These techniques are vital in the neocortex, where diverse neurons are intermingled, and stimulation of specific cell types is difficult without the spatial, temporal, and genetic control available with optogenetic approaches. Channelrhodopsins are effective for mapping excitatory connectivity from one input type to its target. Attempts to use multiple opsins to simultaneously map multiple inputs face the challenge of partially overlapping light spectra for different opsins. This protocol describes one strategy to independently stimulate two comingled inputs in the same brain area to assess convergence and interaction of pathways in neural circuits. This is highly relevant in the neocortex, where pyramidal neurons integrate excitatory inputs from multiple local cell types and long-range corticocortical and thalamocortical projections. © 2018 by John Wiley & Sons, Inc.

Birds use a variety of environmental cues, such as day length, temperature and social interactions, to time reproductive efforts. For most seasonally breeding birds, day length is the most important cue and takes precedence over all others. Experimental manipulation of day length has shown that, in a number of galliformes and passeriformes, exposure to a single long day induces a rise in plasma luteinising hormone (LH). The mechanisms underlying this response are only beginning to be understood. In Japanese quail and Zonotrichia sparrows, one long day causes striking up-regulation of the protein products of immediate early genes (IEGs) in the mediobasal hypothalamus, near gonadotrophin-releasing hormone (GnRH) axons and terminals. Photoperiodic induction of the same proteins in the GnRH somata themselves, however, has not been described in these species. In the present study, we used immunohistochemistry to assay the induction of two IEGs, Fos and Egr-1, in the GnRH somata of male and female white-throated sparrows (Zonotrichia albicollis) exposed to a single long day. We found that immunoreactivity for both proteins increased in a subset of the GnRH neurones of the septo-preoptic area by the morning after the long day. Photo-induced expression of Egr-1 or Fos protein in GnRH neurones was limited to a population of cells in the medial preoptic area. Males showed significantly greater induction of both proteins in this population of GnRH neurones than did females, which is consistent with the hypothesis that males may be more sensitive to photic cues. Overall, the results obtained suggest that photostimulation stimulates new protein synthesis in GnRH neurones on a relatively rapid time scale. Further research is required to determine whether the GnRH somata are themselves integrating photic cues, or whether they are responding rapidly to an increased demand for GnRH synthesis.

To explore the specific contribution of the C1 neurons to blood pressure (BP) control, we used an optogenetic approach to activate these cells in vivo. A lentivirus that expresses channelrhodopsin-2 (ChR2) under the control of the catecholaminergic neuron-preferring promoter PRSx8 was introduced into the rostral ventrolateral medulla (RVLM). After 2–3 weeks, ChR2 was largely confined to Phox2b-expressing neurons (89%). The ChR2-expressing neurons were non-GABAergic, non-glycinergic and predominantly catecholaminergic (∼54%). Photostimulation of ChR2-transfected RVLM neurons (473 nm, 20 Hz, 10 ms, ∼9 mW) increased BP (15 mmHg) and sympathetic nerve discharge (SND; 64%). Light pulses at 0.2–0.5 Hz evoked a large sympathetic nerve response (16 × baseline) followed by a silent period (1–2 s) during which another stimulus evoked a reduced response. Photostimulation activated most (75%) RVLM baroinhibited neurons sampled with 1/1 action potential entrainment to the light pulses and without accommodation during 20 Hz trains. RVLM neurons unaffected by either CO2 or BP were light-insensitive. Botzinger respiratory neurons were activated but their action potentials were not synchronized to the light pulses. Juxtacellular labelling of recorded neurons revealed that, of these three cell types, only the cardiovascular neurons expressed the transgene. In conclusion, ChR2 expression had no discernable effect on the putative vasomotor neurons at rest and was high enough to allow precise temporal control of their action potentials with light pulses. Photostimulation of RVLM neurons caused a sizable sympathoactivation and rise in blood pressure. These results provide the most direct evidence yet that the C1 neurons have a sympathoexcitatory function.

