Organ-on-a-chip

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An organ-on-a-chip (OOC) is a multi-channel 3-D microfluidic cell culture chip that simulates the activities, mechanics and physiological response of entire organs and organ systems, a type of artificial organ.^[1] It constitutes the subject matter of significant biomedical engineering research, more precisely in bio-MEMS. The convergence of labs-on-chips (LOCs) and cell biology has permitted the study of human physiology in an organ-specific context, introducing a novel model of in vitro multicellular human organisms. One day, they will perhaps abolish the need for animals in drug development and toxin testing.

Although multiple publications claim to have translated organ functions onto this interface, the movement towards this microfluidic application is still in its infancy. Organs-on-chips will vary in design and approach between different researchers. As such, validation and optimization of these systems will likely be a long process. Organs that have been simulated by microfluidic devices include the heart, the lung, kidney, artery, bone, cartilage, skin and more.

Nevertheless, building valid artificial organs requires not only a precise cellular manipulation, but a detailed understanding of the human body’s fundamental intricate response to any event. A common concern with organs-on-chips lies in the isolation of organs during testing. "If you don’t use as close to the total physiological system that you can, you’re likely to run into troubles"^[1] says William Haseltine, founder of Human Genome Sciences in Rockville, Maryland. Microfabrication, microelectronics and microfluidics offer the prospect of modeling sophisticated in vitro physiological responses under accurately simulated conditions.

Lab-on-chip

A lab-on-a-chip is a device that integrates one or several laboratory functions on a single chip that deals with handling particles in hollow microfluidic channels. It has been developed for over a decade. Advantages in handling particles at such a small scale include lowering fluid volume consumption (lower reagents costs, less waste), increasing portability of the devices, increasing process control (due to quicker thermo-chemical reactions) and decreasing fabrication costs. Additionally, microfluidic flow is entirely laminar (i.e., no turbulence). Consequently, there is virtually no mixing between neighboring streams in one hollow channel. In cellular biology convergence, this rare property in fluids has been leveraged to better study complex cell behaviors, such as cell motility in response to chemotactic stimuli, stem cell differentiation, axon guidance, subcellular propagation of biochemical signaling and embryonic development.^[2]

Transitioning from 3D cell-culture models to OOCs

3D cell-culture models exceed 2D culture systems by promoting higher levels of cell differentiation and tissue organization. 3D culture systems are more successful because the flexibility of the ECM gels accommodates shape changes and cell-cell connections – formerly prohibited by rigid 2D culture substrates. Nevertheless, even the best 3D culture models fail to mimic an organ’s cellular properties in many aspects,^[2] including tissue-to-tissue interfaces (e.g., epithelium and vascular endothelium), spatiotemporal gradients of chemicals, and the mechanically active microenvironments (e.g. arteries’ vasoconstriction and vasodilator responses to temperature differentials). The application of microfluidics in organs-on-chips enables the efficient transport and distribution of nutrients and other soluble cues throughout the viable 3D tissue constructs. Organs-on-chips are referred to as the next wave of 3D cell-culture models that mimic whole living organs’ biological activities, dynamic mechanical properties and biochemical functionalities.^[1]

Organs

Lung-on-a-chip

Lung-on-a-chips are being designed in an effort to improve the physiological relevance of existing in vitro alveolar-capillary interface models.^[3] Such a multifunctional microdevice can reproduce key structural, functional and mechanical properties of the human alveolar-capillary interface (i.e., the fundamental functional unit of the living lung).

Example

Dongeun Huh from Wyss Institute for Biologically Inspired Engineering at Harvard describes their fabrication of a system containing two closely apposed microchannels separated by a thin (10µm) porous flexible membrane made of PDMS.^[4] The device largely comprises three microfluidic channels, and only the middle one holds the porous membrane. Culture cells were grown on either side of the membrane: human alveolar epithelial cells on one side, and human pulmonary microvascular endothelial cells on the other.

The compartmentalization of the channels facilitates not only the flow of air as a fluid which delivers cells and nutrients to the apical surface of the epithelium, but also allows for pressure differences to exist between the middle and side channels. During normal inspiration in a human’s respiratory cycle, intrapleural pressure decreases, triggering an expansion of the alveoli. As air is pulled into the lungs, alveolar epithelium and the coupled endothelium in the capillaries are stretched. Since a vacuum is connected to the side channels, a decrease in pressure will cause the middle channel to expand, thus stretching the porous membrane and subsequently, the entire alveolar-capillary interface. The pressure-driven dynamic motion behind the stretching of the membrane, also described as a cyclic mechanical strain (valued at approximately 10%), significantly increases the rate of nanoparticle translocation across the porous membrane, when compared to a static version of this device, and to a Transwell culture system.

In order to fully validate the biological accuracy of a device, its whole-organ responses must be evaluated. In this instance, researchers inflicted injuries to the cells:

• Pulmonary inflammation

Pulmonary inflammatory responses entail a multistep strategy, but alongside an increased production of epithelial cells and an early response release of cytokines, the interface should undergo an increased number of leukocyte adhesion molecules.^[5] In Huh’s experiment, the pulmonary inflammation was simulated by introducing medium containing a potent proinflammatory mediator. Only hours after the injury was caused, the cells in the microfluidic device subjected to a cyclic strain reacted in accordance with the previously mentioned biological response.

• Pulmonary infection

Living E-coli bacteria was used to demonstrate how the system can even mimic the innate cellular response to a bacterial pulmonary infection. The bacteria were introduced onto the apical surface of the alveolar epithelium. Within hours, neutrophils were detected in the alveolar compartment, meaning they had transmigrated from the vascular microchannel where the porous membrane had phagocytized the bacteria.

Additionally, researchers believe the potential value of this lung-on-a-chip system will aid in toxicology applications. By investigating the pulmonary response to nanoparticles, researchers hope to learn more about health risks in certain environments, and correct previously oversimplified in vitro models. Because a microfluidic lung-on-a-chip can more exactly reproduce the mechanical properties of a living human lung, its physiological responses will be quicker and more accurate than a Transwell culture system. Nevertheless, published studies admit that responses of a lung-on-a-chip don’t yet fully reproduce the responses of native alveolar epithelial cells.

Heart-on-a-chip

Past efforts to replicate in vivo cardiac tissue environments have proven to be challenging due to difficulties when mimicking contractility and electrophysiological responses. Such features would greatly increase the accuracy of in vitro experiments.

