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Cerebral activity during exposure to non - visible light

Cerebral activity during exposure to non - visible light


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Our eyes only have the ability to sense light within a certain spectrum. My understanding is that particular frequencies energize specific cells in our eyes, each responsible for a different "color". When these cells are energized, they each release an appropriate amount of their chemical transmitter to stimulate additional cells and create the perception of the object from which the photos originate.

At what point does this process not occur for non-visible light?

In other words, do we fail to see non visible light because our eyes lack the receptors for other frequencies, or does our brain receive signals that it just dumps because it has not developed to process the information?

Have there been any experiments done involving a brain scan while exposed to non visible light?


[D]o we fail to see non visible light because our eyes lack the receptors for other frequencies, or does our brain receive signals that it just dumps because it has not developed to process the information?

It is because our eyes lack the receptors for other frequencies.

Humans have 3 'color receptor' called cone cells. We have three types of cone cells, each sensitive to a specific range of wavelengths. Here is a diagram of the sensitivity of each of these three types of cone cells.

As you can see, we do not have receptors for wavelength below 390 nm or above 700 nm which is our visible spectrum.


Vision is based on a protein interaction with a molecule called retinal from vitamin A.

Wavelengths of light in the visual range cause a photoisomerization of retinal (a cis- to trans- change), which is sensed by the protein that is "holding on" to the retinal.

Wavelengths that do not cause this photoisomerization cannot be sensed by the photoreceptors of the retina, so there isn't any signal produced to transmit to the brain.

However, there is some filtering done by the lens of the eye. Without the lens, humans (and other animals) can see further into the UV range. That UV light is also damaging to the retina, though, so it's best to keep the lens intact.

Note that the image in the answer by @Remi.b is actually from a psychometric experiment, so the lens is intact. The responses there are actually human perceptual sensitivity to light for each cone. The blue curve would go off to the left quite a bit further into the 300nm range if the lens were not present. The research subjects probably prefer the experiment as-is, however.

You wouldn't perceive that UV light much differently than a far violet color, however, because differences between different cones are necessary for color vision, and by that end of the spectrum only the cones responsive to the shortest wavelengths are responsive at all.


Cerebral activity during exposure to non - visible light - Biology

Plants have a number of sophisticated uses for light that go far beyond their ability to photosynthesize low-molecular-weight sugars using only carbon dioxide, light, and water. Photomorphogenesis is the growth and development of plants in response to light. It allows plants to optimize their use of light and space. Photoperiodism is the ability to use light to track time. Plants can tell the time of day and time of year by sensing and using various wavelengths of sunlight. Phototropism is a directional response that allows plants to grow towards, or even away from, light.

The sensing of light in the environment is important to plants it can be crucial for competition and survival. The response of plants to light is mediated by different photoreceptors, which are comprised of a protein covalently bonded to a light-absorbing pigment called a chromophore. Together, the two are called a chromoprotein.

The red/far-red and violet-blue regions of the visible light spectrum trigger structural development in plants. Sensory photoreceptors absorb light in these particular regions of the visible light spectrum because of the quality of light available in the daylight spectrum. In terrestrial habitats, light absorption by chlorophylls peaks in the blue and red regions of the spectrum. As light filters through the canopy and the blue and red wavelengths are absorbed, the spectrum shifts to the far-red end, shifting the plant community to those plants better adapted to respond to far-red light. Blue-light receptors allow plants to gauge the direction and abundance of sunlight, which is rich in blue–green emissions. Water absorbs red light, which makes the detection of blue light essential for algae and aquatic plants.


Why are Leaves not Black?

Light must be absorbed for nutrients to be created by Photosynthesis. So why reflect the green and waste the whole middle part of the spectrum? According to Moore, et al., this is a long story in fact ancient history!

It looks like chlorophyll takes the part of the spectrum that bacteriorhodopsin doesn't take. Bacteriorhodopsin is a purple pigment that resembles the light-sensitive pigment in our eyes.

Current understanding is that the earliest photosynthetic organisms were aquatic bacteria, some of which are still around today. One of these, halobacterium halobium, grows in extremely salty water. It makes use of the bacteriorhodopsin pigment. The chlorophyll system developed to use the available light, as if it developed in strata below the purple bacteria and had to use what it could get.

But what about the development of land plants? Why did they stay green? The thoughts are that they had plenty of light and were not pressured to develop more efficient light gathering. That is, the light was not the limiting resource in photosynthesis for plants.

That being said, there is some extension toward the middle of the spectrum with the beta carotene and other pigments.


Antibacterial Activity of Blue Light against Nosocomial Wound Pathogens Growing Planktonically and as Mature Biofilms

The blue wavelengths within the visible light spectrum are intrinisically antimicrobial and can photodynamically inactivate the cells of a wide spectrum of bacteria (Gram positive and negative) and fungi. Furthermore, blue light is equally effective against both drug-sensitive and -resistant members of target species and is less detrimental to mammalian cells than is UV radiation. Blue light is currently used for treating acnes vulgaris and Helicobacter pylori infections the utility for decontamination and treatment of wound infections is in its infancy. Furthermore, limited studies have been performed on bacterial biofilms, the key growth mode of bacteria involved in clinical infections. Here we report the findings of a multicenter in vitro study performed to assess the antimicrobial activity of 400-nm blue light against bacteria in both planktonic and biofilm growth modes. Blue light was tested against a panel of 34 bacterial isolates (clinical and type strains) comprising Acinetobacter baumannii, Enterobacter cloacae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Klebsiella pneumoniae, and Elizabethkingia meningoseptica All planktonic-phase bacteria were susceptible to blue light treatment, with the majority (71%) demonstrating a ≥5-log10 decrease in viability after 15 to 30 min of exposure (54 J/cm(2) to 108 J/cm(2)). Bacterial biofilms were also highly susceptible to blue light, with significant reduction in seeding observed for all isolates at all levels of exposure. These results warrant further investigation of blue light as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications.

Importance: Blue light shows great promise as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications (e.g., wound closure during surgery). This warrants further investigation.

Figures

Survival of planktonic bacteria after…

Survival of planktonic bacteria after exposure to 400-nm blue light. (A) Acinetobacter baumannii…

Comparison of blue light LD…

Comparison of blue light LD 90 values between strains and species. Each individual…

(A) Correlation between survival of…

(A) Correlation between survival of planktonic S. aureus strains following blue light exposure…

Graphs showing the biofilm seeding…

Graphs showing the biofilm seeding results for all isolates. Optical density on the…


Trichromatic Coding

There are three types of cones (with different photopsins), and they differ in the wavelength to which they are most responsive, as shown in Figure 17.21. Some cones are maximally responsive to short light waves of 420 nm, so they are called S cones (“S” for “short”) others respond maximally to waves of 530 nm (M cones, for “medium”) a third group responds maximally to light of longer wavelengths, at 560 nm (L, or “long” cones). With only one type of cone, color vision would not be possible, and a two-cone (dichromatic) system has limitations. Primates use a three-cone (trichromatic) system, resulting in full color vision.

The color we perceive is a result of the ratio of activity of our three types of cones. The colors of the visual spectrum, running from long-wavelength light to short, are red (700 nm), orange (600 nm), yellow (565 nm), green (497 nm), blue (470 nm), indigo (450 nm), and violet (425 nm). Humans have very sensitive perception of color and can distinguish about 500 levels of brightness, 200 different hues, and 20 steps of saturation, or about 2 million distinct colors.

Figure 17.21.
Human rod cells and the different types of cone cells each have an optimal wavelength. However, there is considerable overlap in the wavelengths of light detected.


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Signs and Symptoms of Cerebral Palsy

Reaching the expected developmental benchmarks of infancy and childhood – sitting, rolling over, crawling, standing and walking – are a matter of great joy for parents, but what if a child’s developmental timetable seems delayed? There are many tell-tale signs that a child may have Cerebral Palsy, but those factors can be indicative of many conditions.

Signs and symptoms of Cerebral Palsy

Signs of Cerebral Palsy are different from symptoms of Cerebral Palsy.

Signs are clinically identifiable effects of brain injury or malformation that cause Cerebral Palsy. A doctor will discern signs of a health concern during the exam and testing.

Symptoms, on the other hand, are effects the child feels or expresses symptoms are not necessarily visible.

Impairments resulting from Cerebral Palsy range in severity, usually in correlation with the degree of injury to the brain. Because Cerebral Palsy is a group of conditions, signs and symptoms vary from one individual to the next.

The primary effect of Cerebral Palsy is impairment of muscle tone, gross and fine motor functions, balance, control, coordination, reflexes, and posture. Oral motor dysfunction, such as swallowing and feeding difficulties, speech impairment, and poor facial muscle tone can also indicate Cerebral Palsy.

Associative conditions, such as sensory impairment, seizures, and learning disabilities that are not a result of the same brain injury, occur frequently with Cerebral Palsy. When present, these associative conditions may contribute to a clinical diagnosis of Cerebral Palsy.

Many signs and symptoms are not readily visible at birth, except in some severe cases, and may appear within the first three to five years of life as the brain and child develop.

In these instances, the most apparent early sign of Cerebral Palsy is developmental delay. Delays in reaching key growth milestones, such as rolling over, sitting, crawling and walking are cause for concern. Practitioners will also look for signs such as abnormal muscle tone, unusual posture, persistent infant reflexes, and early development of hand preference.

If the delivery was traumatic, or if significant risk factors were encountered during pregnancy or birth, doctors may suspect Cerebral Palsy immediately. In moderate to mild cases of Cerebral Palsy, parents are often first to notice if the child doesn’t appear to be developing on schedule. If parents do begin to suspect Cerebral Palsy, they will likely want to ask their physician to evaluate their child for Cerebral Palsy.

Most experts agree the earlier a Cerebral Palsy diagnosis can be made, the better.

However, some caution against making a diagnosis too early, and warn that other conditions need to be ruled out first. Because Cerebral Palsy is the result of brain injury, and because the brain continues to develop during the first years of life, early tests may not detect the condition. Later, however, the same test may, in fact, reveal the issue.