Age influences behavioral decisions such as reproductive timing and effort. In photoperiodic species, such age effects may be mediated, in part, by the individual's age-accrued experience with photostimulation. In female European starlings (Sturnus vulgaris) that do not differ in age, experimental manipulation of photostimulation experience (photoexperience) affects hypothalamic, pituitary, and gonadal activity associated with reproductive development. Does photoexperience also affect activity in forebrain regions involved in processing a social cue, the song of males, which can influence mate choice and reproductive timing in females? Female starlings prefer long songs over short songs in a mate-choice context, and, like that in other songbird species, their auditory telencephalon plays a major role in processing these signals. We manipulated the photoexperience of female starlings, photostimulated them, briefly exposed them to either long or short songs, and quantified the expression of the immediate-early gene ZENK (EGR-1) in the caudomedial nidopallium as a measure of activity in the auditory telencephalon. Using an information theoretic approach, we found higher ZENK immunoreactivity in females with prior photostimulation experience than in females experiencing photostimulation for the first time. We also found that long songs elicited greater ZENK immunoreactivity than short songs did. We did not find an effect of the interaction between photoexperience and song length, suggesting that photoexperience does not affect forebrain ZENK-responsiveness to song quality. Thus, photoexperience affects activity in an area of the forebrain that processes social signals, an effect that we hypothesize mediates, in part, the effects of age on reproductive decisions in photoperiodic songbirds. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009

Thyroid hormone and long days interact to programme American tree sparrows (Spizella arborea) for seasonality (i.e. thyroid hormone-dependent photoperiodic gonadal growth, photorefractoriness, and postnuptial moult). This study explored in radiothyroidectomized (THX) males given thyroid hormone replacement therapy whether thyroid hormone acts within the brain and, additionally, the identity of the putative tissue-active thyroid hormone. The minimum dose (30 ng) of L-thyroxine (T4) that restored all components of seasonality when given i.c.v. daily during the first 21 days of photostimulation restored no component of seasonality when given s.c. The same dose of L-triiodothyronine (T3) also was ineffective when administered s.c., but restored photoperiodic testicular growth (though neither photorefractoriness nor postnuptial moult) when admiministered i.c.v. Three of seven birds given a 10-fold lower dose of T4 (3 ng) exhibited thyroid hormone-dependent photoperiodic testicular growth, albeit damped. The other four birds given 3 ng T4 and all birds given 3 ng T3 responded like THX controls, exhibiting only slight thyroid hormone-independent photoperiodic testicular growth. The highest dose (300 ng) of T3 restored all components of seasonality only when administered i.c.v. daily during the first 49 days of photostimulation. This demonstration in American tree sparrows is the first in any species that the thyroid-dependent transition from the breeding season to the non-breeding season can be effected by T3. The same dose of reverse T3 administered daily over the same 49 days restored photoperiodic testicular growth in only half of 10 subjects and photorefractoriness and moult in none. Collectively, the data support the hypothesis that thyroid hormone acts centrally to programme photostimulated male American tree sparrows for all components of seasonality. The most parsimonious interpretation of the data, including the threshold-like effect of 3 ng T4, favours T4 as the tissue-active thyroid hormone for vernal as well as autumnal events, but does not entirely exclude T3.

The annual breeding cycle of ‘unimproved’ breeds of domestic chicken, including the bantam, at temperate latitudes, is terminated by decreasing daylength in autumn and is initiated in late winter, while daylengths are still short. Observations on photoperiodic birds that terminate seasonal breeding by the development of long day photorefractoriness suggest that the photoinduced pattern of prolactin secretion is associated with the pattern of gonadal growth and regression. It was predicted that, if there is a causal relationship between photoinduced changes in prolactin secretion and gonadal function in birds then, in the bantam, the pattern of prolactin secretion observed after photostimulation would not be the same as in birds terminating breeding by the development of long day photorefractoriness. Experiments were carried out on surgically castrated bantams to avoid confounding the effects of photostimulation and the stimulatory actions of testicular hormones on prolactin secretion. Transfer of photosensitive castrated bantams from 8 to 14, 16, 18 or 20 h light/day initially stimulated prolactin release and, subsequently, after 20–30 days, concentrations of plasma prolactin progressively decreased. After 148 days of photostimulation, concentrations of plasma prolactin approached but were still higher than short day controls. Transfer of photosensitive castrated bantam cockerels from 8 to 12 h light/day stimulated a slower increase in plasma prolactin that subsequently remained higher than in other photostimulated groups. A further 4 h increase in photoperiod in the birds exposed for 148 days to 12 or 16 h light/day resulted, respectively, in a transitory increase and no increase in prolactin secretion. Recovery of photosensitivity for prolactin release was observed in the birds transferred to 18 or 20 h light/day for 148 days after treatment with 8 h light/day for 35 days. Attempts to obtain an independent hormonal correlate of the prolactin responses to photostimulation by measurement of plasma luteinizing hormone (LH) were unsuccessful. The concentration of plasma LH in castrated bantams did not change in response to a change in photoperiod. These observations show that the photoinduced pattern of prolactin release in the bantam, a species which terminates seasonal breeding in response to decreasing daylength, is the same as that in birds which terminate seasonal breeding by the development of long day photorefractoriness. It is concluded that the photoinduced pattern of prolactin secretion in birds can be dissociated from the neuroendocrine mechanisms controlling the termination of seasonal breeding.