Microfluidics has already contributed to in vitro experiments on cardiomyocytes, which generate the electrical impulses that control the heart rate.^[6] For instance, researchers have built an array of PDMS microchambers, aligned with sensors and stimulating electrodes as a tool that will electrochemically and optically monitor the cardiomyocytes’ metabolism.^[7] Another lab-on-a-chip similarly combined a microfluidic network in PDMS with planar microelectrodes, this time to measure extracellular potentials from single adult murine cardiomyocytes.^[8]

Example

Preparation of the Heart-on-a-chip substrate and contractility

test samples – After applying a stimulating the contraction of

the myocytes via the field electrodes, strips/teeth in the MTF

start to curl. Researchers have developed a correlation between

tissue stress and the radius of curvature of the MTF strips

during the contractile cycle, validating the demonstrated chip

as a heart-on-a-chip (in the realm of their respective needs).

A reported design of a heart-on-a-chip claims to have built "an efficient means of measuring structure-function relationships in constructs that replicate the hierarchical tissue architectures of laminar cardiac muscle."^[9] This chip determines that the alignment of the myocytes in the contractile apparatus made of cardiac tissue and the gene expression profile (affected by shape and cell structure deformation) contributes to the force produced in cardiac contractility. This heart-on-a-chip is a biohybrid construct: an engineered anisotropic ventricular myocardium is an elastomeric thin film.

The design and fabrication process of this particular microfluidic device entails first covering the edges of a glass surface with tape (or any protective film) such as to contour the substrate’s desired shape. A spin coat layer of PNIPA is then applied. After its dissolution, the protective film is peeled away, resulting in a self-standing body of PNIPA. The final steps involve the spin coating of protective surface of PDMS over the cover slip and curing. Muscular thin films (MTF) enable cardiac muscle monolayers to be engineered on a thin flexible substrate of PDMS.^[10] In order to properly seed the 2D cell culture, a microcontact printing technique was used to lay out a fibronectin "brick wall" pattern on the PDMS surface. Once the ventricular myocytes were seeded on the functionalized substrate, the fibronectin pattern oriented them to generate an anisotropic monolayer.

After the cutting of the thin films into two rows with rectangular teeth, and subsequent placement of the whole device in a bath, electrodes stimulate the contraction of the myocytes via a field-stimulation – thus curving the strips/teeth in the MTF. Researchers have developed a correlation between tissue stress and the radius of curvature of the MTF strips during the contractile cycle, validating the demonstrated chip as a "platform for quantification of stress, electrophysiology and cellular architecture."^[9]

Kidney-on-a-chip

Renal cells and nephrons have already been simulated by microfluidic devices. "Such cell cultures can lead to new insights into cell and organ function and be used for drug screening".^[11] A kidney-on-a-chip device has the potential to accelerate research encompassing artificial replacement for lost kidney function. Nowadays, dialysis requires patients to go to a clinic up to three times per week. A more transportable and accessible form of treatment would not only increase the patient’s overall health (by increasing frequency of treatment), but the whole process would become more efficient and tolerable.^[12] Artificial kidney research is striving to bring transportability, wearability and perhaps implantation capability to the devices through innovative disciplines: microfluidics, miniaturization and nanotechnology.^[13]

Example – nephron-on-a-chip

The nephron is the functional unit of the kidney and is composed of a glomerulus and a tubular component.^[14] Researchers at MIT claim to have designed a bioartificial device that replicates the function of the nephron’s glomerulus, proximal convoluted tubule and loop of Henle.

Each part of the device has its unique design, generally consisting of two microfabricated layers separated by a membrane. The only inlet to the microfluidic device is designed for the entering blood sample. In the glomerulus’ section of the nephron, the membrane allows certain blood particles through its wall of capillary cells, composed by the endothelium, basement membrane and the epithelial podocytes. The fluid that is filtered from the capillary blood into Bowman’s space is called filtrate or primary urine.^[15]

In the tubules, some substances are added to the filtrate as part of the urine formation, and some substances reabsorbed out of the filtrate and back into the blood. The first segment of these tubules is the proximal convoluted tubule. This is where the almost complete absorption of nutritionally important substances takes place. In the device, this section is merely a straight channel, but blood particles going to the filtrate have to cross the previously mentioned membrane and a layer of renal proximal tubule cells. The second segment of the tubules is the loop of Henle where the reabsorption of water and ions from the urine takes place. The device’s looping channels strives to simulate the countercurrent mechanism of the loop of Henle. Likewise, the loop of Henle requires a number of different cell types because each cell type has distinct transport properties and characteristics. These include the descending limb cells, thin ascending limb cells, thick ascending limb cells, cortical collecting duct cells and medullary collecting duct cells.^[14]

One step towards validating the microfluidic device’s simulation of the full filtration and reabsorption behavior of a physiological nephron would include demonstrating that the transport properties between blood and filtrate are identical with regards to where they occur and what is being let in by the membrane. For example, the large majority of passive transport of water occurs in the proximal tubule and the descending thin limb, or the active transport of NaCl largely occurs in the proximal tubule and the thick ascending limb. The device’s design requirements would require the filtration fraction in the glomerulus to vary between 15–20%, or the filtration reabsorption in the proximal convoluted tubule to vary between 65–70%, and finally the urea concentration in urine (collected at one of the two outlets of the device) to vary between 200–400 mM.^[16]

One recent report illustrates a biomimic nephron on hydrogel microfluidic devices with establishing the function of passive diffusion.^[17] The complex physiological function of nephron is achieved on the basis of interactions between vessels and tubules (both are hollow channels).^[18] However, conventional laboratory techniques usually focus on 2D structures, such as petri-dish that lacks capability to recapitulate real physiology that occurs in 3D. Therefore, the authors developed a new method to fabricate functional, cell-lining and perfusable microchannels inside 3D hydrogel. The vessel endothelial and renal epithelial cells are cultured inside hydrogel microchannel and form cellular coverage to mimic vessels and tubules, respectively. They employed confocal microscope to examine the passive diffusion of one small organic molecule (usually drugs) between the vessels and tubules in hydrogel. The study demonstrates the beneficial potential to mimic renal physiology for regenerative medicine and drug screening.