The earlier a diagnosis is made, the sooner a child can be enrolled in early intervention programs and treatment protocols. Early interventions and therapies have proven to help a child maximize their future potential. Early diagnosis also helps families qualify for government benefit programs to pay for such measures.

Eight clinical signs of Cerebral Palsy

Since Cerebral Palsy is most often diagnosed in the first several years of life, when a child is too young to effectively communicate his or her symptoms, signs are the primary method of recognizing the likelihood of Cerebral Palsy.

Cerebral Palsy is a neurological condition which primarily causes orthopedic impairment. Cerebral Palsy is caused by a brain injury or brain abnormality that interferes with the brain cells responsible for controlling muscle tone, strength, and coordination. As a child grows, these changes affect skeletal and joint development, which may lead to impairment and possibly deformities.

The eight clinical signs include muscle tone, movement coordination and control, reflexes, posture, balance, gross motor function, fine motor function and oral motor function. These are detailed below.


Muscle tone
The most noticeable sign of Cerebral Palsy is impairment of muscle tone – the ability of muscles to work together by maintaining proper resistance. Muscles coordinate with other muscles, oftentimes in pairs. As some muscles contract, others must relax. Even something as simple as sitting requires coordination of many muscles some flexing while others relax. The brain injury or malformation that caused Cerebral Palsy impairs the ability of the central nervous system to coordinate muscle movement.

Muscle Tone

Proper muscle tone allows limbs to bend and contract without difficulty, enabling an individual to sit, stand, and maintain posture without assistance. Improper muscle tone occurs when muscles do not coordinate together.

When this happens, those muscles that work in pairs – biceps and triceps, for example – may both contract or relax at the same time, impeding movement and coordination. Trunk muscles might relax too much, making it difficult to maintain a tight core this can result in impaired posture and an inability to sit or to move from a sitting to standing position.

A child with Cerebral Palsy may demonstrate any combination of these signs. Different limbs may be affected by different impairments. The two most common signs of abnormal muscle tone are hypotonia and hypertonia, but tone can be defined in other ways as well:

  • Hypotonia – decreased muscle tone or tension (flaccid, relaxed, or floppy limbs)
  • Hypertonia – increased muscle tone or tension (stiff or rigid limbs)
  • Dystonia – fluctuating muscle tone or tension (too loose at times and too tight at others)
  • Mixed – the trunk of the body may be hypotonic while the arms and legs are hypertonic
  • Muscle spasms – sometimes painful, involuntary muscular contraction
  • Fixed joints – joints that are effectively fused together preventing proper motion
  • Abnormal neck or truncal tone – decreased hypotonic or increased hypertonic, depending on age and Cerebral Palsy type
  • Clonus – muscular spasms with regular contractions
    • Ankle/foot clonus – spasmodic abnormal movement of the foot
    • Wrist clonus – spasmodic movement of the hand


    Movement, coordination and control
    The impairment of muscle tone affects a child’s limbs and body in different ways, although all children with Cerebral Palsy will likely feel some effect on muscle control and coordination. Different muscle control impairments can combine to cause limbs to be perpetually extended, contracted, constantly moving in rhythmic patterns or jerking spastically.

    Movement , coordination and control

    Some signs will be more apparent when the child is under stress. Some may be task related, such as reaching for an object. Sometimes signs will seem to disappear when the child is asleep and muscles are relaxed.

    It is common for a child to experience different types of impaired muscle control in opposite limbs. Coordination and control can likewise be affected differently in each limb.

    The impairment of coordination and control fall under the following types:

    • Spastic movements – hypertonic movements where the muscles are too tight resulting in muscle spasms, scissoring of the legs, clonus, contracture, fixed joints, and over-flexed limbs
    • Athetoid or dyskinetic movements – fluctuating muscle tone causing uncontrolled, sometimes slow, writhing movements which can worsen with stress
    • Ataxic movements – poor coordination and balance making tasks – such as writing, brushing teeth, buttoning shirts, tying shoes, and putting keys into slots – difficult
    • Mixed movements – a mixture of movement impairments, most commonly a combination of spastic and athetoid types, affecting different limbs
    • Gait disturbances – control impairments affecting the way a child walks

    Gait disturbances include:

    • In-toeing – toes angle or rotate inward
    • Out-toeing – toes angle or rotate outward
    • Limping – more weight is placed on one foot than the other, causing a dipping, or wavy stride
    • Toe walking – the weight is unevenly placed on the toes
    • Propulsive gait – a child walks hunched over in a stiff posture with the head and shoulders bent forward
    • Spastic and scissor gait – the hips flex slightly making it look like the child is crouching while knees and thighs slide past one another like scissors
    • Spastic gait – one leg drags due to muscle spasticity
    • Steppage gait – toes drag because the foot drags
    • Waddling gait – duck-like walking pattern that can appear later in life

    Reflex

    Certain abnormal reflexes may also indicate Cerebral Palsy. Hyperreflexia are excessive reflex responses that cause twitching and spasticity. Underdeveloped or lacking postural and protective reflexes are warning signs for abnormal development, including Cerebral Palsy.


    Reflex
    Reflexes are involuntary movements the body makes in response to a stimulus. Certain primitive reflexes are present at or shortly after birth, but disappear at predictable stages of development as the child grows. Specific reflexes that do not fade away – or those that don’t develop as the child grows – can be a sign of Cerebral Palsy.

    Abnormal primitive reflexes may not function properly in children with Cerebral Palsy, or they may not disappear at specific points in development as they do with children with no impairment.

    Common primitive reflexes that may improperly function or persist include, but are not limited to:

    • Asymmetrical tonic reflex – when the head turns, the legs on the same side will extend, and the opposite limbs contract like in a fencing pose. Asymmetrical tonic reflex should disappear around six months of age.
    • Symmetrical tonic neck reflex – the infant assumes a crawling position when the head is extended. Symmetrical tonic neck reflex should disappear between eight and 11 months.
    • Spinal gallant reflexes – when the infant lies on its stomach, the hips will turn towards the side of the body that is touched. Spinal gallant reflexes should disappear between three and nine months.
    • Tonic labyrinthine reflex – when the head is tilted back, the back arches, the legs straighten, and the arms bend. Tonic labyrinthine reflex should disappear by three-and-a-half years of age.
    • Palmer grasp reflex – when stimulating the palm the hand flexes in a grasping motion. Palmer grasp reflex should disappear around four to six months.
    • Placing reflex – when an infant is held upright and the back of a foot touches the surface, the legs will flex. Placing reflex should disappear by five months.
    • Moro (startle) reflex – when the infant is tilted so his or her legs are above their head, the arms will extend. Moro reflex should disappear by six months.

    Early hand preference can also indicate possible impairments. A child normally develops hand preference in his or her second year. As this is a wide timeframe and rough average, development of hand preference, especially if it is early preference, is cause for concern. Various sources state that early hand preference falls between 6-18 months.


    Posture

    Posture

    Cerebral Palsy affects posture and balance. Signs may appear as an infant begins to sit up and learn to move about. Typically, posture is expected to be symmetrical. For example, a baby in a sitting position would normally have both legs in front. When bent, they become mirror images of one another.

    Asymmetrical posture means the right and left limbs will not mirror one another. The hip-joints are one area where this is often prominent in instances of Cerebral Palsy. One leg will bend inward at the hip, and the other will bend outward.

    Much like reflexes, postural responses are expected reactions when putting a baby in certain positions. They typically appear as the baby develops. Impairment may be a possibility if the responses do not develop, or if they are asymmetric.

    Much like reflexes, postural responses are expected reactions when putting a baby in certain positions. They typically appear as the baby develops. Impairment may be a possibility if the responses do not develop, or if they are asymmetric.

    Common postural responses are:

    • Traction
    • Landau reflex – when the infant is supported in a lying position, pushing the head down will cause the legs to drop, and lifting the head will cause them to rise. This response appears around four or five months of age.
    • Parachute response – when the infant is positioned with his or her head towards the ground, the infant should instinctively reach as if bracing for impact. This response appears around eight to 10 months of age.
    • Head righting – when an infant is swayed back and forth, his or her head will remain straight. This response appears around four months of age.
    • Trunk righting – when a sitting infant is quickly pushed to the side, the infant will resist the force and use opposite hand and arm to brace against impact. This response appears around eight months of age.


    Balance

    Balance

    The impairment of gross motor function can affect a child’s ability to balance. Signs become recognizable as a child learns to sit, rise from a sitting position, and begins crawling or walking. Infants need to use their hands often as they learn these skills. They develop the strength, coordination, and balance to accomplish the task when mastering it without the use of their hands.

    A child’s inability to sit without support can be a sign of Cerebral Palsy. The Gross Motor Function Classification System, or GMFCS, a five-level system commonly used to classify function levels, uses balance while sitting as part of its severity level system.

    Signs to look for when a child sits include:

    • Requiring both hands for support
    • Having difficulty balancing when not using hands for support
    • Unable to sit without using hands for support

    Other signs to look for include, but are not limited to:

    • Swaying when standing
    • Unsteady when walking
    • Difficulty making quick movements
    • Needing hands for activities that require balance
    • Walking with abnormal gait

    Balance is often the same whether a child’s eyes are open or closed. Balance impairment is most often associated with ataxic, and to a lesser degree, spastic Cerebral Palsy.


    Gross motor function
    As a child develops, signs of impaired or delayed gross motor function may be noticeable. The ability to make large, coordinating movements using multiple limbs and muscle groups is considered gross motor function.

    Gross motor function

    Gross motor function may be impaired by abnormal muscle tone, especially hypertonia or hypotonia.

    For example, hypertonic limbs can be too tight, or inflexible, to allow proper flexion and movement whereas hypotonic limbs may be too loose to properly support a child’s movements.

    As a baby’s brain and body develop, they are expected to reach developmental milestones. Reaching the milestone later than expected, or reaching it but with low quality of movement (such as favoring one side while crawling), are possible signs of Cerebral Palsy.

    • Impaired gross motor functions – limited capability of accomplishing common physical skills such as walking, running, jumping, and maintaining balance.
    • Delayed gross motor functions – physical skills developed later than expected often used in conjunction with developmental milestones for predictable stages of development.

