Guide Processing of Environmental Information in Vertebrates

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On average, a mammal has a brain roughly twice as large as that of a bird of the same body size, and ten times as large as that of a reptile of the same body size. Size, however, is not the only difference: there are also substantial differences in shape. The hindbrain and midbrain of mammals are generally similar to those of other vertebrates, but dramatic differences appear in the forebrain, which is greatly enlarged and also altered in structure. In non-mammalian vertebrates, the surface of the cerebrum is lined with a comparatively simple three-layered structure called the pallium.

In mammals, the pallium evolves into a complex six-layered structure called neocortex or isocortex. The elaboration of the cerebral cortex carries with it changes to other brain areas. The superior colliculus , which plays a major role in visual control of behavior in most vertebrates, shrinks to a small size in mammals, and many of its functions are taken over by visual areas of the cerebral cortex.

The brains of humans and other primates contain the same structures as the brains of other mammals, but are generally larger in proportion to body size. It takes into account the nonlinearity of the brain-to-body relationship. Dolphins have values higher than those of primates other than humans, [54] but nearly all other mammals have EQ values that are substantially lower.

Most of the enlargement of the primate brain comes from a massive expansion of the cerebral cortex, especially the prefrontal cortex and the parts of the cortex involved in vision.

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It has been estimated that visual processing areas occupy more than half of the total surface of the primate neocortex. It takes up a much larger proportion of the brain for primates than for other species, and an especially large fraction of the human brain. The brain develops in an intricately orchestrated sequence of stages. Neurons are created in special zones that contain stem cells , and then migrate through the tissue to reach their ultimate locations.

Once neurons have positioned themselves, their axons sprout and navigate through the brain, branching and extending as they go, until the tips reach their targets and form synaptic connections. In a number of parts of the nervous system, neurons and synapses are produced in excessive numbers during the early stages, and then the unneeded ones are pruned away. For vertebrates, the early stages of neural development are similar across all species. The neural plate folds inward to form the neural groove , and then the lips that line the groove merge to enclose the neural tube , a hollow cord of cells with a fluid-filled ventricle at the center.

At the front end, the ventricles and cord swell to form three vesicles that are the precursors of the prosencephalon forebrain , mesencephalon midbrain , and rhombencephalon hindbrain. At the next stage, the forebrain splits into two vesicles called the telencephalon which will contain the cerebral cortex, basal ganglia, and related structures and the diencephalon which will contain the thalamus and hypothalamus. At about the same time, the hindbrain splits into the metencephalon which will contain the cerebellum and pons and the myelencephalon which will contain the medulla oblongata.

Each of these areas contains proliferative zones where neurons and glial cells are generated; the resulting cells then migrate, sometimes for long distances, to their final positions. Once a neuron is in place, it extends dendrites and an axon into the area around it. Axons, because they commonly extend a great distance from the cell body and need to reach specific targets, grow in a particularly complex way. The tip of a growing axon consists of a blob of protoplasm called a growth cone , studded with chemical receptors.

These receptors sense the local environment, causing the growth cone to be attracted or repelled by various cellular elements, and thus to be pulled in a particular direction at each point along its path. The result of this pathfinding process is that the growth cone navigates through the brain until it reaches its destination area, where other chemical cues cause it to begin generating synapses.

Considering the entire brain, thousands of genes create products that influence axonal pathfinding.


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The synaptic network that finally emerges is only partly determined by genes, though. In many parts of the brain, axons initially "overgrow", and then are "pruned" by mechanisms that depend on neural activity. In the first stages of development, each axon from the retina is guided to the right general vicinity in the midbrain by chemical cues, but then branches very profusely and makes initial contact with a wide swath of midbrain neurons.

The retina, before birth, contains special mechanisms that cause it to generate waves of activity that originate spontaneously at a random point and then propagate slowly across the retinal layer. These waves are useful because they cause neighboring neurons to be active at the same time; that is, they produce a neural activity pattern that contains information about the spatial arrangement of the neurons.

This information is exploited in the midbrain by a mechanism that causes synapses to weaken, and eventually vanish, if activity in an axon is not followed by activity of the target cell. The result of this sophisticated process is a gradual tuning and tightening of the map, leaving it finally in its precise adult form. Similar things happen in other brain areas: an initial synaptic matrix is generated as a result of genetically determined chemical guidance, but then gradually refined by activity-dependent mechanisms, partly driven by internal dynamics, partly by external sensory inputs.