Artery-on-a-chip

Cardiovascular diseases are often caused by changes in structure and function of small blood vessels. For instance, self-reported rates of hypertension suggest that the rate is increasing, says a 2003 report from the National Health and Nutrition Examination Survey.^[19] A microfluidic platform simulating the biological response of an artery could not only enable organ-based screens to occur more frequently throughout a drug development trial, but also yield a comprehensive understanding of the underlying mechanisms behind pathologic changes in small arteries and develop better treatment strategies. Axel Gunther from the University of Toronto argues that such MEMS-based devices could potentially help in the assessment of a patient’s microvascular status in a clinical setting (personalized medicine).^[20]

Conventional methods used to examine intrinsic properties of isolated resistance vessels (arterioles and small arteries with diameters varying between 30 µm and 300 µm) include the pressure myography technique. However, such methods currently require manually skilled personnel and are not scalable. An artery-on-a-chip could overcome several of these limitations by accommodating an artery onto a platform which would be scalable, inexpensive and possibly automated in its manufacturing.

Example

An organ-based microfluidic platform has been developed as a lab-on-a-chip onto which a fragile blood vessel can be fixed, allowing for determinants of resistance artery malfunctions to be studied.

The artery microenvironment is characterized by surrounding temperature, transmural pressure, and luminal & abluminal drug concentrations. The multiple inputs from a microenvironment cause a wide range of mechanical or chemical stimuli on the smooth muscle cells (SMCs) and endothelial cells (ECs) that line the vessel’s outer and luminal walls, respectively. Endothelial cells are responsible for releasing vasoconstriction and vasodilator factors, thus modifying tone. Vascular tone is defined as the degree of constriction inside a blood vessel relative to its maximum diameter.^[21] Pathogenic concepts currently believe that subtle changes to this microenvironment have pronounced effects on arterial tone and can severely alter peripheral vascular resistance. The engineers behind this design believe that a specific strength lies in its ability to control and simulate heterogeneous spatiotemporal influences found within the microenvironment, whereas myography protocols have, by virtue of their design, only established homogeneous microenvironments.^[20] They proved that by delivering phenylephrine through only one of the two channels providing superfusion to the outer walls, the drug-facing side constricted much more than the drug opposing side.

The artery-on-a-chip is designed for reversible implantation of the sample. The device contains a microchannel network, an artery loading area and a separate artery inspection area. There is a microchannel used for loading the artery segment, and when the loading well is sealed, it is also used as a perfusion channel, to replicate the process of nutritive delivery of arterial blood to a capillary bed in the biological tissue.^[22] Another pair of microchannels serves to fix the two ends of the arterial segment. Finally, the last pair of microchannels is used to provide superfusion flow rates, in order to maintain the physiological and metabolic activity of the organ by delivering a constant sustaining medium over the abluminal wall. A thermoelectric heater and a thermoresistor are connected to the chip and maintain physiological temperatures at the artery inspection area.

The protocol of loading and securing the tissue sample into the inspection zone helps understand how this approach acknowledges whole organ functions. After immersing the tissue segment into the loading well, the loading process is driven by a syringe withdrawing a constant flow rate of buffer solution at the far end of the loading channel. This causes the transport of the artery towards its dedicated position. This is done with closed fixation and superfusion in/outlet lines. After stopping the pump, sub-atmospheric pressure is applied through one of the fixation channels. Then after sealing the loading well shut, the second fixation channel is subjected to a sub-atmospheric pressure. Now the artery is symmetrically established in the inspection area, and a transmural pressure is felt by the segment. The remaining channels are opened and constant perfusion and superfusion are adjusted using separate syringe pumps.^[20]

Skin-on-a-chip

A skin model on a biochip has been created by researchers at cellasys.^[23]

Human-on-a-chip

Researchers are working towards building a multi-channel 3D microfluidic cell culture system that compartmentalizes microenvironments in which 3D cellular aggregates are cultured to mimic multiple organs in the body.^[24] Most organ-on-a-chip models today only culture one cell type, so even though they may be valid models for studying whole organ functions, the systemic effect of a drug on the human body is not verified.

Conceptual schematic of a human-on-a-chip – Designing a

whole body biomimetic device will potentially correct one

of the most significant limitations on organs-on-chips: the

isolation of organs.

In particular, an integrated cell culture analog (µCCA) was developed and included lung cells, drug-metabolizing liver and fat cells. The cells were linked in a 2D fluidic network with culture medium circulating as a blood surrogate, thus efficiently providing a nutritional delivery transport system, while simultaneously removing wastes from the cells.^[25] "The development of the µCCA laid the foundation for a realistic in vitro pharmacokinetic model and provided an integrated biomimetic system for culturing multiple cell types with high fidelity to in vivo situations", claim C. Zhang et al. They have developed a microfluidic human-on-a-chip, culturing four different cell types to mimic four human organs: liver, lung, kidney and fat.^[26] They focused on developing a standard serum-free culture media that would be valuable to all cell types included in the device. Optimized standard media are generally targeted to one specific cell-type, whereas a human-on-a-chip will evidently require a common medium (CM). In fact, they claim to have identified a cell culture CM that, when used to perfuse all cell cultures in the microfluidic device, maintains the cells’ functional levels. Heightening the sensitivity of the in vitro cultured cells ensures the validity of the device, or that any drug injected into the microchannels will stimulate an identical physiological and metabolic reaction from the sample cells as whole organs in humans.

With more extensive development of this kind of chip, pharmaceutical companies will potentially be able to measure direct effects of one organ’s reaction on another. For instance, the delivery of biochemical substances would be screened to confirm that even though it may benefit one cell type, it does not compromise the functions of others. It is probably already possible to print these organs with 3D printers, but the cost is too high. Designing whole body biomimetic devices addresses a major reservation that pharmaceutical companies have towards organs-on-chips, namely the isolation of organs.^{[citation needed]} As these devices become more and more accessible, the complexity of the design increases exponentially. Systems will soon have to simultaneously provide mechanical perturbation and fluid flow through a circulatory system. "Anything that requires dynamic control rather than just static control is a challenge", says Takayama from the University of Michigan.^[27]

Replacing animal testing

In the early phase of drug development, animal models were the only way of obtaining in vivo data that would predict the human pharmacokinetic responses. However, experiments on animals are lengthy, expensive and controversial. For example, animal models are often subjected to mechanical or chemical techniques that simulate human injuries. There are also concerns with regards to the validity of such animal models, due to deficiency in cross-species extrapolation.^[28] Moreover, animal models offer very limited control of individual variables and it can be cumbersome to harvest specific information.