    Significant milestones of gross motor function include:

    • Rolling
    • Sitting up
    • Crawling
    • Standing
    • Walking
    • Balancing

    These should be monitored to note when the baby reaches the milestone, and the quality of movement.


    Fine motor function

    Fine motor function

    Executing precise movements defines the category of fine motor function. Fine motor control encompasses many activities that are learned, and involves a combination of both mental (planning and reasoning) and physical (coordination and sensation) skills to master.

    Impaired or delayed fine motor skills are an indicator of possible Cerebral Palsy. Intention tremors, where a task becomes more difficult as it gets closer to completion, is one such sign.

    Examples of fine motor function development are:

    • Grasping small objects
    • Holding objects between thumb and forefinger
    • Setting objects down gently
    • Using crayons
    • Turning pages in a book


    Oral motor function

    Oral motor function

    Difficulty in using the lips, tongue, and jaw indicate impaired oral motor function this is a sign that may be present in up to 90% of preschool-aged children diagnosed with Cerebral Palsy. Signs of oral motor function impairment include, but are not limited to difficulty with:

    • Speaking
    • Swallowing
    • Feeding/chewing
    • Drooling

    Speech requires proper intellectual and physical development. Cerebral Palsy impairs the physical aspects of speaking by improperly controlling the muscles required to speak. Oral motor impairment can affect:

    • Breathing – the lungs, and specifically the muscles controlling inhalation and exhalation necessary for proper speech patterns. The diaphragm and abdominal muscles are important for proper air flow and posture.
    • Articulating – muscles controlling the face, throat, mouth, tongue, jaw, and palate all must work together to form the proper shape necessary for pronunciation of words and syllables.
    • Voicing – vocal cords are controlled by muscles that essentially stretch the vocal folds between two regions of cartilage.

    Apraxia, an inability of the brain to effectively transmit proper signals to the muscles used in speaking, is one type of speech impairment common to Cerebral Palsy. It is divided into two types:

    • Verbal apraxia – affects the articulation muscles, especially regarding the specific sequence of movements needed to carry out proper pronunciation. It is common in children with hypotonia.
    • Oral apraxia – affects the ability to make nonspeaking movements of the mouth, but is not related solely to speaking. Examples of oral apraxia would be the inability to lick the lips or inflate the cheeks.

    Dysarthria is another speech impairment common to Cerebral Palsy. Like apraxia, it is a neurological impairment, as opposed to a muscular condition. It is often found in Cerebral Palsy that results in hypertonia and hypotonia. Dysarthria is broken into the following subgroups:

    • Ataxic dysarthria – slow, erratic, inarticulate speech caused by poor breathing and muscular coordination
    • Flaccid dysarthria – nasal, whiny, breathy speech caused by the inability of the vocal chords to open and close properly. There may be difficulty with consonants.
    • Spastic dysarthria – slow, strenuous, monotone speech and difficulty with consonants
    • Mixed dysarthria – all three may be present.

    Drooling is another sign of Cerebral Palsy that results from muscles in the face and mouth not being able to properly control coordination. Some specific factors which can contribute to drooling are impairments in:

    • Swallowing
    • Closing the mouth
    • Positioning the teeth
    • Inability to move saliva to back of mouth
    • Tongue thrusting

    Feeding difficulties can be present with Cerebral Palsy. They typically manifest as decreased ability to chew and swallow, and may also involve choking, coughing, gagging, and vomiting.

    RESOURCES

    Signs of Cerebral Palsy

    For other sources with general information on the signs and symptoms of Cerebral Palsy, MyChild recommends the following:

    National Dissemination Center for Children with Developmental Disabilities


    Conclusion

    In this manuscript the effect of multiple exposure of skin to VL were explored for pro-pigmentation activity. Interestingly multiple exposure with VL was able to induce pigmentation in explants extracted from Caucasian skin. Further exploration at biological endpoints suggested that besides pigment formation due to photo-oxidation activities, VL was able to activate the whole melanogenesis process. The clinical result of the current study confirm the ex-vivo studies and demonstrate that VL is able to induce pigmentation after multiple exposures, which suggests that preconditioning is required to activate the melanogenesis process. The findings also align very well with the findings from Mahmud et al., since both studies demonstrate that VL cannot produce persistent pigmentation with just one VL exposure, especially in subjects with Caucasian skin.

    Taken together these results demonstrate that in addition to UV, VL can have significant impact on producing uneven pigmentation in skin which is a main factor in photoaging. Furthermore this is the first report that preconditioning of the skin with VL, followed by multiple exposures to VL, can result in pigment formation. Thus photoexposure and photodamage should not be considered strictly as a result of UV exposure since the skin is exposed to whole spectra of wavelengths including VL, and VL can induce photodamage pathways in a manner similar to UV.


    Introduction

    Lighting sources and technology have experienced a revolution in the last 15� years. Lighting sources and technology, especially in non-commercial or industrial illumination applications, have traditionally been slow to change [1]. In most homes, the incandescent bulb and Edison socket have been omnipresent. In the past 10 years, we have seen significant use of other technologies, such as compact fluorescent lamps (CFLs), replacing incandescent sources. However, this transition has often been driven by legislation, which has focused on energy-efficient sources instead of consumer desire for different light sources. The general user quickly noted the difference in the quality of CFL source but not necessarily in the specifics of its power spectrum. Simultaneously, the development and performance of high brightness light-emitting diodes (LEDs) have experienced tremendous advances [2]. The coupling of a blue-light LED with a phosphor has also been used to produce a white light source, the white-light LED. This solid-state fluorescent analog has become known as solid-state lighting (SSL). This approach is now considered the next generation of illumination due to the many inherent and potential advantages over current technologies.

    In addition to use for general illumination, LEDs quickly became the choice for mobile devices, such as smart phones [3]. The small size of LEDs and the limited screen size make them ideal for these applications. The potential for the use of LEDs for backlighted liquid crystal displays (LCDs) in laptop computers was also quickly realized. This transition was driven by the fragility of the microfluorescent lamps used for illumination and consumer desire for thinner screens. LEDs have now become the dominant technology for backlighted tablet displays, such as iPads and e-readers, and large LCD television sets. This now means that blue light prevails in red, green, and blue (RGB) and SSL illumination systems that did not exist a decade ago. The ways in which people read have also changed. Light is now being used directly for illumination in smart phones, tablets, and readers instead of for reflection, which is typical for reading from paper.

    The white-light LED (i.e., the most common type of LED) is essentially a bichromatic source that couples the emission from a blue LED (peak of emission around 450� nm with a full width at half max of 30� nm) [4] with a yellow phosphor (peak of emission around 580 nm with a full width at half max of 160 nm) that appears white to the eye when viewed directly [5]. The specific pump wavelength of the phosphor in the range 450� nm depends critically on the absorption properties of the phosphor. Although the white-light LED can be considered the SSL analog of the fluorescent source, the power spectrum of the white-light LED is considerably different from traditional, fluorescent, or incandescent white light sources [6] ( Figure 1 ).

    A comparison of the power spectrum of a standard white-light LED, a tricolor fluorescent lamp, and an incandescent source. The radically different power spectrums can look similar when viewed directly by the eye, irrespective of how much blue emission is present.

    Early commercial devices lacked sophistication, adopting the currently available LED technology that was small, 350󗍐 mm 2 , and operated at low drive currents, typically 20 mA, producing 1� mW of power. The last decade has seen the scaling of LEDs to larger areas, 1൱ μm 2 , and higher drive currents of 𾍐 mA with significantly increased power output ϡ,000 mW [2]. During this period, LED devices were also optimized for use in illumination applications, and reflected from a surface instead of emitted directly.

    In addition, white-light LEDs degrade over time primarily through bleaching of phosphors so that they no longer efficiently absorb blue light [7]. This shifts the color temperature of the device over time, with a corresponding change in the color-rendering index but, more importantly, an increasing blue emission from the device with time.

    In this review, we summarize the current knowledge of the effects of blue light on the regulation of physiologic function and the effects of blue light exposure on ocular health. Finally, we discuss the available data to determine whether long-term exposure to blue light is safe or whether additional studies are needed to fully understand the effects of blue light exposure on ocular health.

    Non-image-forming photoreception

    In mammals, photoreception occurs only in the retina [8] by three types of photoreceptor: cones, rods, and the intrinsically photosensitive retinal ganglion cells (ipRGCs). The classical photoreceptors (e.g., rods and cones) are mostly responsible for the image-forming vision, whereas the ipRGCs play a major role in non-image-forming photoreception, that is, the photoreceptive system that regulates circadian photic entrainment, pupillary light response, and other important biologic functions ( Figure 2 ).

    In addition to the classical photoreceptors (rods and cones), ipRGCs are present in the retina. Recent studies have shown that at least two types of intrinsically photosensitive retinal ganglion cells (ipRGCs) have been identified: M1 and M2. Most of the M1 cells project to the suprachiasmatic nucleus (SCN) of the hypothalamus whereas the number of M1 and M2 projecting to the olivary pretectal nucleus (OPN) is similar (55% from M1 cells versus 45% from M2 cells). The M1 cells are considerably smaller but respond with significantly larger depolarizations and light-induced currents than do the M2 cells. Other neural targets of ipRGCs not shown in the figure include the preoptic area, sub-paraventricular zone, anterior hypothalamic nucleus, lateral hypothalamus, medial amygdaloid nucleus, lateral habenula, lateral geniculate nucleus (dorsal division), bed nucleus of the stria terminalis, periaqueductal gray, and superior colliculus. OS=outer segments IS=inner segments ONL=outer nuclear layer OPL=outer plexiform layer INL=inner nuclear layer IPL=inner plexiform layer GCL=ganglion cell layer from [31] with permission.