In some cases, as with the retina-midbrain system, activity patterns depend on mechanisms that operate only in the developing brain, and apparently exist solely to guide development. In humans and many other mammals, new neurons are created mainly before birth, and the infant brain contains substantially more neurons than the adult brain. The two areas for which adult neurogenesis is well established are the olfactory bulb, which is involved in the sense of smell, and the dentate gyrus of the hippocampus, where there is evidence that the new neurons play a role in storing newly acquired memories.

With these exceptions, however, the set of neurons that is present in early childhood is the set that is present for life. Glial cells are different: as with most types of cells in the body, they are generated throughout the lifespan. There has long been debate about whether the qualities of mind , personality, and intelligence can be attributed to heredity or to upbringing—this is the nature and nurture controversy. Genes determine the general form of the brain, and genes determine how the brain reacts to experience.

Experience, however, is required to refine the matrix of synaptic connections, which in its developed form contains far more information than the genome does.

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In some respects, all that matters is the presence or absence of experience during critical periods of development. The functions of the brain depend on the ability of neurons to transmit electrochemical signals to other cells, and their ability to respond appropriately to electrochemical signals received from other cells. The electrical properties of neurons are controlled by a wide variety of biochemical and metabolic processes, most notably the interactions between neurotransmitters and receptors that take place at synapses.

Neurotransmitters are chemicals that are released at synapses when an action potential activates them—neurotransmitters attach themselves to receptor molecules on the membrane of the synapse's target cell, and thereby alter the electrical or chemical properties of the receptor molecules. With few exceptions, each neuron in the brain releases the same chemical neurotransmitter, or combination of neurotransmitters, at all the synaptic connections it makes with other neurons; this rule is known as Dale's principle.

The great majority of psychoactive drugs exert their effects by altering specific neurotransmitter systems. This applies to drugs such as cannabinoids , nicotine , heroin, cocaine , alcohol, fluoxetine , chlorpromazine , and many others. The two neurotransmitters that are used most widely in the vertebrate brain are glutamate , which almost always exerts excitatory effects on target neurons, and gamma-aminobutyric acid GABA , which is almost always inhibitory. Neurons using these transmitters can be found in nearly every part of the brain.

Some general anesthetics act by reducing the effects of glutamate; most tranquilizers exert their sedative effects by enhancing the effects of GABA. There are dozens of other chemical neurotransmitters that are used in more limited areas of the brain, often areas dedicated to a particular function. Serotonin , for example—the primary target of antidepressant drugs and many dietary aids—comes exclusively from a small brainstem area called the raphe nuclei. As a side effect of the electrochemical processes used by neurons for signaling, brain tissue generates electric fields when it is active.

When large numbers of neurons show synchronized activity, the electric fields that they generate can be large enough to detect outside the skull, using electroencephalography EEG [72] or magnetoencephalography MEG. EEG recordings, along with recordings made from electrodes implanted inside the brains of animals such as rats, show that the brain of a living animal is constantly active, even during sleep.

In mammals, the cerebral cortex tends to show large slow delta waves during sleep, faster alpha waves when the animal is awake but inattentive, and chaotic-looking irregular activity when the animal is actively engaged in a task. During an epileptic seizure , the brain's inhibitory control mechanisms fail to function and electrical activity rises to pathological levels, producing EEG traces that show large wave and spike patterns not seen in a healthy brain.

Relating these population-level patterns to the computational functions of individual neurons is a major focus of current research in neurophysiology. All vertebrates have a blood—brain barrier that allows metabolism inside the brain to operate differently from metabolism in other parts of the body. Glial cells play a major role in brain metabolism by controlling the chemical composition of the fluid that surrounds neurons, including levels of ions and nutrients. Brain tissue consumes a large amount of energy in proportion to its volume, so large brains place severe metabolic demands on animals.

The need to limit body weight in order, for example, to fly, has apparently led to selection for a reduction of brain size in some species, such as bats. Information from the sense organs is collected in the brain. There it is used to determine what actions the organism is to take. The brain processes the raw data to extract information about the structure of the environment. Next it combines the processed information with information about the current needs of the animal and with memory of past circumstances.