Therefore, mimicking a human's physiological responses in an in vitro model needs to be made more affordable, and needs to offer cellular level control in biological experiments: biomimetic microfluidic systems could replace animal testing. The development of MEMS-based biochips that reproduce complex organ-level pathological responses could revolutionize many fields, including toxicology and the developmental process of pharmaceuticals and cosmetics that rely on animal testing and clinical trials.^[29]

Recently, physiologically based perfusion in vitro systems have been developed to provide cell culture environment close to in vivo cell environment. A new testing platforms based on multi-compartmental perfused systems have gained a remarkable interest in pharmacology and toxicology. It aims to provide a cell culture environment close to the in vivo situation to reproduce more reliably in vivo mechanisms or ADME processes that involve its absorption, distribution, metabolism, and elimination. Perfused in vitro systems combined with kinetic modelling are promising tools for studying in vitro the different processes involved in the toxicokinetics of xenobiotics.

Efforts made toward the development of micro fabricated cell culture systems that aim to create models that replicate aspects of the human body as closely as possible and give examples that demonstrate their potential use in drug development, such as identifying synergistic drug interactions as well as simulating multi-organ metabolic interactions. Multi compartment micro fluidic-based devices, particularly those that are physical representations of physiologically based pharmacokinetic (PBPK) models that represent the mass transfer of compounds in compartmental models of the mammalian body, may contribute to improving the drug development process.

Mathematical pharmacokinetic (PK) models aim to estimate concentration-time profiles within each organ on the basis of the initial drug dose. Such mathematical models can be relatively simple, treating the body as a single compartment in which the drug distribution reaches a rapid equilibrium after administration. Mathematical models can be highly accurate when all parameters involved are known. Models that combine PK or PBPK models with PD models can predict the time-dependent pharmacological effects of a drug. We can nowadays predict with PBPK models the PK of about any chemical in humans, almost from first principles. These models can be either very simple, like statistical dose-response models, or sophisticated and based on systems biology, according to the goal pursued and the data available. All we need for those models are good parameter values for the molecule of interest.

Microfluidic cell culture systems such as micro cell culture analogs (μCCAs) could be used in conjunction with PBPK models. These μCCAs scaled-down devices, termed also body-on-a-chip devices, can simulate multi-tissue interactions under near-physiological fluid flow conditions and with realistic tissue-to-tissue size ratios . Data obtained with these systems may be used to test and refine mechanistic hypotheses. Microfabricating devices also allows us to custom-design them and scale the organs' compartments correctly with respect to one another.

Because the device can be used with both animal and human cells, it can facilitate cross-species extrapolation. Used in conjunction with PBPK models, the devices permit an estimation of effective concentrations that can be used for studies with animal models or predict the human response. In the development of multicompartment devices, representations of the human body such as those in used PBPK models can be used to guide the device design with regard to the arrangement of chambers and fluidic channel connections to augment the drug development process, resulting in increased success in clinical trials.

Molecular neuroscience

From Wikipedia, the free encyclopedia

 

Molecular neuroscience is a branch of neuroscience that observes concepts in molecular biology applied to the nervous systems of animals. The scope of this subject covers topics such as molecular neuroanatomy, mechanisms of molecular signaling in the nervous system, the effects of genetics and epigenetics on neuronal development, and the molecular basis for neuroplasticity and neurodegenerative diseases.^[1] As with molecular biology, molecular neuroscience is a relatively new field that is considerably dynamic.

Locating neurotransmitters

In molecular biology, communication between neurons typically occurs by chemical transmission across gaps between the cells called synapses. The transmitted chemicals, known as neurotransmitters, regulate a significant fraction of vital body functions.^[2] It is possible to anatomically locate neurotransmitters by labeling techniques. It is possible to chemically identify certain neurotransmitters such as catecholamines by fixing neural tissue sections with formaldehyde. This can give rise to formaldehyde-induced fluorescence when exposed to ultraviolet light. Dopamine, a catecholamine, was identified in the nematode C. elegans by using this technique.^[3]  Immunocytochemistry, which involves raising antibodies against targeted chemical or biological entities, includes a few other techniques of interest. A targeted neurotransmitter could be specifically tagged by primary and secondary antibodies with radioactive labeling in order to identify the neurotransmitter by autoradiography. The presence of neurotransmitters (though not necessarily the location) can be observed in enzyme-linked immunocytochemistry or enzyme--linked immunosorbent assays (ELISA) in which substrate-binding in the enzymatic assays can induce precipitates, fluorophores, or chemiluminescence. In the event that neurotransmitters cannot be histochemically identified, an alternative method is to locate them by their neural uptake mechanisms.^[1]

Voltage-gated ion channels

Structure of eukaryotic voltage-gated potassium ion channels

Excitable cells in living organisms have voltage-gated ion channels. These can be observed throughout the nervous system in neurons. The first ion channels to be characterized were the sodium and potassium ion channels by A.L. Hodgkin and A.F. Huxley in the 1950s upon studying the giant axon of the squid genus Loligo. Their research demonstrated the selective permeability of cellular membranes, dependent on physiological conditions, and the electrical effects that result from these permeabilities to produce action potentials.^[4]

Sodium ion channels

Sodium channels were the first voltage-gated ion channels to be isolated in 1984 from the eel Electrophorus electricus by Shosaku Numa. The pufferfish toxin tetrodotoxin (TTX), a sodium channel blocker, was used to isolate the sodium channel protein by binding it using the column chromatography technique for chemical separation. The amino acid sequence of the protein was analyzed by Edman degradation and then used to construct a cDNA library which could be used to clone the channel protein. Cloning the channel itself allowed for applications such as identifying the same channels in other animals.^[1] Sodium channels are known for working in concert with potassium channels during the development of graded potentials and action potentials. Sodium channels allow an influx of Na⁺ ions into a neuron, resulting in a depolarization from the resting membrane potential of a neuron to lead to a graded potential or action potential, depending on the degree of depolarization.^[5]

Potassium ion channels

Potassium channels come in a variety of forms, are present in most eukaryotic cells, and typically tend to stabilize the cell membrane at the potassium equilibrium potential. As with sodium ions, graded potentials and action potentials are also dependent on potassium channels. While influx of Na⁺ ions into a neuron induce cellular depolarization, efflux of influx of K⁺ ions out of a neuron causes a cell to repolarize to resting membrane potential. The activation of potassium ion channels themselves are dependent on the depolarization resulting from Na⁺ influx during an action potential.^[1] As with sodium channels, the potassium channels have their own toxins that block channel protein action. An example of such a toxin is the large cation, tetraethylammonium (TEA), but it is notable that the toxin does not have the same mechanism of action on all potassium channels, given the variety of channel types across species. The presence of potassium channels was first identified in Drosophila melanogaster mutant flies that shook uncontrollably upon anesthesia due to problems in cellular repolarization that led to abnormal neuron and muscle electrophysiology. Potassium channels were first identified by manipulating molecular genetics (of the flies) instead of performing channel protein purification because there were no known high-affinity ligands for potassium channels (such as TEA) at the time of discovery.^[1][6]