    The idea that the mammalian retina is capable of non-image-forming photoreception emerged during the 1990s when a series of studies indicated that mice lacking rod photoreceptors (rd/rd) have a normal phase response curve (PRC) to light [9], with an action spectrum that peaks around 480 nm [10]. This result suggested that a photo pigment different from rhodopsin (λmax 498 nm), short wavelength sensitive opsin (λmax 460 nm), and middle wavelength sensitive opsin (λmax 508 nm) [11] was responsible for the entrainment of circadian rhythms. Additional studies reported that mice lacking rods and cones were still capable of synchronizing their circadian rhythms to light-dark cycles [12], thus demonstrating that an undiscovered photo pigment/photoreceptor in the mammalian retina was responsible for the photoentrainment of circadian rhythms.

    The most likely candidate to emerge as the circadian retinal photo pigment is a mammalian homolog of Xenopus melanopsin (aka Opn4) [13-15]. In mammals, melanopsin mRNA (and protein) is expressed only in a small population (about 3𠄵%) of the RGCs [14,16] that are directly photosensitive and have an absorption peak around 470� nm [17-19]. These RGCs express pituitary adenylate cyclase-activating polypeptide (PACAP) [20] and form the retinohypothalamic tract (RHT) [16,21]. The RHT is responsible for conveying the light information from RGCs to the part of the brain that controls circadian rhythms within the whole body [22,23]. The RGCs that express melanopsin were named intrinsically photosensitive RGCs (ipRGCs), and these cells were no longer intrinsically photosensitive in melanopsin knockout (KO) mice, although the cell number, morphology, and projections remained unchanged [24].

    Additional studies have also shown that melanopsin KO mice entrained to light-dark photoperiods, albeit the response to light was attenuated in the KO animals as the magnitude of the phase-shift is about half (40%) of that of wild-type mice at each of the three non-saturating irradiance levels [25]. A saturating white light pulse also produced a diminished phase shift in the KO animals [26]. The length of the free-running period that follows the exposure to constant light is reduced (to about 55�% of that of controls) in melanopsin KO animals [25,26].

    Melanopsin has also been implicated in regulation of the pupillary light reflex (PLR). Transgenic mice lacking rod and cone photoreceptors (rdcl) retain a PLR, and this response is driven by a photo pigment with peak sensitivity of around 479 nm [27]. Melanopsin KO animals showed a PLR indistinguishable from that of the wild-type mice at low irradiances, but at high irradiances, the reflex was incomplete. This result suggests that the melanopsin-associated system and the classical rod/cone system are complementary functions [28,29]. Thus, the current view is that no single photoreceptor type is necessary for the synchronization of circadian rhythms with external light-dark cycles [30,31].

    Finally, mice with the melanopsin gene ablated only in ipRGCs have normal outer retinal function but lack non-image-forming visual responses, such as circadian photoentrainment, light modulation of activity, and PLR [32]. Thus, the ipRGCs represent the site of integration of non-image-forming photo responses in mammals.

    Further studies have also shown that melanopsin-based photoreception is involved in the modulation of sleep [33-36] and mood and learning [37], and recent data have also indicated that melanopsin-based photoreception may be involved in the regulation of metabolism [38]. Finally, it has been reported that loss of the melanopsin gene abolishes circadian control in some parameters of cone electroretinogram, causing significant attenuation of the diurnal variation in cone vision [39]. Melanopsin signaling may influence intraretinal signaling by acting on dopaminergic neurons [40]. Therefore, these data suggest melanopsin-dependent regulation of visual processing within the retina.

    Melanopsin also plays an important role in mediating human circadian rhythms. Several studies have reported that in humans, the action spectra for melatonin suppression has a lambda max (λmax) of around 460 nm, suggesting that melanopsin is a key player in the photic regulation of melatonin levels [41-43]. Additional studies have also shown that blue light in the range of 460� nm is more effective compared to monochromatic light of 555 nm in phase-shifting the human circadian clock [44,45]. Finally, a recent study expanded these previous results by showing that light in the 555 nm range may significantly affect the synchronization of the circadian system to light exposure of short duration or to low irradiance, whereas light in the 460 nm range is more effective in phase-shifting the circadian system than exposure to light of longer duration and higher irradiance [46]. Additional studies have also shown that exposure to blue light can increase alertness [47-50] and stimulate cognitive functions [51-53]. A recent study reported that exposure to light-emitting e-readers at bedtime may negatively affect sleep and the circadian system [54]. Finally, blue light may also be used to treat seasonal affective disorders [55], and mutations in the melanopsin gene may increase the susceptibility to developing seasonal affective disorders [56,57]. However, another study reported that exposure to blue-enriched light was less effective compared to full-spectrum light in the treatment of seasonal affective disorder [58].

    With age, the lens becomes more yellowish, and thus, the spectrum of blue light transmission dramatically decreases through the years. It is suspected that one reason older individuals experience sleep problems is the lack of blue light during the daytime. Ayaki et al. [59] reported that after cataract extraction, sleep quality improved dramatically because more blue light could pass through the intraocular lens. In addition, there has been a discussion on whether a clear or yellow lens is preferable [60]. Of course, the yellow lens may protect the retina, but the clear lens provides more blue light during the day, providing better quality of sleep [61]. Consistent with this result, Sletten et al. [62] reported that in older people, acute exposure to blue light, but not to green light, significantly decreased their alertness and suppressed their sleep and melatonin production compared to young people. However, another study reported that in older patients with decreased lens transmittance, melatonin was not significantly suppressed following blue light exposure [43]. Thus, whether the yellowing of the lens associated with aging really affects the non-image-forming photoreception is still a matter of debate.

    Light-induced damage to the retina

    Several investigations have shown that exposure to light of specific wavelengths or intensity may induce severe damage to the retina [63,64]. This type of damage is called light-induced damage. Light can induce damage via three mechanisms: photomechanical, photothermal, and photochemical. Photomechanical damage is due to a rapid increase in the amount of energy captured by the RPE, which may cause irreversible damage to the RPE and lead to photoreceptor damage. This type of retinal damage depends on the amount of energy absorbed and not on the spectral composition of the light. Photothermal damage occurs when the retina and the RPE are exposed to brief (100 ms to 10 s) but intense light that induce a significant increase in the temperature of these tissues [63,64].

    A more common type of retinal/RPE damage is photochemical damage, which occurs when the eyes are exposed to light of high intensity in the visible range (390� nm). The current view suggests that there are two distinct types of photochemical damage. The first type is associated with short but intense exposure to light affecting the RPE, and the second type is associated with longer but less intense light exposure, affecting the outer segment of the photoreceptors. Short (up to 12 h) exposure to blue light may induce damage in the RPE of the rhesus monkey [65], and a clear relationship has been found between the extent of the damage and the oxygen concentration [66,67]. The fact that many different antioxidants can reduce the damage suggests that this type of damage is associated with oxidative processes [68,69]. Experimental data suggest that lipofuscin is the chromophore involved in the mediation of light-induced retinal damage following the exposure to blue light [70-73].

    The second type of light-induced photochemical damage occurs with longer (12� h) but less intense light exposure. This type of damage was initially observed in albino rats [74] but has also been observed in other species. The cones seem to be more vulnerable compared to the rods [75]. Several lines of evidence suggest that the visual photo pigments (e.g., rhodopsin and cone opsins) are involved in this type of damage. Early studies [76-78] also provided evidence that the action spectrum for light-induced photoreceptor damage is similar to the absorption spectrum of rhodopsin, but later studies indicated that blue light (400� nm) might be more damaging [79-81]. Grimm et al. [82] provided an explanation for this phenomenon, demonstrating that in vivo bleached rhodopsin may be regenerated not only via a metabolic pathway (e.g., via the visual cycle) but also via a photochemical reaction called photoreversal of bleaching [83] that is most effective with blue light. Photoreversal of bleaching augments the capability of rhodopsin molecules to absorb photons by several orders of magnitude, thus allowing the molecules to reach the critical number of photons required to induce damage in the retinal cells [84].

    This process can further increase the potential production of reactive oxygen species (ROS) thus, the oxidative damage can lead to the accumulation and build-up of lipofuscin in the RPE. The build-up of lipofuscin in the RPE can affect the ability of the RPE to provide nutrients to the photoreceptors, affecting photoreceptor viability [85]. Moreover, when lipofuscin absorbs blue light, the material becomes phototoxic, which can lead to further damage in the RPE and in the photoreceptors [70]. The data from our laboratory indicate that in albino rats, exposure to blue light (λmax 474 nm, 1휐 𢄡 μW/cm 2 ) acutely suppressed melatonin levels [6] while exposure to blue light for 4 h/day for 30 days did not produce significant effects on photoreceptor viability ( Figure 3 ). However, this treatment produced a small (10�%) but significant reduction in the levels of melanopsin and short wavelength opsin mRNAs in rats exposed to white or green (λmax513 nm) light ( Figure 4 ).

    Top panels. The exposure to blue light (λmax 474), green light (λmax 513), or fluorescent light at the intensity of 1휐 𢄡 μW/cm 2 for 4 h/day for 30 days did not produce a significant change in the number of cells in the photoreceptor layers of the Sprague-Dawley rats (n=6 see [121] for details about the methods used to quantify cells in the photoreceptor layer). Lower panels. The exposure to blue or green light-emitting diodes (LEDs) for 4 h in the middle of the day did not induce apoptosis. Terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay: 4- to 6-week-old Sprague-Dawley rats (n=6) were anesthetized (75 mg/kg ketamine and 23 mg/kg xylazine), kept on heating pads (37 ଌ), and exposed to blue or green light for 4 h. The pupils were dilated with 1% atropine and 2.5% phenylephrine eye drops 45 min before the light exposure. Rats were then killed 16 h after the exposure to blue light or green light. The eyes were explanted and fixed using freshly prepared 4% polyformaldehyde in PBS, pH 7.4 for 20 min at room temperature. They were washed 3X with PBS, permeabilized with freshly prepared 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice (2𠄸 ଌ), and then the TUNEL reaction was performed according to the instructions included in the manual (In Situ cell Death Detection kit). The slides were incubated in a humidified container for 60 min at 37 ଌ in the dark. Slides were rinsed 3X with PBS, and samples were analyzed under a fluorescence microscope (Zeiss Axioskop).