The substantia gelatinosa (lamina II) of the spinal dorsal horn contains inhibitory and excitatory interneurons that are thought to play a critical role in the modulation of nociception. However, the organization of the intrinsic circuitry within lamina II remains poorly understood. We used glutamate uncaging by laser scanning photostimulation to map the location of neurons that give rise to local synaptic inputs to islet cells, a major class of inhibitory interneuron in lamina II. We also mapped the distribution of sites on the islet cells that exhibited direct (non-synaptic) responses to uncaging of excitatory and inhibitory transmitters. Local synaptic inputs to islet cells arose almost entirely from within lamina II, and these local inputs included both excitatory and inhibitory components. Furthermore, there was a striking segregation in the location of sites that evoked excitatory versus inhibitory synaptic inputs, such that inhibitory presynaptic neurons were distributed more proximal to the islet cell soma. This was paralleled in part by a differential distribution of transmitter receptor sites on the islet cell, in that inhibitory sites were confined to the peri-somatic region while excitatory sites were more widespread. This differential organization of excitatory and inhibitory inputs suggests a principle for the wiring of local circuitry within the substantia gelatinosa.

Gambel's white-crowned sparrow is a long distance migrant that undergoes spontaneous gonadal regression as a result of long day exposure. This termination of breeding is caused by the development of photorefractoriness and the birds become insensitive to long days, including continuous light. The present study investigated its possible mechanisms by examining the activity of the gonadotrophin-releasing hormone (GnRH) system under different photoperiodic regimes. We investigated the localisation and distribution of GnRH-I, its precursor pro-GnRH-GAP and GnRH-II in Gambel's white-crowned sparrow brain using immunocytochemistry with specific antibodies during photostimulation and the development of photorefractoriness. The study revealed that photoperiodic treatment, including the onset of photorefractoriness, had no significant effect on the size or number of GnRH-I, pro-GnRH-GAP or GnRH II immunoreactive cells, or the density of the GnRH-I, pro-GnRH-GAP immunoreactive fibres at the median eminence. GnRH-II was not found in the median eminence, suggesting that it does not regulate pituitary gonadotrophin secretion. GnRH-I measurement in hypothalamic extracts by radioimmunoassay did not reveal any significant difference between birds that were photostimulated or in the early stages of photorefractoriness. Furthermore, the action of the excitatory amino acid glutamate agonist N-methyl-D-aspartate on GnRH neurones in photorefractory birds was demonstrated by the significant blockade of luteinising hormone release with a specific GnRH antagonist. Taken together, these results suggest that, in Gambel's white-crowned sparrow, a decrease in GnRH-I secretion is the initial step for the onset of photorefractoriness and not a decrease in GnRH-I biosynthesis.

We examined whether sensory deprivation during formation of the cortical circuitry influences the pattern of intracortical single-cell connections in rat barrel cortex. Excitatory postsynaptic potentials (EPSPs) from layer 2/3 (L2/3) pyramidal neurons were recorded in vitro using patch-clamp techniques. In order to evoke EPSPs, presynaptic neurons were stimulated by photolytically applied glutamate, thus generating action potentials. Synaptic connections between the stimulated and the recorded neuron were identified by the occurrence of PSPs following photostimulation. Sensory deprivation changed the pattern of projections from L4 and L2/3 neurons to L2/3 pyramidal cells. In slices of non-deprived rats 86% of the total presynaptic neurons were located in the first and only 10% in the second barrel column. Deprivation changed these values to 67% and 26%, respectively. Therefore, the probability of presynaptic cells projecting to L2/3 neurons was shifted from adjacent to more remote barrel columns. These results indicate that deprivation of sensory input influences the pattern of intracortical connections.