Finally, on the basis of the results, it generates motor response patterns. These signal-processing tasks require intricate interplay between a variety of functional subsystems. The function of the brain is to provide coherent control over the actions of an animal. A centralized brain allows groups of muscles to be co-activated in complex patterns; it also allows stimuli impinging on one part of the body to evoke responses in other parts, and it can prevent different parts of the body from acting at cross-purposes to each other.

The human brain is provided with information about light, sound, the chemical composition of the atmosphere, temperature, head orientation, limb position, the chemical composition of the bloodstream, and more. In other animals additional senses are present, such as the infrared heat-sense of snakes, the magnetic field sense of some birds, or the electric field sense of some types of fish. Each sensory system begins with specialized receptor cells, [8] such as light-receptive neurons in the retina of the eye, or vibration-sensitive neurons in the cochlea of the ear.

The axons of sensory receptor cells travel into the spinal cord or brain, where they transmit their signals to a first-order sensory nucleus dedicated to one specific sensory modality. This primary sensory nucleus sends information to higher-order sensory areas that are dedicated to the same modality. Eventually, via a way-station in the thalamus , the signals are sent to the cerebral cortex, where they are processed to extract the relevant features, and integrated with signals coming from other sensory systems.

Motor systems are areas of the brain that are involved in initiating body movements , that is, in activating muscles. Except for the muscles that control the eye, which are driven by nuclei in the midbrain, all the voluntary muscles in the body are directly innervated by motor neurons in the spinal cord and hindbrain. The intrinsic spinal circuits implement many reflex responses, and contain pattern generators for rhythmic movements such as walking or swimming. The descending connections from the brain allow for more sophisticated control.

The brain contains several motor areas that project directly to the spinal cord. At the lowest level are motor areas in the medulla and pons, which control stereotyped movements such as walking, breathing, or swallowing. At a higher level are areas in the midbrain, such as the red nucleus , which is responsible for coordinating movements of the arms and legs.

At a higher level yet is the primary motor cortex , a strip of tissue located at the posterior edge of the frontal lobe. The primary motor cortex sends projections to the subcortical motor areas, but also sends a massive projection directly to the spinal cord, through the pyramidal tract. This direct corticospinal projection allows for precise voluntary control of the fine details of movements. Other motor-related brain areas exert secondary effects by projecting to the primary motor areas. Among the most important secondary areas are the premotor cortex, basal ganglia, and cerebellum.

In addition to all of the above, the brain and spinal cord contain extensive circuitry to control the autonomic nervous system , which works by secreting hormones and by modulating the "smooth" muscles of the gut. Many animals alternate between sleeping and waking in a daily cycle. Arousal and alertness are also modulated on a finer time scale by a network of brain areas. A key component of the arousal system is the suprachiasmatic nucleus SCN , a tiny part of the hypothalamus located directly above the point at which the optic nerves from the two eyes cross.

The SCN contains the body's central biological clock. Neurons there show activity levels that rise and fall with a period of about 24 hours, circadian rhythms : these activity fluctuations are driven by rhythmic changes in expression of a set of "clock genes". The SCN continues to keep time even if it is excised from the brain and placed in a dish of warm nutrient solution, but it ordinarily receives input from the optic nerves, through the retinohypothalamic tract RHT , that allows daily light-dark cycles to calibrate the clock.

The SCN projects to a set of areas in the hypothalamus, brainstem, and midbrain that are involved in implementing sleep-wake cycles. An important component of the system is the reticular formation , a group of neuron-clusters scattered diffusely through the core of the lower brain. Reticular neurons send signals to the thalamus, which in turn sends activity-level-controlling signals to every part of the cortex. Damage to the reticular formation can produce a permanent state of coma. Sleep involves great changes in brain activity. During deep NREM sleep, also called slow wave sleep , activity in the cortex takes the form of large synchronized waves, whereas in the waking state it is noisy and desynchronized.

Levels of the neurotransmitters norepinephrine and serotonin drop during slow wave sleep, and fall almost to zero during REM sleep; levels of acetylcholine show the reverse pattern. For any animal, survival requires maintaining a variety of parameters of bodily state within a limited range of variation: these include temperature, water content, salt concentration in the bloodstream, blood glucose levels, blood oxygen level, and others.

The basic principle that underlies homeostasis is negative feedback : any time a parameter diverges from its set-point, sensors generate an error signal that evokes a response that causes the parameter to shift back toward its optimum value. In vertebrates, the part of the brain that plays the greatest role is the hypothalamus , a small region at the base of the forebrain whose size does not reflect its complexity or the importance of its function.