    Different light treatments did not affect rhodopsin mRNA levels (one-way ANOVA, pϠ.1). Exposure to blue light (λmax 474) at the intensity of 1휐 𢄡 μW/cm 2 for 4 h/day for 30 days produced significant changes in the mRNA levels of short wavelength sensitive (SW) opsin, melanopsin, and medium wavelength sensitive opsin (* one-way ANOVA followed by Holm-Sidak tests, pπ.05). Rats were exposed to blue, green, or white light-emitting diodes (LEDs) every day (4 h) for 30 days in the middle of the day (11:00 to 15:00) and then returned to a 12 h:12 h light-dark cycle. The intensity of the light during the light phase of the 12 h:12 h light-dark cycle was about 400� lux. Every day, the pupils were dilated with 1% atropine and 2.5% phenylephrine eye drops 45 min before exposure to blue, green, or white light-emitting diodes (LEDs). After 30 days, the rats were killed, and the retinas were explanted, immediately frozen, and stored at � ଌ. mRNA was then extracted, and mRNA levels were measured using real-time quantitative PCR (qPCR see [122] for details about primers and qPCR conditions).

    In this context, two recent studies on the effect of blue light exposure on the RPE and cone-like cells (661W, murine photoreceptor-derived cells [86]) should be mentioned. In the first study, Arnault et al. [87] reported that in the primary porcine RPE, exposure to light with irradiance similar to that of natural sunlight, that is, light in the range of 415� nm, was the most effective in reducing cell viability.

    In the second study, Kuse et al. [88] reported that 661W cells are more sensitive to light-induced damage when exposed to light emitted by blue (464 nm) LEDs than when exposed to green (522 nm) or white LEDs (wavelength peak at 456 and 553 nm) of the same intensity (0.38 mW/cm 2 ). The exposure to blue light, unlike the exposure to white and green LEDs, also produced a significant increase in ROS and induced cell damage. Similar results were also observed in primary retinal cells [88]. These data support the idea that exposure to blue light in the range of 400� nm (even at low levels) may damage photoreceptors and retinal pigment epithelium cells.

    Although most studies have focused on the acute effect of light exposure, several have also investigated the cumulative effect of light. For example, Noell [89] reported that a single 5 min exposure to light did not induce significant damage in photoreceptor cells, whereas a series of 5 min exposures led to significant photoreceptor damage. Furthermore, the time between exposures affects the cumulative effect of light [90-92]. In some cases, intermittent light exposure may produce even more pronounced damage than an equivalent amount of light in a single exposure [93]. In addition, the type of illumination to which the animals had been exposed before the experimental treatment influenced the extent of the retinal damage following light exposure. For example, rats raised in complete darkness showed greater susceptibility to light-induced retinal damage [89], and rats raised in an 800 lux light-dark cycle were more resistant to light-induced retinal damage compared to animals raised in a 5 lux light-dark cycle [94]. Finally, light-induced damage to photoreceptors increases with age. The exposure to light that might affect adult animals might not induce retinal damage in young animals [95]. In this context, with age, superoxide dismutase 1 (SOD1) and protective enzymes do not function as well due to zinc deficiency. SOD1 does not function well because the enzyme activity is controlled by zinc. Imamura et al. have shown that even with normal light that contains some blue light, fluorescent light damaged the retina tremendously in the SOD1 knockout mouse, which is similar to an aging mouse [96]. However, nothing happened in the normal mouse. The protective mechanism of the retina is important. From that point of view, the protective function of lutein, or blue-blocking pigment, on the retina is also considered. Ozawa et al. published research showing that when lutein was given, retina photodamage was alleviated [97].

    Finally, the severity of light-induced retinal damage changes with the time of the day [98-102]. For example, rats are three to four times more susceptible to light damage at night (01:00) than during the day (09:00 and 17:00). The circadian dependency of light-induced photoreceptor damage appears to involve mechanisms that control cAMP and c-fos levels (see [63] for a review), both of which are under the control of the retinal circadian clock [103,104]. Exposure to blue light during the night might have more negative effects compared to the same exposure during the daytime. However, in this case, this assumption is based on the experimental data obtained from nocturnal rodents. Thus, it is difficult to determine whether light-induced retinal damage has a daily rhythm in humans, and further studies on diurnal animal models (e.g., non-human primates) are required to address this important point.

    Experimental evidence indicates that wavelengths in the blue part of the spectrum (400� nm) can induce damage in the retina, and although the initial damage following exposure to blue light may be confined to the RPE, a damaged RPE eventually leads to photoreceptor death. Although most studies on the effects of blue light have focused on the mechanisms responsible for the damage to the photoreceptors following an acute exposure to high intensity light, some studies have reported that sub-threshold exposure to blue light can also induce damage in photoreceptors [105-107]. In addition, several authors have proposed that the amount of blue light received during an individual’s entire lifespan can be an important factor in the development of age-related macular degeneration (AMD). The use of lenses (intra- and extraocular) that block blue light (𠇋lue-blockers”) may provide some protection against the development of AMD [60,108].

    The mechanism through which long-term exposure to blue light may induce photoreceptor damage is mostly unknown. Several studies have indicated lipofuscin (absorption peak around 450 nm) is a possible mediator of the risk associated with long-term exposure to blue light–induced retinal damage [109,110]. Lipofuscin accumulates in the RPE in the form of granules located in the lysosomes of the RPE. The formation of lipofuscin begins in photoreceptors’ outer segments as a byproduct of the degradation of rod photoreceptor discs [105]. When lipofuscin absorbs blue light, ROS are produced, and these free radicals are responsible for the oxidative damage that occurs in the retina. The number of reactive oxygen species produced by lipofuscin is directly related to the spectral composition of the light, and it steadily decreases from 400 to 490 nm [73]. The accumulation of lipofuscin in the RPE, particularly in the macula, has been linked to photoreceptor death and to AMD [109]. Furthermore, the amount of lipofuscin present in the RPE increases with age (i.e., the amount of lipofuscin is low in young and high in old animals) thus, the potential for blue light to damage the retina may increase with age [111]. Finally, it has been reported that chronic exposure to blue light may accelerate photoreceptor degeneration in an animal model in the study of retinal degeneration [112].

    Thus, experimental evidence obtained from different experimental models indicates that exposure to blue light in the 470� nm range may be less damaging to the eye compared to blue light in the 400� nm range. We believe that the development of LEDs with a peak emission of around 470� nm may represent an important advancement in the safety of LEDs for ocular health [6] ( Figure 3 ).

    Light exposure and age-related macular degeneration in humans

    A series of studies in many animal models have shown that exposure to blue light may represent a risk for the development of AMD or other retinal pathologies [113,114]. However, the real risk from artificial light (white or blue) exposure in humans is difficult to assess, since light therapy has been in use for only a few years and in a small number of individuals. In addition, individual susceptibility to blue light damage varies significantly among individuals, making the assessment of the risk associated with repeated exposure to blue light in the etiology of AMD difficult.

    Previous epidemiological studies have indicated that chronic exposure to visible and blue light may be a cofactor in the development of AMD [115-117]. However, Darzin et al. [118] found no significant relationships between blue light and the development of AMD. Okuno et al. [119] evaluated the blue-light hazards from many different light sources and reported that the exposure (even for less than a minute) to blue light from the sun, arc-welding lamps, and the arc of discharge lamps is hazardous to the retina, whereas the exposure to blue light from fluorescent lamps or LEDs does not pose a significant hazard.

    Thus, it is clear that many different factors are involved in the pathogenesis of AMD. This observation, together with the limited data in terms of number of subjects or length of treatment, makes it difficult to predict the association between blue light exposure and the development of AMD.

    Finally, ultraviolet (UV) light is a risk factor for age-related macular degeneration. UV is mostly blocked by the cornea or lens therefore, only visible light can penetrate the eye and reach the retina. A recent study by Narimatsu et al. [120] conducted with an animal model reported that blocking UV light and blue light with yellow-tinted intraocular lenses materials (400� nm) could protect the retina [120]. Thus, reducing the amount of blue light reaching the retina in the range 400� nm may also be important for the protection of the retina.


    Genes' effect on mood and depression

    Every part of your body, including your brain, is controlled by genes. Genes make proteins that are involved in biological processes. Throughout life, different genes turn on and off, so that — in the best case — they make the right proteins at the right time. But if the genes get it wrong, they can alter your biology in a way that results in your mood becoming unstable. In a person who is genetically vulnerable to depression, any stress (a missed deadline at work or a medical illness, for example) can then push this system off balance.

    Mood is affected by dozens of genes, and as our genetic endowments differ, so do our depressions. The hope is that as researchers pinpoint the genes involved in mood disorders and better understand their functions, depression treatment can become more individualized and more successful. Patients would receive the best medication for their type of depression.

    Another goal of gene research, of course, is to understand how, exactly, biology makes certain people vulnerable to depression. For example, several genes influence the stress response, leaving us more or less likely to become depressed in response to trouble.

    Perhaps the easiest way to grasp the power of genetics is to look at families. It is well known that depression and bipolar disorder run in families. The strongest evidence for this comes from the research on bipolar disorder. Half of those with bipolar disorder have a relative with a similar pattern of mood fluctuations. Studies of identical twins, who share a genetic blueprint, show that if one twin has bipolar disorder, the other has a 60% to 80% chance of developing it, too. These numbers don't apply to fraternal twins, who — like other biological siblings — share only about half of their genes. If one fraternal twin has bipolar disorder, the other has a 20% chance of developing it.

    The evidence for other types of depression is more subtle, but it is real. A person who has a first-degree relative who suffered major depression has an increase in risk for the condition of 1.5% to 3% over normal.

    One important goal of genetics research — and this is true throughout medicine — is to learn the specific function of each gene. This kind of information will help us figure out how the interaction of biology and environment leads to depression in some people but not others.

    Temperament shapes behavior

    Genetics provides one perspective on how resilient you are in the face of difficult life events. But you don't need to be a geneticist to understand yourself. Perhaps a more intuitive way to look at resilience is by understanding your temperament. Temperament — for example, how excitable you are or whether you tend to withdraw from or engage in social situations — is determined by your genetic inheritance and by the experiences you've had during the course of your life. Some people are able to make better choices in life once they appreciate their habitual reactions to people and to life events.