During the developmental stage, the brain undergoes anatomic, functional, and metabolic changes necessary to support the complex adaptive behavior of a mature individual. Estimation of developmental changes occurring in different regions of the brain would provide a means of relating various behavioral phenomena to maturation‐specific brain structures, thereby providing useful information on structure‐function relationships in both normal and disease states. We used multichannel near‐infrared spectroscopy (MNIRS), a new noninvasive imaging technique for revealing the course of neural activity in selected brain regions, to monitor the activities of the visual cortex as mirrored by hemodynamic responses in infants subjected to photostimulation during natural sleep. In the infants, oxyhemoglobin and total hemoglobin decreased and deoxyhemoglobin increased in the visual cortex with photostimulation. This pattern of responses was different from the response pattern in adults reported previously. The different patterns of responses to photostimulation in the visual cortices of infants and adults might reflect developmental and behavioral differences. It may reflect a different functional organization of the visual cortex in infants or ongoing retinal development. Our results demonstrated that regional hemodynamic change could be detected in a small area around the visual cortex. MNIRS offers considerable potential for research and noninvasive clinical applications. Hum. Brain Mapping 22:124–134, 2004. © 2004 Wiley‐Liss, Inc.

Although the gonadal photoperiodic response and its influence upon the song control system in canaries have been extensively studied, photoperiodic regulation of the GnRH system has not been investigated. To examine the relationship between photoperiod and the reproductive neuroendocrine system in male and female canaries, three groups of canaries were exposed to chronic short days (8L:16D; Phsens), acute long days (18L:6D; Phstim) and chronic long days (also 18L:6D; Phrefr) to induce the reproductive states of photosensitivity, photostimulation, and photorefractoriness, respectively. Brain sections were processed for GnRH immunocytochemistry. The canaries in this study did not demonstrate consistent or uniform responses to different photoperiodic treatments. In males, gonad size varied with photoperiod; Phstim males had larger gonads than either the Phsens or Phrefr males. In contrast, there was no difference between groups in female gonad size as a result of photoperiodic treatment. Brain GnRH cell number, cell size, and fiber density were similar in all groups. The results suggest that canaries are not as obligatory photoperiodic as previously thought (or at least not all varieties of domestic canaries are). This could be a result of many years of domestication, the natural history of the species, phylogenetic constraint, or a combination of these factors.

It is well known that, under exposure to bright light, eggs tend to hatch earlier than control, without any damage to the birds. This report aims to systematically show the effect and establishes a proposal for a possible application to accelerate chicken egg formation, which could be extrapolated or adapted as a great advance in premature human newborns. Comparing several protocols, the experiments show that lower doses of light slowly delivered for 24 h promote higher efficiency in embryo development, increasing on average 25% of its size and more than 70% in weight when compared to the control. This weight difference shows promising results compared to rates of up to 17% found in the literature. These results can be a first step to reduce the stay of premature human infants in hospitals because light, when applied in very low doses, can accelerate the natural biological processes without risks.

Acute hypoxia causes hyperventilation, sighs and arousal from sleep. The role of C1 neurons in mediating hypoxia counter-regulation in a behaving animal remains untested. We determined sufficiency by optogenetic methods. Channelrhodopsin-2 was selectively introduced into C1 neurons of Tyrosine Hydroxylase Cre-driver rats (Cre/0) with a Cre-dependant adeno-associated viral vector (AAV2 DIO-EF1alpha-ChR2-EYFP). Sleep stages were identified from EEG and neck EMG recordings. Breathing was measured using whole body plethysmography and blood pressure by telemetry. During non-rapid eye movement sleep (nREM), unilateral photostimulation of C1 neurons produced arousal in 84 ± 4% of trials and sighs in 68 ± 7% of trials and increased respiratory frequency by 26 ± 5 bpm (n=5). During quiet awake states, C1 stimulation also caused sighs and increased breathing rate. This increase in breathing by C1 stimulation was occluded by hypoxia (12% O2), but not hypercapnia (6% CO2), despite the significantly higher basal venti...