Calcium ion channels

Calcium channels are important for certain cell-signaling cascades as well as neurotransmitter release at axon terminals. A variety of different types of calcium ion channels are found in excitable cells. As with sodium ion channels, calcium ion channels have been isolated and cloned by chromatographic purification techniques. It is notable, as with the case of neurotransmitter release, that calcium channels can interact with intracelluar proteins and plays a strong role in signaling, especially in locations such as the sarcoplasmic reticulum of muscle cells.^[1]

Receptors

Various types of receptors can be used for cell signaling and communication and can include ionotropic receptors and metabotropic receptors. These cell surface receptor types are differentiated by the mechanism and duration of action with ionotropic receptors being associated with fast signal transmission and metabotropic receptors being associated with slow signal transmission. Metabotropic receptors happen to cover a wide variety of cell-surface receptors with notably different signaling cascades.^[1][5]

Ionotropic receptors

Prototypical depiction of ionotropic receptor in the case of Ca²⁺ ion flow

Ionotropic receptors, otherwise known as ligand-gated ion channels, are fast acting receptors that mediate neural and physiological function by ion channel flow with ligand-binding. Nicotinic, GABA, and Glutamate receptors are among some of the cell surface receptors regulated by ligand-gated ion channel flow. GABA is the brain's main inhibitory neurotransmitter and glutamate is the brain's main excitatory neurotransmitter.^[1]

GABA receptors

GABA_A and GABA_C receptors are known to be ionotropic, while the GABA_B receptor is metabotropic. GABA_A receptors mediate fast inhibitory responses in the central nervous system (CNS) and are found on neurons, glial cells, and adrenal medulla cells. It is responsible for inducing Cl⁻ ion influx into cells, thereby reducing the probability that membrane depolarization will occur upon the arrival of a graded potential or an action potential. GABA receptors can also interact with non-endogenous ligands to influence activity. For example, the compound diazepam (marketed as Valium) is an allosteric agonist which increases the affinity of the receptor for GABA. The increased physiological inhibitory effects resulting from increased GABA binding make diazepam a useful tranquilizer or anticonvulsant (antiepileptic drugs). On the other hand, GABA receptors can also be targeted by decreasing Cl⁻ cellular influx with the effect of convulsants like picrotoxin. The antagonistic mechanism of action for this compound is not directly on the GABA receptor, but there are other compounds that are capable of allosteric inactivation, including T-butylbicyclophorothionate (TBPS) and pentylenetetrazole (PZT). Compared with GABA_A, GABA_C receptors have a higher affinity for GABA, they are likely to be longer-lasting in activity, and their responses are likely to be generated by lower GABA concentrations.^[1]

Glutamate receptors

Ionotropic glutamate receptors can include NMDA, AMPA, and kainate receptors. These receptors are named after agonists that facilitate glutamate activity. NMDA receptors are notable for their excitatory mechanisms to affect neuronal plasticity in learning and memory, as well as neuropathologies such as stroke and epilepsy. NDMA receptors have multiple binding sites just like ionotropic GABA receptors and can be influenced by co-agonists such the glycine neurotransmitter or phencyclidine (PCP). The NMDA receptors carry a current by Ca²⁺ ions and can be blocked by extracellular Mg²⁺ ions depending on voltage and membrane potential. This Ca²⁺ influx is increased by excitatory postsynaptic potentials (EPSPs) produced by NMDA receptors, activating Ca²⁺-based signaling cascades (such as neurotransmitter release). AMPA generate shorter and larger excitatory postsynaptic currents than other ionotropic glutamate receptors.^[5]

Nicotinic ACh receptors

Nicotinic receptors bind the acetylcholine (ACh) neurotransmitter to produce non-selective cation channel flow that generates excitatory postsynaptic responses. Receptor activity, which can be influenced by nicotine consumption, produces feelings of euphoria, relaxation, and inevitably addiction in high levels.^[5]

Metabotropic receptors

G-protein-linked receptor signaling cascade

Metabotropic receptors, are slow response receptors in postsynaptic cells. Typically these slow responses are characterized by more elaborate intracellular changes in biochemistry. Responses of neurotransmitter uptake by metabotropic receptors can result in the activation of intracellualar enzymes and cascades involving second messengers, as is the case with G protein-linked receptors. Various metabotropic receptors can include certain glutamate receptors, muscarinic ACh receptors, GABA_B receptors, and receptor tyrosine kinases.

G protein-linked receptors

The G protein-linked signaling cascade can significantly amplify the signal of a particular neurotransmitter to produce hundreds to thousands of second messengers in a cell. The mechanism of action by which G protein-linked receptors cause a signaling cascade is as follows:

1. Neurotransmitter binds to the receptor
2. The receptor undergoes a conformational change to allow G-protein complex binding
3. GDP is exchanged with GTP upon G protein complex binding to the receptor
4. The α-subunit of the G protein complex is bound to GTP and separates to bind with a target protein such as adenylate cyclase
5. The binding to the target protein either increases or decreases the rate of second messenger (such as cyclic AMP) production
6. GTPase hydrolyzes the α-subunit so that is bound to GDP and the α-subunit returns to the G protein complex inactive

Neurotransmitter release

Structure of a synapse where neurotransmitter release and uptake occurs

Neurotransmitters are released in discrete packets known as quanta from the axon terminal of one neuron to the dendrites of another across a synapse. These quanta have been identified by electron microscopy as synaptic vesicles. Two types of vesicles are small synaptic vessicles (SSVs), which are about 40-60nm in diameter, and large dense-core vesicles (LDCVs), electron-dense vesicles approximately 120-200nm in diameter.^[1] The former is derived from endosomes and houses neurotransmitters such as acetylcholine, glutamate, GABA, and glycine. The latter is derived from the Golgi apparatus and houses larger neurotransmitters such as catecholamines and other peptide neurotransmitters.^[7] Neurotransmitters are released from an axon terminal and bind to postsynaptic dendrites in the following procession:^[5]