Some of these functions relate to arousal or to social interactions such as sexuality, aggression, or maternal behaviors; but many of them relate to homeostasis. Several hypothalamic nuclei receive input from sensors located in the lining of blood vessels, conveying information about temperature, sodium level, glucose level, blood oxygen level, and other parameters. These hypothalamic nuclei send output signals to motor areas that can generate actions to rectify deficiencies. Some of the outputs also go to the pituitary gland , a tiny gland attached to the brain directly underneath the hypothalamus.

The pituitary gland secretes hormones into the bloodstream, where they circulate throughout the body and induce changes in cellular activity. The individual animals need to express survival-promoting behaviors, such as seeking food, water, shelter, and a mate. The motivational system works largely by a reward—punishment mechanism. When a particular behavior is followed by favorable consequences, the reward mechanism in the brain is activated, which induces structural changes inside the brain that cause the same behavior to be repeated later, whenever a similar situation arises.

Conversely, when a behavior is followed by unfavorable consequences, the brain's punishment mechanism is activated, inducing structural changes that cause the behavior to be suppressed when similar situations arise in the future. Most organisms studied to date utilize a reward—punishment mechanism: for instance, worms and insects can alter their behavior to seek food sources or to avoid dangers.

Rewards and punishments function by altering the relationship between the inputs that the basal ganglia receive and the decision-signals that are emitted. The reward mechanism is better understood than the punishment mechanism, because its role in drug abuse has caused it to be studied very intensively. Research has shown that the neurotransmitter dopamine plays a central role: addictive drugs such as cocaine, amphetamine, and nicotine either cause dopamine levels to rise or cause the effects of dopamine inside the brain to be enhanced.

Almost all animals are capable of modifying their behavior as a result of experience—even the most primitive types of worms. Because behavior is driven by brain activity, changes in behavior must somehow correspond to changes inside the brain. Neuroscientists currently distinguish several types of learning and memory that are implemented by the brain in distinct ways:. The field of neuroscience encompasses all approaches that seek to understand the brain and the rest of the nervous system. The brain is also the most important organ studied in psychiatry , the branch of medicine that works to study, prevent, and treat mental disorders.

The oldest method of studying the brain is anatomical , and until the middle of the 20th century, much of the progress in neuroscience came from the development of better cell stains and better microscopes. Neuroanatomists study the large-scale structure of the brain as well as the microscopic structure of neurons and their components, especially synapses. Among other tools, they employ a plethora of stains that reveal neural structure, chemistry, and connectivity.

In recent years, the development of immunostaining techniques has allowed investigation of neurons that express specific sets of genes. Also, functional neuroanatomy uses medical imaging techniques to correlate variations in human brain structure with differences in cognition or behavior. Neurophysiologists study the chemical, pharmacological, and electrical properties of the brain: their primary tools are drugs and recording devices. Thousands of experimentally developed drugs affect the nervous system, some in highly specific ways.

Recordings of brain activity can be made using electrodes, either glued to the scalp as in EEG studies, or implanted inside the brains of animals for extracellular recordings, which can detect action potentials generated by individual neurons. The same techniques have occasionally been used to study brain activity in human patients suffering from intractable epilepsy , in cases where there was a medical necessity to implant electrodes to localize the brain area responsible for epileptic seizures.


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  • Another approach to brain function is to examine the consequences of damage to specific brain areas. Even though it is protected by the skull and meninges , surrounded by cerebrospinal fluid , and isolated from the bloodstream by the blood—brain barrier, the delicate nature of the brain makes it vulnerable to numerous diseases and several types of damage.

    In humans, the effects of strokes and other types of brain damage have been a key source of information about brain function. Because there is no ability to experimentally control the nature of the damage, however, this information is often difficult to interpret. In animal studies, most commonly involving rats, it is possible to use electrodes or locally injected chemicals to produce precise patterns of damage and then examine the consequences for behavior.

    Computational neuroscience encompasses two approaches: first, the use of computers to study the brain; second, the study of how brains perform computation. On one hand, it is possible to write a computer program to simulate the operation of a group of neurons by making use of systems of equations that describe their electrochemical activity; such simulations are known as biologically realistic neural networks. On the other hand, it is possible to study algorithms for neural computation by simulating, or mathematically analyzing, the operations of simplified "units" that have some of the properties of neurons but abstract out much of their biological complexity.