Catecholaminergic neurons located in the rostroventrolateral medulla (CA-RVLM) contribute to sympathetic, parasympathetic, and neuroendocrine responses and are activated by hypotension, hypoglycemia, hypoxia, infection, pain, and psychological stress. Most CA-RVLM express vGluT2, and work on slices suggests they release glutamate but the significance of vGluT2 in these neurons in vivo is unestablished. To investigate the role of vGluT2 in CA-RVLM in vivo, we crossed floxed vGluT2 mice with dopamine-β-hydroxylase (DβH) Cre mice, creating mice with vGluT2 selectively deleted from DβH neurons (cKO mice). We then injected a Cre-dependent AAV2 that expresses channelrhodopsin fused to mCherry into the RVLM of cKO or control mice. In both experimental and control groups, photostimulation activated the channelrhodopsin+ CA-RVLM. Photostimulation of CA-RVLM in control mice increased respiratory rate in conscious mice and efferent vagal nerve activity in anesthetized mice, while in cKO mice, photostimulation had no...

Melatonin was recently identified as playing a role in fine-tuning the effects of gonadal steroids in the regulation of seasonal neuroplasticity within the telencephalic song control system of European starlings. The present study investigated possible seasonal regulation of melatonin receptors (MelR) within the starling song control system, in the presence or absence of gonadal steroids. Brains were sampled from photosensitive starlings exposed to short days, photostimulated starlings exposed to long days and photorefractory starlings also exposed to long days. Each condition contained a group of gonad-intact birds and a group of castrated birds. Melatonin receptor distribution was assessed in vitro by 125-iodomelatonin (IMEL) receptor autoradiography. In general, MelR distribution was similar to that described in other songbird species. However, there was a striking downregulation of MelR in the song control nucleus Area X of intact and castrated photostimulated birds on long days compared to their photorefractory counterparts on the same long days and to the short-day groups. Downregulation of MelR occurred independently of gonadal steroids. Nevertheless, superimposed on this general pattern of MelR downregulation during photostimulation, IMEL binding was observed in a medial subdivision of Area X when gonadal steroids were present. Downregulation of MelR in Area X during the short breeding season has implications for seasonal regulation of the song control system. Subsequent upregulation of MelR as birds become photorefractory, in the absence of any change in photoperiod, gonadal steroids or melatonin signal is the first description of photoperiod-independent regulation of MelR in adults of any vertebrate class.

Background and Objective To gain some insight into the photostimulation of isolated hepatocytes irradiated with Helium-Neon (He-Ne) laser light certain biochemical events were studied with respect to two mechanisms: i) the direct light dependent activation of certain biochemical events investigated in intact cells and isolated mitochondria, ii) the indirect stimulation of processes per se light independent. Study Design/Materials and Methods Irradiation of either isolated hepatocytes or isolated rat liver mitochondria was carried out with He-Ne laser (wavelength, 632.8 nm; fluence, 0.24 J cm−2; fluence rate, 12 mW cm−2). Changes in mitochondrial membrane potential in isolated hepatocytes were monitored using the cationic probe safranine. The c-fos expression was studied by Northern blot and immunoblot analysis. Results As a result of irradiation, increase of the mitochondrial membrane potential was found to occur in irradiated hepatocytes both in the presence or in the absence of CaCl2. The hyperpolarization of the mitochondrial membrane is assumed to cause an increase in mitochondrial Ca2+ uptake that was measured in isolated mitochondria. Finally, an increase in c-fos expression was found in irradiated hepatocytes when incubated in the presence of CaCl2. Conclusion This paper gives additional information on the mechanism by which He-Ne laser light, either directly or in a cascade-like effect dependent on increase in cell Ca2+, can cause cell stimulation. Lasers Surg. Med. 29:433–441, 2001. © 2001 Wiley-Liss, Inc.

The unicellular green alga Chlamydomonas displays two distinct kinds of behavioral response to light: phototaxis, in which cells swim toward or away from the light source under constant illumination; and photophobic responses (also called stop responses or photoshock responses), in which cells transiently convert their flagellar waveform and swim backward upon sudden increase in light intensity. It has been suggested that the two responses partly share a common signal transduction pathway, but exactly how the different responses are produced has not been established. In this study, to help understand the molecular and cellular mechanisms that bring about the photophobic response, we isolated novel mutants (ppr1, ppr2, ppr3, and ppr4) that do not show the photophobic response. Importantly, these mutants retain the ability to display phototaxis, with almost the same sensitivities as in the wild type cell. Demembranated and reactivated flagellar axonemes of the ppr mutants were found to convert the bending patterns depending on the Ca2+ concentration, indicating that the axonemal mechanism for waveform conversion required for the photophobic response was unaffected by the mutations. In addition, measurements of electric currents in cell suspensions showed that these mutants generate normal photoreceptor currents (PRC) upon photostimulation, suggesting that they retain the normal activity of photoreception and the ionic channels that produce PRCs. However, the all-or-none flagellar current (FC), a Ca2+ current generated by PRC-induced depolarization of flagellar membrane, was absent or seriously impaired in the mutants. These findings clearly indicate that the all-or-none FC is necessary for the photophobic response but not for phototaxis. The isolation of the four genetically independent ppr mutants suggests that the generation of the FC is based on multiple components that are not used in the mechanism for phototaxis, and implies that the Chlamydomonas flagellar membrane possesses a voltage-dependent Ca2+-channel specifically used for generation of photophobic responses.