1. Mobilization/recruitment of synaptic vesicle from cytoskeleton
2. Docking of vesicle (binding) to presynaptic membrane
3. Priming of vesicle by ATP (relatively slow step)
4. Fusion of primed vesicle with presynaptic membrane and exocytosis of the housed neurotransmitter
5. Uptake of neurotransmitters in receptors of a postsynaptic cell
6. Initiation or inhibition of action potential in postsynaptic cell depending on whether the neurotransmitters are excitatory or inhibitory (excitatory will result in depolarization of the postsynaptic membrane)

Neurotransmitter release is calcium-dependent

Neurotransmitter release is dependent on an external supply of Ca²⁺ ions which enter axon terminals via voltage-gated calcium channels. Vesicular fusion with the terminal membrane and release of the neurotransmitter is caused by the generation of Ca²⁺ gradients induced by incoming action potentials. The Ca²⁺ ions cause the mobilization of newly synthesized vesicles from a reserve pool to undergo this membrane fusion. This mechanism of action was discovered in squid giant axons.^[8] Lowering intracellular Ca²⁺ ions provides a direct inhibitory effect on neurotransmitter release.^[1] After release of the neurotransmitter occurs, vesicular membranes are recycled to their origins of production. Calcium ion channels can vary depending on the location of incidence. For example, the channels at an axon terminal differ from the typical calcium channels of a cell body (whether neural or not). Even at axon terminals, calcium ion channel types can vary, as is the case with P-type calcium channels located at the neuromuscular junction.^[1]

Neuronal gene expression

Sex differences

Differences in sex determination are controlled by sex chromosomes. Sex hormonal releases have a significant effect on sexual dimorphisms (phenotypic differentiation of sexual characteristics) of the brain. Recent studies seem to suggest that regulating these dimorphisms has implications for understanding normal and abnormal brain function. Sexual dimorphisms may be significantly influenced by sex-based brain gene expression which varies from species to species.

Animal models such as rodents, Drosophila melanogaster, and Caenorhabditis elegans, have been used to observe the origins and/or extent of sex bias in the brain versus the hormone-producing gonads of an animal. With the rodents, studies on genetic manipulation of sex chromosomes resulted in an effect on one sex that was completely opposite of the effect in the other sex. For example, a knockout of a particular gene only resulted in anxiety-like effects in males. With studies on D. menlanogaster it was found that a large brain sex bias of expression occurred even after the gonads were removed, suggesting that sex bias could be independent of hormonal control in certain aspects.^[9]

Observing sex-biased genes has the potential for clinical significance in observing brain physiology and the potential for related (whether directly or indirectly) neurological disorders. Examples of diseases with sex biases in development include Huntington's disease, cerebral ischemia, and Alzheimer's disease.^[9]

Epigenetics of the brain

Many brain functions can be influenced at the cellular and molecular level by variations and changes in gene expression, without altering the sequence of DNA in an organism. This is otherwise known as epigenetic regulation. Examples of epigenetic mechanisms include histone modifications and DNA methylation. Such changes have been found to be strongly influential in the incidence of brain disease, mental illness, and addiction.^[10] Epigenetic control has been shown to be involved in high levels of plasticity in early development, thereby defining its importance in the critical period of an organism.^[11] Examples of how epigenetic changes can affect the human brain are as follows:

• Higher methylation levels in rRNA genes in the hippocampus of the brain results in a lower production of proteins and thus limited hippocampal function can result in learning and memory impairment and resultant suicidal tendencies.^[12]
• In a study comparing genetic differences between healthy people and psychiatric patients 60 different epigenetic markers associated with brain cell signaling were found.^[12]
• Environmental factors such as child abuse appears to cause the expression of an epigenetic tag on glucocorticoid receptors (associated with stress responses) that was not found in suicide victims.^[12] This is an example of experience-dependent plasticity.
• Environmental enrichment in individuals is associated with increased hippocampal gene histone acetylation and thus improved memory consolidation (notably spatial memory).^[11]

Molecular mechanisms of neurodegenerative diseases

Excitotoxicity and glutamate receptors

Excitotoxicity is phenomenon in which glutamate receptors are inappropriately activated. It can be caused by prolonged excitatory synaptic transmission in which high levels of glutamate neurotransmitter cause excessive activation in a postsynaptic neuron that can result in the death of the postsynaptic neuron. Following brain injury (such as from ischemia), it has been found that excitotoxicity is a significant cause of neuronal damage. This can be understandable in the case where sudden perfusion of blood after reduced blood flow to the brain can result in excessive synaptic activity caused by the presence of increased glutamate and aspartate during the period of ischemia.^[5][13]

Alzheimer's disease

Alzheimer's disease is the most common neurodegenerative disease and is the most common form of dementia in the elderly. The disorder is characterized by progressive loss of memory and various cognitive functions. It is hypothesized that the deposition of amyloid-β peptide (40-42 amino acid residues) in the brain is integral in the incidence of Alzheimer's disease. Accumulation is purported to block hippocampal long-term potentiation. It is also possible that a receptor for amyloid-β oligomers could be a prion protein.^[14]

Parkinson's disease

Parkinson's disease is the second most common neurodegenerative disease after Alzheimer's disease. It is a hypokinetic movement basal ganglia disease caused by the loss of dopaminergic neurons in the substantia nigra of the human brain. The inhibitory outflow of the basal ganglia is thus not decreased, and so upper motor neurons, mediated by the thalamus, are not activated in a timely manner. Specific symptoms include rigidity, postural problems, slow movements, and tremors. Blocking GABA receptor input from medium spiny neurons to reticulata cells, causes inhibition of upper motor neurons similar to the inhibition that occurs in Parkinson's disease.^[5]

Huntington's disease

Huntington's disease is a hyperkinetic movement basal ganglia disease caused by lack of normal inhibitory inputs from medium spiny neurons of the basal ganglia. This poses the opposite effects of those associated with Parkinson's disease, including inappropriate activation of upper motor neurons. As with the GABAergic mechanisms observed in relation to Parkinson's disease, a GABA agonist injected into the substantia nigra pars reticulata decreases inhibition of upper motor neurons, resulting in ballistic involuntary motor movements, similar to symptoms of Huntington's disease.^[5]