    The computational functions of the brain are studied both by computer scientists and neuroscientists. Computational neurogenetic modeling is concerned with the study and development of dynamic neuronal models for modeling brain functions with respect to genes and dynamic interactions between genes. Recent years have seen increasing applications of genetic and genomic techniques to the study of the brain [] and a focus on the roles of neurotrophic factors and physical activity in neuroplasticity.

    It is now possible with relative ease to "knock out" or mutate a wide variety of genes, and then examine the effects on brain function.

    Processing Of Environmental Information In Vertebrates

    More sophisticated approaches are also being used: for example, using Cre-Lox recombination it is possible to activate or deactivate genes in specific parts of the brain, at specific times. The oldest brain to have been discovered was in Armenia in the Areni-1 cave complex. The brain, estimated to be over 5, years old, was found in the skull of a 12 to year-old girl. Although the brains were shriveled, they were well preserved due to the climate found inside the cave.

    Early philosophers were divided as to whether the seat of the soul lies in the brain or heart. Aristotle favored the heart, and thought that the function of the brain was merely to cool the blood. Democritus, the inventor of the atomic theory of matter, argued for a three-part soul, with intellect in the head, emotion in the heart, and lust near the liver. Men ought to know that from nothing else but the brain come joys, delights, laughter and sports, and sorrows, griefs, despondency, and lamentations.

    And by the same organ we become mad and delirious, and fears and terrors assail us, some by night, and some by day, and dreams and untimely wanderings, and cares that are not suitable, and ignorance of present circumstances, desuetude, and unskillfulness. All these things we endure from the brain, when it is not healthy The Roman physician Galen also argued for the importance of the brain, and theorized in some depth about how it might work. Galen traced out the anatomical relationships among brain, nerves, and muscles, demonstrating that all muscles in the body are connected to the brain through a branching network of nerves.

    He postulated that nerves activate muscles mechanically by carrying a mysterious substance he called pneumata psychikon , usually translated as "animal spirits".

    Descartes, like Galen, thought of the nervous system in hydraulic terms. He believed that the highest cognitive functions are carried out by a non-physical res cogitans , but that the majority of behaviors of humans, and all behaviors of animals, could be explained mechanistically. The first real progress toward a modern understanding of nervous function, though, came from the investigations of Luigi Galvani — , who discovered that a shock of static electricity applied to an exposed nerve of a dead frog could cause its leg to contract. Since that time, each major advance in understanding has followed more or less directly from the development of a new technique of investigation.

    Until the early years of the 20th century, the most important advances were derived from new methods for staining cells.

    Biomonitoring of marine vertebrates in Monterey Bay using eDNA metabarcoding

    Without such a stain, brain tissue under a microscope appears as an impenetrable tangle of protoplasmic fibers, in which it is impossible to determine any structure. In the first half of the 20th century, advances in electronics enabled investigation of the electrical properties of nerve cells, culminating in work by Alan Hodgkin , Andrew Huxley , and others on the biophysics of the action potential, and the work of Bernard Katz and others on the electrochemistry of the synapse.

    Reflecting the new understanding, in Charles Sherrington visualized the workings of the brain waking from sleep:. The great topmost sheet of the mass, that where hardly a light had twinkled or moved, becomes now a sparkling field of rhythmic flashing points with trains of traveling sparks hurrying hither and thither. The brain is waking and with it the mind is returning. It is as if the Milky Way entered upon some cosmic dance.

    Swiftly the head mass becomes an enchanted loom where millions of flashing shuttles weave a dissolving pattern, always a meaningful pattern though never an abiding one; a shifting harmony of subpatterns. The invention of electronic computers in the s, along with the development of mathematical information theory , led to a realization that brains can potentially be understood as information processing systems.

    This concept formed the basis of the field of cybernetics , and eventually gave rise to the field now known as computational neuroscience. One of the most influential early contributions was a paper titled What the frog's eye tells the frog's brain : the paper examined the visual responses of neurons in the retina and optic tectum of frogs, and came to the conclusion that some neurons in the tectum of the frog are wired to combine elementary responses in a way that makes them function as "bug perceivers". Theorists have worked to understand these response patterns by constructing mathematical models of neurons and neural networks , which can be simulated using computers.