    Cognitive psychologists point out that your view of the world and, in particular, your unacknowledged assumptions about how the world works also influence how you feel. You develop your viewpoint early on and learn to automatically fall back on it when loss, disappointment, or rejection occurs. For example, you may come to see yourself as unworthy of love, so you avoid getting involved with people rather than risk losing a relationship. Or you may be so self-critical that you can't bear the slightest criticism from others, which can slow or block your career progress.

    Yet while temperament or world view may have a hand in depression, neither is unchangeable. Therapy and medications can shift thoughts and attitudes that have developed over time.

    Stressful life events

    At some point, nearly everyone encounters stressful life events: the death of a loved one, the loss of a job, an illness, or a relationship spiraling downward. Some must cope with the early loss of a parent, violence, or sexual abuse. While not everyone who faces these stresses develops a mood disorder — in fact, most do not — stress plays an important role in depression.

    As the previous section explained, your genetic makeup influences how sensitive you are to stressful life events. When genetics, biology, and stressful life situations come together, depression can result.

    Stress has its own physiological consequences. It triggers a chain of chemical reactions and responses in the body. If the stress is short-lived, the body usually returns to normal. But when stress is chronic or the system gets stuck in overdrive, changes in the body and brain can be long-lasting.

    How stress affects the body

    Stress can be defined as an automatic physical response to any stimulus that requires you to adjust to change. Every real or perceived threat to your body triggers a cascade of stress hormones that produces physiological changes. We all know the sensations: your heart pounds, muscles tense, breathing quickens, and beads of sweat appear. This is known as the stress response.

    The stress response starts with a signal from the part of your brain known as the hypothalamus. The hypothalamus joins the pituitary gland and the adrenal glands to form a trio known as the hypothalamic-pituitary-adrenal (HPA) axis, which governs a multitude of hormonal activities in the body and may play a role in depression as well.

    When a physical or emotional threat looms, the hypothalamus secretes corticotropin-releasing hormone (CRH), which has the job of rousing your body. Hormones are complex chemicals that carry messages to organs or groups of cells throughout the body and trigger certain responses. CRH follows a pathway to your pituitary gland, where it stimulates the secretion of adrenocorticotropic hormone (ACTH), which pulses into your bloodstream. When ACTH reaches your adrenal glands, it prompts the release of cortisol.

    The boost in cortisol readies your body to fight or flee. Your heart beats faster — up to five times as quickly as normal — and your blood pressure rises. Your breath quickens as your body takes in extra oxygen. Sharpened senses, such as sight and hearing, make you more alert.

    CRH also affects the cerebral cortex, part of the amygdala, and the brainstem. It is thought to play a major role in coordinating your thoughts and behaviors, emotional reactions, and involuntary responses. Working along a variety of neural pathways, it influences the concentration of neurotransmitters throughout the brain. Disturbances in hormonal systems, therefore, may well affect neurotransmitters, and vice versa.

    Normally, a feedback loop allows the body to turn off "fight-or-flight" defenses when the threat passes. In some cases, though, the floodgates never close properly, and cortisol levels rise too often or simply stay high. This can contribute to problems such as high blood pressure, immune suppression, asthma, and possibly depression.

    Studies have shown that people who are depressed or have dysthymia typically have increased levels of CRH. Antidepressants and electroconvulsive therapy are both known to reduce these high CRH levels. As CRH levels return to normal, depressive symptoms recede. Research also suggests that trauma during childhood can negatively affect the functioning of CRH and the HPA axis throughout life.

    Early losses and trauma

    Certain events can have lasting physical, as well as emotional, consequences. Researchers have found that early losses and emotional trauma may leave individuals more vulnerable to depression later in life.

    Profound early losses, such as the death of a parent or the withdrawal of a loved one's affection, may resonate throughout life, eventually expressing themselves as depression. When an individual is unaware of the wellspring of his or her illness, he or she can't easily move past the depression. Moreover, unless the person gains a conscious understanding of the source of the condition, later losses or disappointments may trigger its return.

    Journal of the American Medical Association showed that women who were abused physically or sexually as children had more extreme stress responses than women who had not been abused. The women had higher levels of the stress hormones ACTH and cortisol, and their hearts beat faster when they performed stressful tasks, such as working out mathematical equations or speaking in front of an audience.

    Many researchers believe that early trauma causes subtle changes in brain function that account for symptoms of depression and anxiety. The key brain regions involved in the stress response may be altered at the chemical or cellular level. Changes might include fluctuations in the concentration of neurotransmitters or damage to nerve cells. However, further investigation is needed to clarify the relationship between the brain, psychological trauma, and depression.

    Seasonal affective disorder: When winter brings the blues

    Many people feel sad when summer wanes, but some actually develop depression with the season's change. Known as seasonal affective disorder (SAD), this form of depression affects about 1% to 2% of the population, particularly women and young people.

    SAD seems to be triggered by more limited exposure to daylight typically it comes on during the fall or winter months and subsides in the spring. Symptoms are similar to general depression and include lethargy, loss of interest in once-pleasurable activities, irritability, inability to concentrate, and a change in sleeping patterns, appetite, or both.

    To combat SAD, doctors suggest exercise, particularly outdoor activities during daylight hours. Exposing yourself to bright artificial light may also help. Light therapy, also called phototherapy, usually involves sitting close to a special light source that is far more intense than normal indoor light for 30 minutes every morning. The light must enter through your eyes to be effective skin exposure has not been proven to work. Some people feel better after only one light treatment, but most people require at least a few days of treatment, and some need several weeks. You can buy boxes that emit the proper light intensity (10,000 lux) with a minimal amount of ultraviolet light without a prescription, but it is best to work with a professional who can monitor your response.

    There are few side effects to light therapy, but you should be aware of the following potential problems:

    • Mild anxiety, jitteriness, headaches, early awakening, or eyestrain can occur.
    • There is evidence that light therapy can trigger a manic episode in people who are vulnerable.
    • While there is no proof that light therapy can aggravate an eye problem, you should still discuss any eye disease with your doctor before starting light therapy. Likewise, since rashes can result, let your doctor know about any skin conditions.
    • Some drugs or herbs (for example, St. John's wort) can make you sensitive to light.
    • If light therapy isn't helpful, antidepressants may offer relief.

    Medical problems

    Certain medical problems are linked to lasting, significant mood disturbances. In fact, medical illnesses or medications may be at the root of up to 10% to 15% of all depressions.

    Among the best-known culprits are two thyroid hormone imbalances. An excess of thyroid hormone (hyperthyroidism) can trigger manic symptoms. On the other hand, hypothyroidism, a condition in which your body produces too little thyroid hormone, often leads to exhaustion and depression.

    Heart disease has also been linked to depression, with up to half of heart attack survivors reporting feeling blue and many having significant depression. Depression can spell trouble for heart patients: it's been linked with slower recovery, future cardiovascular trouble, and a higher risk of dying within about six months. Although doctors have hesitated to give heart patients older depression medications called tricyclic antidepressants because of their impact on heart rhythms, selective serotonin reuptake inhibitors seem safe for people with heart conditions.

    The following medical conditions have also been associated with depression and other mood disorders:

    • degenerative neurological conditions, such as multiple sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease
    • stroke
    • some nutritional deficiencies, such as a lack of vitamin B12
    • other endocrine disorders, such as problems with the parathyroid or adrenal glands that cause them to produce too little or too much of particular hormones
    • certain immune system diseases, such as lupus
    • some viruses and other infections, such as mononucleosis, hepatitis, and HIV
    • cancer
    • erectile dysfunction in men.

    When considering the connection between health problems and depression, an important question to address is which came first, the medical condition or the mood changes. There is no doubt that the stress of having certain illnesses can trigger depression. In other cases, depression precedes the medical illness and may even contribute to it. To find out whether the mood changes occurred on their own or as a result of the medical illness, a doctor carefully considers a person's medical history and the results of a physical exam.

    If depression or mania springs from an underlying medical problem, the mood changes should disappear after the medical condition is treated. If you have hypothyroidism, for example, lethargy and depression often lift once treatment regulates the level of thyroid hormone in your blood. In many cases, however, the depression is an independent problem, which means that in order to be successful, treatment must address depression directly.


    Materials and methods

    Mutagenesis and molecular cloning

    Synthetic DNA oligonucleotides used for cloning and library construction were purchased from Integrated DNA Technologies (Coralville, Iowa, United States of America). Random mutagenesis of NIR-GECO variants was performed using Taq DNA polymerase (New England Biolabs, Ipswich, Massachusetts, USA) with conditions that resulted in a mutation frequency of 1 to 2 mutations per 1,000 base pairs. Gene fragments for NIR-GECO libraries were then inserted between restriction sites XhoI and HindIII of pcDuex2 for expression. The DNA sequences encoding miRFP1 to 172, CaM-RS20 (from NIR-GECO1), and miRFP179 to 311 were amplified by PCR amplification separately and then used as DNA templates for the assembly of miRFP1 to 172—CaM-RS20—miRFP179 to 311 by overlap extension PCR. The resulting DNA sequence was then digested and ligated into the pcDNA3.1(-) vector for mammalian expression and into a pBAD-MycHisC (Invitrogen, Waltham, Massachusetts, USA) vector for bacterial expression. Q5 high-fidelity DNA polymerase (New England Biolabs) was used for routine PCR amplification and overlap extension PCR. PCR products and products of restriction digests were routinely purified using preparative agarose gel electrophoresis followed by DNA isolation with the GeneJET gel extraction kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Restriction endonucleases were purchased from Thermo Fisher Scientific. Ligations were performed using T4 ligase in Rapid Ligation Buffer (Thermo Fisher Scientific).