Neuroscience and intelligence

From Wikipedia, the free encyclopedia

Neuroscience and intelligence refers to the various neurological factors that are partly responsible for the variation of intelligence within a species or between different species. A large amount of research in this area has been focused on the neural basis of human intelligence. Historic approaches to study the neuroscience of intelligence consisted of correlating external head parameters, for example head circumference, to intelligence.^[1] Post-mortem measures of brain weight and brain volume have also been used.^[1] More recent methodologies focus on examining correlates of intelligence within the living brain using techniques such as magnetic resonance imaging (MRI), functional MRI (fMRI), electroencephalography (EEG), positron emission tomography and other non-invasive measures of brain structure and activity.^[1]

Researchers have been able to identify correlates of intelligence within the brain and its functioning. These include overall brain volume,^[2] grey matter volume,^[3] white matter volume,^[4] white matter integrity,^[5] cortical thickness^[3] and neural efficiency.^[6] Although the evidence base for our understanding of the neural basis of human intelligence has increased greatly over the past 30 years, even more research is needed to fully understand it.^[1]

The neural basis of intelligence has also been examined in animals such as primates, cetaceans and rodents.^[7]

Humans

Brain volume

One of the main methods used to establish a relationship between intelligence and the brain is to use measures of Brain volume.^[1] The earliest attempts at estimating brain volume were done using measures of external head parameters, such as head circumference as a proxy for brain size.^[1] More recent methodologies employed to study this relationship include post-mortem measures of brain weight and volume. These have their own limitations and strengths.^[8] The advent of MRI as a non-invasive highly-accurate measure of living brain structure and function (using fMRI) made this the pre-dominant and preferred method for measuring brain volume.^[1]

Overall, larger brain size and volume is associated with better cognitive functioning and higher intelligence.^[1] The specific regions that show the most robust correlation between volume and intelligence are the frontal, temporal and parietal lobes of the brain.^[9][10][11] A large number of studies have been conducted with uniformly positive correlations, leading to the generally safe conclusion that larger brains predict greater intelligence.^[12][13] In healthy adults, the correlation of total brain volume and IQ is ~ 0.4 ^[14]

Less is known about variation on scales less than total brain volume. A meta-analytic review by McDaniel found that the correlation between Intelligence and in vivo brain size was larger for females (0.40) than for males (0.25).^[15] The same study also found that the correlation between brain size and Intelligence increased with age, with children showing smaller correlations.^[15] It has been suggested that the link between larger brain volumes and higher intelligence is related to variation in specific brain regions: a whole-brain measure would under-estimate these links.^[9] For functions more specific than general intelligence, regional effects may be more important. For instance evidence suggests that in adolescents learning new words, vocabulary growth is associated with gray matter density in bilateral posterior supramarginal gyri.^[16] Small studies have shown transient changes in gray-matter associated with developing a new physical skill (juggling) occipito-temporal cortex. ^[17]

Brain volume is not a perfect account of intelligence: the relationship explains a modest amount of variance in intelligence – 12% to 36% of the variance.^[8][9] The amount of variance explained by brain volume may also depend on the type of intelligence measured.^[8] Up to 36% of variance in verbal intelligence can be explained by brain volume, while only approximately 10% of variance in visuospatial intelligence can be explained by brain volume.^[8] A 2015 study by researcher Stuart J Ritchie found that brain size explained 12% of the variance in intelligence among individuals.^[18] These caveats imply that there are other major factors influencing how intelligent an individual is apart from brain size.^[1] In a large meta-analysis consisting of 88 studies Pietschnig et al. (2015) estimated the correlation between brain volume and intelligence to be about correlation coefficient of 0.24 which equates to 6% variance.^[19] Taking into account measurement quality, and sample type and IQ-range, the meta-analytic association of brain volume in appears to be ~ .4 in normal adults.^[14] Researcher Jakob Pietschnig argued that the strength of the positive association of brain volume and IQ remains robust, but has been overestimated in the literature. He has stated that "It is tempting to interpret this association in the context of human cognitive evolution and species differences in brain size and cognitive ability, we show that it is not warranted to interpret brain size as an isomorphic proxy of human intelligence differences".^[19]

Grey matter

Grey matter has been examined as a potential biological foundation for differences in intelligence. Similarly to brain volume, global grey matter volume is positively associated with intelligence.^[1] More specifically, higher intelligence has been associated with larger cortical grey matter in the prefrontal and posterior temporal cortex in adults.^[3] Furthermore, both verbal and nonverbal intelligence have been shown to be positively correlated with grey matter volume across the parietal, temporal and occipital lobes in young healthy adults, implying that intelligence is associated with a wide variety of structures within the brain.^[20]

There appear to be sex differences between the relationship of grey matter to intelligence between men and women.^[21] Men appear to show more intelligence to grey matter correlations in the frontal and parietal lobes, while the strongest correlations between intelligence and grey matter in women can be found in the frontal lobes and Broca's area.^[21] However, these differences do not seem to impact overall Intelligence, implying that the same cognitive ability levels can be attained in different ways.^[21]

One specific methodology used to study grey matter correlates of intelligence in areas of the brain is known as voxel-based morphometry (VBM). VBM allows researchers to specify areas of interest with great spatial resolution, allowing the examination of grey matter areas correlated with intelligence with greater special resolution. VBM has been used to correlate grey matter positively with intelligence in the frontal, temporal, parietal, and occipital lobes in healthy adults.^[22] VBM has also been used to show that grey matter volume in the medial region of the prefrontal cortex and the dorsomedial prefrontal cortex correlate positively with intelligence in a group of 55 healthy adults.^[23] VBM has also been successfully used to establish a positive correlation between grey matter volumes in the anterior cingulate and intelligence in children aged 5 to 18 years old.^[24]

Grey matter has also been shown to positively correlate with intelligence in children.^[24][25][26] Reis and colleagues^[26] have found that grey matter in the prefrontal cortex contributes most robustly to variance in Intelligence in children between 5 and 17, while subcortical grey matter is related to intelligence to a lesser extent. Frangou and colleagues^[25] examined the relationship between grey matter and intelligence in children and young adults aged between 12 and 21, and found that grey matter in the orbitofrontal cortex, cingulate gyrus, cerebellum and thalamus was positively correlated to intelligence, while grey matter in the caudate nucleus is negatively correlated with intelligence. However, the relationship between grey matter volume and intelligence only develops over time, as no significant positive relationship can be found between grey matter volume and intelligence in children under 11.^[24]

An underlying caveat to research into the relationship of grey matter volume and intelligence is demonstrated by the hypothesis of neural efficiency.^[6][27] The findings that more intelligent individuals are more efficient at using their neurons might indicate that the correlation of grey matter to intelligence reflects selective elimination of unused synapses, and thus a better brain circuitry.^[28]