Background and Objective Low energy laser photostimulation at certain wavelengths can enhance tissue repair by releasing growth factors from fibroblasts and stimulate the healing process. This study was designed to evaluate the influence of laser photostimulation on collagen production in experimentally tenotomized and repaired rabbit Achilles tendons. Study Design/Materials and Methods: A total of 24 male New Zealand rabbits, ages 10–12 weeks, were used. Following tenotomy and repair, the surgical hind limbs of the rabbits were immobilized in customized polyurethane casts. The experimental animals were treated with a 632.8 nm He:Ne laser daily at 1.0 J cm−2 for 14 days. Control animals were sham treated with the laser head. On the fifth day after repair, the casts were removed to allow the animals to bear weight on the lower extremity. The animals were euthanized on the 15th postoperative day, then, the Achilles tendons were excised, processed and analyzed. Results Biochemical analyses of the tendons revealed a 26% increase in collagen concentration with laser photostimulation indicating a more rapid healing process in treated tendons compared to controls. Sequential extractions of collagen from regenerating tissues revealed that the laser photostimulated tendons had 32% and 33% greater concentrations of neutral salt soluble collagen and insoluble collagen, respectively, than control tendons suggesting an accelerated production of collagen with laser photostimulation. A significant decrease (9%) in pepsin soluble collagen was observed in laser-treated tendons compared to controls. There were no statistically significant differences recorded in the concentrations of hydroxypyridinium crosslinks and acid soluble collagen between treated and control tendons. Conclusion This study of laser photostimulation on tendon healing in rabbits suggests that such therapy facilitates collagen production in a manner that enhances tendon healing. Lasers Surg. Med. 22:281–287, 1998. © 1998 Wiley-Liss, Inc.

Background and Objective Low-level laser irradiation at certain fluences and wavelengths can enhance the release of growth factors from fibroblasts and stimulate cell proliferation in vitro. We evaluated whether low-level laser irradiation can improve wound healing in diabetes mellitus. Study Design/Materials and Methods Genetically diabetic mice (C57BL/Ksj/db/db) were used as the animal model for this wound healing study. The experimental animals were divided among four groups: negative control, positive control (topical basic fibroblast growth factor [bFGF] on wound), laser therapy group; and a combination group of laser therapy and topical bFGF. An argon dye laser (Lexel Auora Model 600) at a wavelength of 630 nm and an output of 20 mW/cm2 was used as the light source. The speed of wound closure and histological evaluation were used to analyze the experimental results. Results Laser irradiation enhanced the percentage of wound closure over time as compared to the negative control group (58.4 ± 2.6 vs. 40.8 ± 3.4 at day 10 and 95.7 ± 2 vs. 82.3 ± 3.6 at day 20, P < .01). Histological evaluation showed that laser irradiation improved wound epithelialization, cellular content, granulation tissue formation, and collagen deposition in laser-treated wounds as compared to the negative control group (6.4 ± 0.16 vs. 3.8 ± 0.13 at day 10 and 12 ± 0.21 vs. 8.2 ± 0.31, P < .01). Conclusion This study of laser biostimulation on wound healing in diabetic mice suggests that such therapy may be of great benefit in the treatment of chronic wounds that occur as a complication of diabetes mellitus. Lasers Surg Med 20:56–63, 1997. © 1997 Wiley-Liss, Inc.