    Genes or gene libraries in expression plasmids were electroporated into E. coli strain DH10B (Thermo Fisher Scientific). The transformed cells were then plated on 10-cm Lysogeny broth (LB) agar Petri dishes supplemented with 400 μg/mL ampicillin (Sigma, St. Louis, Missouri, USA) and 0.0004% (wt/vol) L-arabinose (Alfa Aesar, Haverhill, Massachusetts, USA) at 37°C overnight. For library screening, bright bacterial colonies expressing NIR-GECO variants were picked and cultured. Proteins were extracted using B-PER (Thermo Fisher Scientific) from overnight cultures of bacteria growing in LB media supplemented with 100 μg/mL ampicillin and 0.0016% L-arabinose and then tested for fluorescence and Ca 2+ response in 384-well plates. Variants with the highest brightness and Ca 2+ response were selected, and the corresponding plasmids were purified. HeLa cells were transfected with the selected plasmids, and live-cell fluorescence imaging was used to reevaluate both brightness and Ca 2+ response. Small-scale isolation of plasmid DNA was done with GeneJET miniprep kit (Thermo Fisher Scientific).

    For the construction of Opto-CRAC-EYFP, a synthetic double-stranded DNA fragment consisting of fused EYFP, LOV2, and STIM1-CT fragments (residues 336–486) [16], flanked with NotI and XhoI restriction sites, was cloned into the pcDNA3.1(-) vector.

    Protein purification and in vitro characterization

    The genes for the miRFP-based Ca 2+ indicators NIR-GECO1, NIR-GECO2, and NIR-GECO2G, with a poly-histidine tag on the C-terminus, were expressed from a pBAD-MycHisC (Invitrogen) vector containing the gene of cyanobacteria Synechocystis HO1 as previously described [23,24]. Bacteria were lysed with a cell disruptor (Constant Systems, Northants, United Kingdom) and then centrifuged at 15,000g for 30 minutes, and proteins were purified by Ni-NTA affinity chromatography (Agarose Bead Technologies, Doral, Florida, USA). The buffer was exchanged to 10 mM MOPS, 100 mM KCl (pH 7.2) with centrifugal concentrators (GE Healthcare Life Sciences, Vancouver, British Columbia, Canada). The spectra of miRFP-based Ca 2+ indicator prototype, with and without Ca 2+ , were measured in a 384-well plate. Briefly, purified proteins were loaded into 384-well plates and then supplied with either 10 mM EGTA or 5 mM CaCl2 before measuring emission spectra. The ECs, QY, and pKa for NIR-GECO variants were determined as previously described [8]. Ca 2+ titrations of NIR-GECO variants were performed with EGTA-buffered Ca 2+ solutions. We prepared buffers by mixing a CaEGTA buffer (30 mM MOPS, 100 mM KCl, 10 mM EGTA, and 10 mM CaCl2) and an EGTA buffer (30 mM MOPS, 100 mM KCl, and 10 mM EGTA) to give free Ca 2+ concentrations ranging from 0 nM to 39 μM at 25°C. Fluorescence intensities were plotted against Ca 2+ concentrations and fitted by a sigmoidal binding function to determine the Hill coefficient and Kd. To determine koff for NIR-GECO variants, an SX20 stopped-flow spectrometer (Applied Photophysics, Surrey, UK) was used. Proteins samples with 10-μM CaCl2 (30 mM MOPS, 100 mM KCl, and pH 7.2) were rapidly mixed with 10 mM EGTA (30 mM MOPS, 100 mM KCl, and pH 7.2) at room temperature, and fluorescence growth curve was measured and fitted by a single exponential equation.

    Imaging of miRFP-based Ca 2+ indicator prototype in HeLa cells

    HeLa cells (40% to 60% confluent) on 24-well glass bottom plate (Cellvis, Mountain View, California, USA) were transfected with 0.5 μg of plasmid DNA and 2 μl TurboFect (Thermo Fisher Scientific). Following 2-hour incubation, the media was changed to DEME (Gibco, Waltham, Massachusetts, USA) with 10% fetal bovine serum (FBS) (Sigma), 2 mM GlutaMax (Thermo Fisher Scientific), and 1% penicillin-streptomycin (Gibco), and the cells were incubated for 48 hours at 37°C in a CO2 incubator before imaging. Prior to imaging, culture medium was changed to Hank’s Balanced Salt Solution (HBSS). Wide-field imaging was performed on a Nikon Eclipse Ti microscope that was equipped with a 75 W Nikon xenon lamp, a 16-bit 512SC QuantEM EMCCD (Photometrics, Tucson, Arizona, USA), and a 60× objective and was driven by a NIS-Elements AR 4.20 software package (Nikon, Tokyo, Japan). For time-lapse imaging, HeLa cells were treated with 4 mM EGTA (with 5-μM ionomycin) and then 10 mM CaCl2 (with 5-μM ionomycin). Images were taken every 5 seconds using a filter set with 650/60-nm excitation and 720/60-nm emission.

    Imaging of NIR-GECO1 and NIR-GECO2G in HeLa cells with Opto-CRAC

    HeLa cells were co-transfected with pcDNA3.1-NIR-GECO1 or pcDNA3.1-NIR-GECO2 and pcDNA3.1-Opto-CRAC-EYFP using transfection reagent Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions. An inverted microscope (D1, Zeiss, Oberkochen, Germany) equipped with a 63× objective lens (NA 1.4, Zeiss) and a multiwavelength LED light source (pE-4000, CoolLED, Andover, UK) was used. Blue (470 nm) and red (635 nm) excitations were used to illuminate Opto-CRAC-EYFP and image NIR-GECO variants, respectively. The GFP filter set (BP 470–490, T495lpxr dichroic mirror, and HQ525/50 emission filter) and the NIR filter set (ET 650/45x, T685lpxr dichroic mirror, and ET720/60 emission filter) were used to confirm the expression of Opto-CRAC-EYFP and NIR-GECO variants. The filter set (T685lpxr dichroic mirror, and ET720/60 emission filter) was used to stimulate Opto-CRAC and to acquire fluorescence imaging of NIR-GECO1 and NIR-GECO2G. Optical stimulation was achieved with the 470-nm LED light at a power density of 1.9 mW/mm 2 . Photoconversion testing of NIR-GECO2 and NIR-GECO2G was performed with 470-nm LED light at a power density of 6.2 mW/mm 2 . Fluorescence signals were recorded by a sCMOS camera (ORCA-Flash4.0LT, Hamamatsu, Hamamatsu City, Japan) and controlled by a software (HC Image or NIS-Elements Advanced Research).

    Imaging of NIR-GECO2G in Human iPSC-derived cardiomyocytes

    Human iPSC-derived cardiomyocytes (human iPSC cardiomyocytes—male | ax2505) were purchased from Axol Bioscience (Cambridge, UK). The 96-well glass bottom plate was first coated with fibronectin and gelatin (0.5% and 0.1%, respectively) at 37°C for at least 1 hour. The cells were then plated and cultured for 3 days in Axol’s Cardiomyocyte Maintenance Medium. IPSC-CMs were then transfected with pcDNA3.1-NIR-GECO2 with or without pcDNA3.1-hChR2-EYFP using Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions. The medium was switched to Tyrode’s buffer right before imaging. Imaging was performed with an inverted microscope (D1, Zeiss) equipped with a 63× objective lens (NA 1.4, Zeiss) and a multiwavelength LED light source (pE-4000, CoolLED) using the same settings described above.

    Imaging of NIR-GECO1, NIR-GECO2, NIR-GECO2G, and miRFP720 in cultured neurons

    For dissociated hippocampal mouse neuron culture preparation, postnatal day 0 or 1 Swiss Webster mice (Taconic Biosciences, Albany, New York, USA) were used as previously described [21]. Briefly, dissected hippocampal tissue was digested with 50 units of papain (Worthington Biochem, Lakewood, New Jersey, USA) for 6 to 8 minutes at 37°C, and the digestion was stopped by incubating with ovomucoid trypsin inhibitor (Worthington Biochem) for 4 minutes at 37°C. Tissue was then gently dissociated with Pasteur pipettes, and dissociated neurons were plated at a density of 20,000 to 30,000 per glass coverslip coated with Matrigel (BD Biosciences, San Jose, California, USA). Neurons were seeded in 100 μL plating medium containing Minimum Essential Medium (MEM) (Thermo Fisher Scientific), glucose (33 mM, Sigma), transferrin (0.01%, Sigma), HEPES (10 mM, Sigma), Glutagro (2 mM, Corning), insulin (0.13%, Millipore, Burlington, Massachusetts, USA), B27 supplement (2%, Gibco), and heat-inactivated FBS (7.5%, Corning, Corning, New York, USA). After cell adhesion, additional plating medium was added. AraC (0.002 mM, Sigma) was added when glia density was 50% to 70% of confluence. Neurons were grown at 37°C and 5% CO2 in a humidified atmosphere.

    To express each of NIR-GECO variants in primary hippocampal neurons and compare their brightness and photostability, the gene encoding NIR-GECO(1,2,2G)-T2A-GFP was constructed using overlap-extension PCR followed by subcloning into pAAV-CAG vector (Addgene no. 108420) using BamHI and EcoRI sites. The gene for miRFP720-P2A-GFP was synthesized de novo by GenScript, based on the reported sequence [13], and cloned into the pAAV-CAG vector. Cultured neurons were transfected with plasmids (1.5 μg of plasmid DNA per well) at 4 to 5 days in vitro (DIV) using a commercial calcium phosphate transfection kit (Thermo Fisher Scientific) as previously described [21,25].

    Wide-field fluorescence microscopy of cultured neurons was performed using an epifluorescence inverted microscope (Eclipse Ti-E, Nikon) equipped with an Orca-Flash4.0 V2 sCMOS camera (Hamamatsu) and a SPECTRA X light engine (Lumencor, Beaverton, Oregon, USA). The NIS-Elements Advanced Research (Nikon) was used for automated microscope and camera control. Cells were imaged with a 20× NA 0.75 air objective lens (Nikon) at room temperature for quantification of brightness or a 40× NA 1.15 for photobleaching experiments (excitation: 631/28 nm emission: 664LP).