White matter

Similar to grey matter, white matter has been shown to correlate positively with intelligence in humans.^[1][4] White matter consists mainly of myelinated neuronal axons, responsible for delivering signals between neurons. The pinkish-white color of white matter is actually a result of these myelin sheaths that electrically insulate neurons that are transmitting signals to other neurons. White matter connects different regions of grey matter in the cerebrum together. These interconnections make transport more seamless and allow us to perform tasks easier. Significant correlations between intelligence and the corpus callosum have been found, as larger callosal areas have been positively correlated with cognitive performance.^[1] However, there appear to be differences in importance for white matter between verbal and nonverbal intelligence, as although both verbal and nonverbal measures of intelligence correlate positively with the size of the corpus callosum, the correlation for intelligence and corpus callosum size was larger (.47) for nonverbal measures than that for verbal measures (.18).^[29] Anatomical mesh-based geometrical modelling^[30][31][32] has also shown positive correlations between the thickness of the corpus callosum and Intelligence in healthy adults.^[33]

White matter integrity has also been found to be related to Intelligence.^[5] White matter tract integrity is important for information processing speed, and therefore reduced white matter integrity is related to lower intelligence.^[5] The effect of white matter integrity is mediate entirely through information processing speed.^[5] These findings indicate that the brain is structurally interconnected and that axonal fibres are integrally important for fast information process, and thus general intelligence.^[5]

Contradicting the findings described above, VBM failed to find a relationship between the corpus callosum and intelligence in healthy adults.^[22] This contradiction can be viewed to signify that the relationship between white matter volume and intelligence is not as robust as that of grey matter and intelligence.^[1]

Cortical thickness

Cortical thickness has also been found to correlate positively with intelligence in humans.^[3] However, the rate of growth of cortical thickness is also related to intelligence.^[28] In early childhood, cortical thickness displays a negative correlation with intelligence, while by late childhood this correlation has shifted to a positive one.^[28] More intelligent children were found to develop cortical thickness more steadily and over longer periods of time than less bright children.^[28] Studies have found cortical thickness to explain 5% in the variance of intelligence among individuals.^[18] In a study conducted to find associations between cortical thickness and general intelligence between different groups of people, sex did not play a role in intelligence.^[34] Although it is hard to pin intelligence on age based on cortical thickness due to different socioeconomic circumstances and education levels, older subjects (17 - 24) tended to have less variances in terms of intelligence than when compared to younger subjects (19 - 17).^{[34][dubious – discuss]}

Cortical convolution

Cortical convolution has increased the folding of the brain’s surface over the course of human evolution. It has been hypothesized that the high degree of cortical convolution may be a neurological substrate that supports some of the human brain's most distinctive cognitive abilities. Consequently, individual intelligence within the human species might be modulated by the degree of cortical convolution.^[35]

Neural efficiency

The neural efficiency hypothesis postulates that more intelligent individuals display less activation in the brain during cognitive tasks, as measured by Glucose metabolism.^[6] A small sample of participants (N=8) displayed negative correlations between intelligence and absolute regional metabolic rates ranging from -0.48 to -0.84, as measured by PET scans, indicating that brighter individuals were more effective processors of information, as they use less energy.^[6] According to an extensive review by Neubauer & Fink^[36] a large number of studies (N=27) have confirmed this finding using methods such as PET scans,^[37] EEG^[38] and fMRI.^[39]

fMRI and EEG studies have revealed that task difficulty is an important factor affecting neural efficiency.^[36] More intelligent individuals display neural efficiency only when faced with tasks of subjectively easy to moderate difficulty, while no neural efficiency can be found during difficult tasks.^[40] In fact, more able individuals appear to invest more cortical resources in tasks of high difficulty.^[36] This appears to be especially true for the Prefrontal Cortex, as individuals with higher intelligence displayed increased activation of this area during difficult tasks compared to individuals with lower intelligence.^[41][42] It has been proposed that the main reason for the neural efficiency phenomenon could be that individuals with high intelligence are better at blocking out interfering information than individuals with low intelligence.^[43]

Further research

Some scientists prefer to look at more qualitative variables to relate to the size of measurable regions of known function, for example relating the size of the primary visual cortex to its corresponding functions, that of visual performance.^[44][45]

In a study of the head growth of 633 term-born children from the Avon Longitudinal Study of Parents and Children cohort, it was shown that prenatal growth and growth during infancy were associated with subsequent IQ. The study’s conclusion was that the brain volume a child achieves by the age of 1 year helps determine later intelligence. Growth in brain volume after infancy may not compensate for poorer earlier growth.^[46]

There is an association between IQ and myopia. One suggested explanation is that one or several pleiotropic gene(s) affect the size of the neocortex part of the brain and eyes simultaneously.^[47]

Parieto-frontal integration theory

In 2007, Behavioral and Brain Sciences published a target article that put forth a biological model of intelligence based on 37 peer-reviewed neuroimaging studies (Jung & Haier, 2007). Their review of a wealth of data from functional imaging (functional magnetic resonance imaging and positron emission tomography) and structural imaging (diffusion MRI, voxel-based morphometry, in vivo magnetic resonance spectroscopy) argues that human intelligence arises from a distributed and integrated neural network comprising brain regions in the frontal and parietal lobes.^[48]

A recent lesion mapping study conducted by Barbey and colleagues provides evidence to support the P-FIT theory of intelligence.^[49][50][51]

Brain injuries at an early age isolated to one side of the brain typically results in relatively spared intellectual function and with IQ in the normal range.^[52]

Primates

Brain size

Another theory of brain size in vertebrates is that it may relate to social rather than mechanical skill. Cortical size relates directly to a pairbonding life style and among primates cerebral cortex size varies directly with the demands of living in a large complex social network. Compared to other mammals, primates have significantly larger brain size. Additionally, most primates are found to be polygynandrous, having many social relationships with others. Although inconclusive, some studies have shown that this polygnandrous statue correlates to brain size.^[53]

Health

Several environmental factors related to health can lead to significant cognitive impairment, particularly if they occur during pregnancy and childhood when the brain is growing and the blood–brain barrier is less effective. Developed nations have implemented several health policies regarding nutrients and toxins known to influence cognitive function. These include laws requiring fortification of certain food products and laws establishing safe levels of pollutants (e.g. lead, mercury, and organochlorides). Comprehensive policy recommendations targeting reduction of cognitive impairment in children have been proposed.^[54]