    The essential difficulty is that sophisticated computation by neural networks requires distributed processing in which hundreds or thousands of neurons work cooperatively—current methods of brain activity recording are only capable of isolating action potentials from a few dozen neurons at a time. Furthermore, even single neurons appear to be complex and capable of performing computations. However, the Human Brain Project is trying to build a realistic, detailed computational model of the entire human brain.

    The wisdom of this approach has been publicly contested, with high-profile scientists on both sides of the argument. In the second half of the 20th century, developments in chemistry, electron microscopy, genetics, computer science, functional brain imaging, and other fields progressively opened new windows into brain structure and function. In the United States, the s were officially designated as the " Decade of the Brain " to commemorate advances made in brain research, and to promote funding for such research.

    In the 21st century, these trends have continued, and several new approaches have come into prominence, including multielectrode recording , which allows the activity of many brain cells to be recorded all at the same time; [] genetic engineering , which allows molecular components of the brain to be altered experimentally; [] genomics , which allows variations in brain structure to be correlated with variations in DNA properties [] and neuroimaging.

    Animal brains are used as food in numerous cuisines. Some archaeological evidence suggests that the mourning rituals of European Neanderthals also involved the consumption of the brain. The Fore people of Papua New Guinea are known to eat human brains. In funerary rituals, those close to the dead would eat the brain of the deceased to create a sense of immortality.

    A prion disease called kuru has been traced to this. From Wikipedia, the free encyclopedia. This article is about the brains of all types of animals, including humans. For information specific to the human brain, see Human brain. For other uses, see Brain disambiguation and Brains disambiguation. Not to be confused with Brane or Brian. A common chimpanzee brain. Main article: Evolution of the brain. See also: List of regions in the human brain.

    See also: Human brain. Main article: Neural development. See also: Sleep. Main article: Neuroscience. For the scientific journal, see Brain Research. See also: History of neuroscience. Brain—computer interface Central nervous system disease List of neuroscience databases Neurological disorder Optogenetics Outline of neuroscience.

    Human anatomy 3rd ed. All researchers must wear laboratory gloves and eye protection at all times. If the solution touches skin, the affected area should be washed immediately with running water. Solution volume can be adjusted depending on the sample processing requirements. It is recommended to prepare the solution in a beaker or conical flask with a lid. Close the lid and gently mix until pellets are dissolved, this can be carried out by shaking by hand or using a magnetic stirrer depending on the volume being prepared. Once pellets have been dissolved, the solution will have turned colorless, leave for at least 15 min to cool.

    The final solution should be filtered using a Buchner filter or similar Table 1 to ensure that there are no residual microplastics in the solution, either from the KOH pellets, water or from procedural contamination. Laboratory preparation The laboratory should be scrubbed before use to remove any sources of airborne contamination. There should be limited access to reduce external contamination.

    Researchers in the laboratory should wear white cotton laboratory coat and gloves. All equipment must be rinsed at least three times with pre-filtered water immediately before use. Glass or metal equipment is recommended, however, if unavoidable see Table 1. The water supply used for flushing the samples must be filtered using a sieve with a mesh size smaller than target microplastics. Any variation on the color of the equipment should be noted. Set up nested metal sieves in a sink connected to the pre-filtered water source.

    The sieve at the top will have the largest mesh size e. Using consecutive sieves will allow removal of larger pieces of plastics, food remains and parasites. If researchers wish to target smaller plastics, they can add additional sieves. This allows for size differentiation and speeds up processing in later steps. The sieves should be covered with a lid when not in use to prevent contamination. Table 1. Troubleshooting advice and alternative methodological approaches Sample preparation If samples have been frozen, it is recommended that samples be defrosted slowly in a fridge h , to avoid the fast decomposition of the tissues.

    It is also possible to defrost samples at room temperature in a clean laboratory. Note: Depending on the sample collected there are different initial processing steps required. For digestive tract samples, proceed with Steps D2-D10, for regurgitate and scat samples proceed from Steps D Digestive tracts Digestive tracts must be rinsed externally before processing and removing the ties to remove external contamination. Note: Ties are used to stop contents leaking. Digestive tracts of marine organisms vary in length and number of compartments esophagus, stomachs and intestines. Each compartment should be processed and rinsed independently e.

    If different sections of fish digestive tracts cannot be differentiated, it can be analyzed without divisions. The most appropriate number is 20 equal pieces. The division of this part of the digestive tracts will allow the researcher to investigate if microplastics tend to concentrate in any specific area as some parasites do Lawlor et al. Each compartment or intestine section must be opened using clean and sterilized scissors and forceps. Transfer the remaining material to the nested sieve column avoiding touching the mucosal surface.