    Field stimulation

    Neurons expressing NIR-GECO variants (driven by CAG promoter) were imaged and stimulated in 24-well plates with 300 μL growth medium in each well at room temperature. Field stimuli (83 Hz, 50 V, and 1 ms) were delivered in trains of 1, 2, 3, 5, 10, 20, 40, and 80 via 2 platinum electrodes with a width of 6.5 mm to neurons. Neurons were imaged simultaneously while delivering trains of field stimuli with a 40× NA 1.15, a 631/28-nm LED (Spectra X light engine, Lumencor). Fluorescence was collected through 664LP using a sCMOS camera (Orca-Flash4.0, Hamamatsu) at 2 Hz.

    Ethics statement

    All experimental manipulations performed at MIT were in accordance with protocols approved by the Massachusetts Institute of Technology Committee on Animal Care (protocol number: 1218-100-21), following guidelines described in the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures performed at McGill University were in accordance with the Canadian Council on Animal Care guidelines for the use of animals in research and approved by the Montreal Neurological Institute Animal Care Committee (protocol number: 2015–7728).

    IUE and acute brian slice imaging

    In utero electroporation (IUE) was used to deliver the DNA encoding NIR-GECO2 and CoChR or NIR-GECO2G to the mouse brain. Briefly, embryonic day (E) 15.5 timed-pregnant female Swiss Webster (Taconic Biosciences) mice were deeply anesthetized with 2% isoflurane. Uterine horns were exposed and periodically rinsed with warm sterile PBS. A mixture of plasmids pAAV-CAG-NIR-GECO2-WPRE and pCAG-CoChR-mTagBFP2-Kv2.2motif-WPRE or plasmid pAAV-CAG-NIR-GECO2G (at total DNA concentration approximately 1 to 2 μg/μL) were injected into the lateral ventricle of 1 cerebral hemisphere of an embryo. Five voltage pulses (50 V, 50-ms duration, and 1 Hz) were delivered using round plate electrodes (ECM™ 830 electroporator, Harvard Apparatus, Holliston, Massachusetts, USA). Injected embryos were placed back into the dam and allowed to mature to delivery.

    Acute brain slices were obtained from Swiss Webster (Taconic Biosciences) mice at postnatal day (P) P11 to P22, using standard techniques. Mice were used without regard for sex. Mice were anesthetized by isoflurane inhalation, decapitated, and cerebral hemispheres were quickly removed and placed in cold choline-based cutting solution consisting of (in mM) 110 choline chloride, 25 NaHCO3, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4, 25 glucose, 11.6 ascorbic acid, and 3.1 pyruvic acid (339 to 341 mOsm/kg pH 7.75 adjusted with NaOH) for 2 minutes, blocked, and transferred into a slicing chamber containing ice-cold choline-based cutting solution. Coronal slices (300-μm thick) were cut with a Compresstome VF-300 slicing machine, transferred to a holding chamber with artificial cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 2.5 KCl, 25 NaHCO3, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, and 11 glucose (300 to 310 mOsm/kg pH 7.35 adjusted with NaOH), and recovered for 10 minutes at 34 °C, followed by another 30 minutes at room temperature. Slices were subsequently maintained at room temperature until use. Both cutting solution and ACSF were constantly bubbled with 95% O2 and 5% CO2.

    Individual slices were transferred to a recording chamber mounted on an inverted microscope (Eclipse Ti-E, Nikon) and continuously superfused (2 to 3 mL/min) with ACSF at room temperature. Cells were visualized through a 10× (0.45 NA) or 20× (0.75 NA) air objective with epifluorescence to identify positive cells. The fluorescence of NIR-GECO2 or NIR-GECO2G was excited by a SPECTRA X light engine (Lumencor) with 631/28-nm excitation and was collected through a 664LP emission filter, and imaged onto an Orca-Flash4.0 V2 sCMOS camera (Hamamatsu). Optical stimulation of slices expressing NIR-GECO2 and CoChR was performed using a 470-nm LED (M470L3, ThorLabs, Newton, New Jersey, USA) at 0.157 mW/mm 2 . A 4-AP stimulation was done by adding 4-AP solution to the imaging chamber at a final concentration of 1 mM.

    Imaging of NIR-GECO2 in C. elegans

    Worms were cultured and maintained following standard protocols [26]. The genes of NIR-GECO2, NIR-GECO2G, HO1, CoChR, and jGCaMP7s for expression in C. elegans were codon-optimized using SnapGene codon-optimization tool and synthesized by GenScript. Transgenic worms expressing NIR-GECO2(G) and jGCaMP7s pan-neuronally or NIR-GECO2 in AVA and CoChR-GFP in ASH were generated by injecting the plasmids tag-168::NLS-NIR-GECO2(G)-T2A-HO1(or NIR-GECO2) and tag-168::NLS-jGCaMP7s or plasmids flp-18::NIR-GECO2-T2A-HO1, sra-6::CoChR-SL2-GFP, and elt-2::NLS-GFP into N2 background worms, respectively, picking those with the strongest expression of green fluorescence (in neurons for the pan-neuronal strain and in the gut for optogenetic strain). NLS sequence used in this experiment was PKKKRKV.

    Hermaphrodite transgenic worms were picked at L4 stage of development and put onto NGM plates with freshly seeded OP50 lawns 12 to 24 hours before experiments, with or without 100-μM all-trans-retinal (ATR) for optogenetic experiments. Worms were mounted on 2% agarose pads on microscope slides, immobilized with 5 mM tetramisole, covered by a coverslip, and imaged using a Nikon Eclipse Ti inverted microscope equipped with a confocal spinning disk (CSU-W1), a 40×, 1.15 NA water-immersion objective, and a 5.5 Zyla camera (Andor, Belfast, Northern Ireland), controlled by NIS-Elements AR software. To acquire data shown in Fig 3 and S8 Fig, the fluorescence of NIR-GECO2 was imaged with 640-nm excitation provided by a 41.9-mW laser and a 685/40-nm emission filter jGCaMP7s/GFP fluorescence was imaged with a 488-nm excitation provided by 59.9-mW laser and a 525/50-nm emission filter. Optogenetic stimulation was performed with 488-nm illumination at 20 mW/mm 2 . For 200 mM NaCl stimulation, worms were imaged using the same optical setup as above, using a microfluidic device that was described previously [20].

    For brightness and SBR comparison of NLS-jGCaMP7s and NIR-GECO2, as shown in Fig 4A, 4B and 4D, the plasmids tag168::NLS-jGCaMP7s and tag168::NIR-GECO2-T2A-HO1 were mixed at a ratio of 1:1 before injection. NLS-jGCaMP7s was imaged with 488-nm excitation at a power of 17.2 mW/mm 2 , and a 525/50-nm emission filter. NIR-GECO2 was imaged with 640-nm excitation at a power of 12.6 mW/mm 2 ,and a 660LP emission filter. All other instrument settings were the same for NLS-jGCaMP7s and NIR-GECO2. The data for brightness from each ROI were averaged by ROI area. SBR was obtained via dividing fluorescence intensity from neurons by averaged autofluorescence from the intestine area. The imaging conditions in Fig 4C and 4E are the same as those in Fig 3. The data for SNR of NLS-jGCaMP7s and NLS-NIR-GECO2 were quantified from spontaneously spiking neurons. SNR was calculated by dividing fluorescence change associated with a spike by the standard deviation (SD) of the baseline fluorescence over the 2-second period immediately before the spike. The imaging conditions in S9 Fig are the same as those in Fig 4.

    Imaging of NIR-GECO2G in Xenopus laevis tadpoles

    NIR-GECO2G and GCaMP6s were cloned into the pCS2+ vector and the plasmid was linearized with NotI. Capped mRNA of NIR-GECO2G and GCaMP6s was transcribed with the SP6 mMessageMachine Kit (Ambion, Thermo Fisher Scientific). The RNA (500 pg of each sample) was injected in 1 blastomere at the 2-cell stage resulting in animals expressing NIR-GECO2G and GCaMP6s protein in 1 lateral half of the animal. The animals were kept at 20°C until stage 47. Immediately before imaging, the tadpole was paralyzed with pancuronium bromide (1.5 mg/mL in 0.1× MSBH) and embedded in 1% low-melt agarose.

    Light-sheet imaging was performed on a Zeiss Z1 located in the McGill Advanced BioImaging Facility. The instrument was equipped with a sCMOS camera (1920 × 1920 pixel PCO.Edge, Kelheim, Germany), excitation lasers with wavelengths of 488 nm (75-mW max. output) and 640 nm (50-mW max. output). For acquisition, we excited the sample with 10% intensity of each wavelength through 5× LSFM excitation objectives (NA = 0.1) resulting in a 4.53-μm thick light sheet. We utilized a water immersion objective for detection (Zeiss 10× PLAN APOCHROMAT, NA = 0.5, UV-VIS-IR, ND = 1.336), allowing the sample to be immersed in 0.1× MBSH. The fluorescence was directed via a dichroic mirror LBF 405/488/640 nm and filtered depending on the probe with 505- to 545-nm bandpass or 660-nm long-pass emission filters.

    The imaging data (Fig 5) was acquired with 50-ms integration time and 2.39 seconds cycle time through the volume. The instrument has a short dead time to home the axial position leading to a scanning frequency of 3.0 seconds for the entire volume. The raw data were corrected for drift and rapid movement with the ImageJ plugin TurboReg [27]. The image in Fig 5A is a volume projection of a 45-μm thick volume capturing the spontaneous Ca 2+ responses in the olfactory bulb. The cells were manually selected if they showed a spontaneous Ca 2+ response as detected with both NIR-GECO2G and GCaMP6s (Fig 5B). The fluorescence response was measured as the mean fluorescence intensity per cell and normalized by Inorm = (Im-Imin)/(Imax-Imin). Im indicates the measured mean value per area and Imax and Imin the maximal and minimal value measured for the specific ROI. The normalized response of NIR-GECO2G and GCaMP6s of a cell is plotted over time in the graph in Fig 5C.

    Data and image analysis

    All images in the manuscript were processed and analyzed using either ImageJ (NIH) or NIS-Elements Advanced Research software (Nikon). Traces and graphs were generated using GraphPad prism 8, Origin (OriginLab, Wellesley, Massachusetts, USA), and Matlab. Data are presented as mean ± SD or mean ± standard error of the mean (SEM) as indicated.


    Watch the video: Neuronal activity during perception (May 2022).


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