Purpose Central serous chorioretinopathy (CSCR) is a disease in which a serous detachment of the neurosensory retina occurs over an area of leakage from the choriocapillaris through the retinal pigment epithelium (RPE). CSCR can be acute or chronic. Classic image studies for CSCR are Fluorescine Angiography (FA) and Optical Coherence Tomography (OCT).No medical therapy is currently indicated for CSCR despite a range of potential medical treatments evaluated in many case reports. Argon laser photocoagulation can be considered only with a single leak located more than 300 µm from the center of the fovea. Autofluorescence (AF) with ultra-widefield scanning laser and Subthreshold Micropulse (SDM) photostimulation with true yellow 577nm diode laser are the newer diagnostic and therapeutic options. Methods AF with ultra-widefield scanning laser (Daytona™, Optos plc UK) was performed in fifteen patients affected by acute and chronic CSCR. All the areas of serous retinal detachment visible at AF as hyperfluorescence were treated with SDM photostimulation (IQ 577™ true yellow laser, Iridex CA). Controls were made at 15 days, 1 and three months after the treatment. Results Ultra-widefield AF allowed to identify several zones of serous detachment not detected with FA and out of range of OCT scans. In most of cases we obtained the complete resolution of serous detachment and visual acuity improvement. Gain in visual acuity was better in acute cases of CSCR. Conclusion Ultra-widefield AF followed by SDM photostimulation can be an effective diagnostic and therapeutic option for patients with acute and chronic CSCR.

The present study was designed to investigate the photoperiodic responses of blackheaded buntings to various T-photocycles [T =the period length of light:dark (LD) cycle]. Two series of experiments were performed. In series I, groups of buntings (n =8-12) were exposed to LD cycles of 22 to 26 hr with photoperiods of 6, 10, or 11 hr (T22 =6L:16D, 10L:12D, 11L:11D; T24 =6L:18D, 11L:13D; T26 =6L:20D, 11L:15D). Among these groups, birds subjected to 11L:11D showed heavy deposition of fat, weight gain, and testicular recrudescence, a response that is characteristic of long day (≥ 12 hr photoperiod per day) photostimulation, indicating that a T-photocycle of less than 24 hr (T =22 hr) was photoinductive in buntings when presented with a photophase long enough to interact with the photoinducible phase (ϕi), located in the middle of the photocycle. Experiments in series II included an interrupted-night lighting protocol with a main photophase of 6 hr and 1-hr light break in photocycles of T =22 hr, T =24 hr, and T =26 hr. The results revealed that a 1-hr light pulse was inductive when it was introduced in the middle of the photocycles, and, thus, was coincident with the ϕi. It is suggested that the critical daylength for photoperiodic induction in buntings is altered as a result of entrainment to various T-photocycles. © 1995 Wiley-Liss, Inc.

Previously, stress induced sudden cardiac death (SCD) was discussed along with methods that might help prevent its occurrence. In this article, emphasis is on one aspect of prevention — a recently reintroduced technique to induce the relaxation response: photostimulation dark goggles that purportedly act as a brain wave synchronizer (BWS). Relative to BWS, first a review of the literature is given; then a summary of a recently completed clinical study is presented. Next, the results of an ongoing clinical study are given. Following this, case studies are presented of assessment by galvanic skin resistance, pulse rate, and electroencephalograph. Finally, future directions are discussed.

We report on the cognitive-behavioral treatment of a 12-year-old boy with photogenic partial seizures with secondary generalization who had developed phobic avoidant behavior toward all kinds of situations with potential photostimulation, leading to serious impairments of life quality. Based on a behavioral analysis of seizure and anxiety reaction, a habituation training (systematic desensitization procedure) was developed and performed, while maintaining protection against seizures with antiepileptic pharmacotherapy. The treatment was to gradually increase exposure to computer monitor and television screen photostimulation, closely adjusted to the level of subjective distress and tolerance. In addition to exercising control of photogenic input by regulating the monitor control button, rapid relaxation and imagery techniques were taught and applied as countermeasures at the onset of seizure precipitants. The treatment resulted in complete remission of phobic anxieties and responses and may also have facilitated ongoing seizure control after termination of antiepileptic medication.

The reaction of 4-hydroxycoumarins with a series of quinonic chlorides under photostimulation was shown to provide an efficient approach to 3-alkylated coumarin derivatives. Alkylation reactions of this type proceeded via an electron transfer mechanism (SRN1).

2,3-Bis(chloromethyl)-1,4-anthraquinone reacts with primary nitroalkanes in a one-pot synthesis under standard SRN1 reaction conditions (inert atmosphere, photostimulation) and in a phase-transfer system to give original 2,3-dialkylnaphthacene-5,12-diones in fairly good yields.