    Examination of the gastric and intestinal mucosal surface should be carried out with care to prevent damage to the surface by rubbing it with fingers or other material. If any pathology is detected, a sample of that area should be collected and stored with the fixed chemical required following the standard protocols. Compartments should be processed individually under the filtered water supply through nested sieves following Step E1 onwards. Scats and regurgitates solid samples The container where scats and regurgitates are stored must be rinsed externally with pre-filtered water before processing.

    This will avoid procedural contamination.

    The rest of the samples should be poured and processed in the nested sieves following Step E1 onwards. If the scat or the regurgitated sample is not fluid, they can be poured into a container with pre-filter water for few hours to hydrate before processing.

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    Samples must always be covered with a lid to prevent procedural contamination. Sample processing Rinse each sample or compartment into the nested sieves separately Figure 1. Store hard food remains e. Material kept in the smaller sieve will be transferred to a glass container with pre-filtered water to obtain a suspension for the microplastic sample Figure 1. The suspension should be as concentrated as possible. This will reduce the volume of KOH solution required in the following step. Note: Eye protection must be worn from this step.

    Cover samples loosely with aluminum foil, or screw top lids to prevent contamination and evaporation. Remove samples from the incubator and leave to cool before further processing. Alternatively, if this equipment is unavailable, glass funnels with a microfiber filter covered during the filtration can be used. Note : Filter papers must be checked under a microscope for contamination before use.

    When large amounts of undigested organic material e. Great care is required when using density separation as there are some less dense dietary remains that can float, such as crustacean carapaces. NaCl is the recommended density separation solution 1. More costly solutions include NaI 1. Another option to remove undigested organic matter involves rinsing the solution through a sieve once more before filtering; this reduces the likelihood of filter papers clogging.

    Visual characterization Visual characterization is valuable when researchers are categorizing and sorting samples. Visual characterization of plastics must be used with other more robust identification techniques such as chemical analysis e. If more robust techniques are not available, Steps F4-F5 can aid researchers in reducing the likelihood of misidentification.

    Use a stereomicroscope to investigate particles retained on the filter paper. It is recommended that a camera be attached to the microscope to allow researchers to record visual images of all particles. Visually inspect each filter paper for potential plastic microparticles. Carry out visual characterization following existing criteria Lusher et al. Record shape and color Figure 1. Shape categories include: fiber, fragment and spherical beads. Fragment can be further divided into films and foams depending on research objectives, but this division is not always necessary.

    Color is subjective and therefore not recommended as a stand-alone classification, but it does help researchers when categorizing samples. Particles observed need to be visually inspected for the following characteristics, otherwise they should be rejected or tested with other chemical techniques Lusher et al. No natural structures such as cells.

    Processing of Environmental Information in Vertebrates

    Unnatural bending. Fibers should have a consistent thickness throughout length. There should be no fraying at ends of fibers. A hot needle can be used during visual characterization to aid in the presence of plastic particles, which in case of plastic will react to the heat through bending or melting.

    Therefore, some research groups are advised to take this step with caution. Chemical characterization Note: Chemical characterization should only be carried out on clean and dry suspected plastic particles. Chemical characterization should be carried out on a representative subsample.

    The specific instrumentation used for chemical classification will depend on the research facilities available. Here we describe FT-IR because this instrumentation is available at most of research laboratories Figure 1. Trained personnel should conduct chemical analysis. Sufficient knowledge of polymer identification and spectra interpretation is required. Spectral analysis should follow the protocols available at individual laboratories to produce an output spectrum. All output spectra should be compared to a polymer library database. Caution should be taken for such interpretation.

    One example is water absorbance which can alter spectra and that have clear polymeric characteristics. Data analysis This protocol is a standard protocol for the collection of microplastics. Acknowledgments The authors obtained no external funding for the elaboration of this protocol, A. Competing interests The authors declare no conflict of interest.

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    Nat Ecol Evol 1 5 : Hernandez-Milian, G. Mar Pollut Bull 33 1 : Jepson, P. PCB pollution continues to impact populations of orcas and other dolphins in European waters. Karami, A. Microplastic and mesoplastic contamination in canned sardines and sprats. Sci Total Environ The use of potassium hydroxide KOH solution as a suitable approach to isolate plastics ingested by marine organisms.

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