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What are the factors that control the speed of propagation of neuronal signals?

What are the factors that control the speed of propagation of neuronal signals?


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If we consider an analogy between a wire and a neuron there may be some resemblance between the factors controlling the data flow rate. For example the increased width of wire leads to decreased resistance etc; similarly increasing or decreasing the thickness of myelin sheath may have an effect on the conductivity or data transfer between neurons.

Are there additional factors that can control the speed of nervous conduction?


The diameter of axons also makes a difference, the thicker the axon, the faster the rate of action potential conduction. This is seen in invertebrates, such as squid. Squid have giant axons (up to 2mm in diameter), which exhibit rapid conduction rates. See Wikipedia 'squid giant axon', in which it says up to 1mm, but I have measured 2mm myself. These are not found in mammals.

Original research on squid giant axon was by Cole & Curtis (The Journal of general physiology, 1939). For more recent research see Hartline & Colman (Curr Biol. 2007 Jan 9;17(1):R29-35.).

Work by Hodgkin, Huxley and Katz (The Journal of physiology, 1952, 116(4): 424-448) using squid giant axons lead to the elucidation of the mechanism of action potential conduction and a Nobel prize. Academic summary here: http://jp.physoc.org/content/538/1/2.full

Wikipedia: see the Goldman equation (Goldman shared the Nobel Prize with Hodgkins and Katz). This is more frequently known as the Goldman-Hodgkin-Katz equation in UK publications.


The myelin sheath of a neuron affects conduction of action potentials.

Things to read through at wikipedia for example are the Nodes of Ranvier, the saltatory conduction and the Schwann cells.

There might be other features changing nervous conduction, but those are the ones I remember from my study times.


Speed of Nerve Impulses

The nervous system is responsible for transmitting impulses throughout the body. The function of our bodies throughout our lives are primarily supported by the nervous system. If not for the nervous system we will not be able to control our muscles, and our tissues and organs will no longer be able to function. Sense organs provide the nervous system with information about the environment through such senses as sight hearing, smell, taste, touch, pressure, and pain. Nerves are connected throughout the body leading up to the brain. They carry the information through the body in the form of electrochemical signals called impulses impulses. These signals travel from the brain to the spinal cord, through the nerves to the organs, tissues and muscles.

The speed of a nerve impulse varies with the type of nerve impulse the nervous system is sending. Some signals such as those for muscle position, travel at speeds up to 119m/s. Nerve impulses such as pain signals travel slower at 0.61m/s. Touch signals travel at speeds of 76.2m/s. If you are reading this at this moment and thinking at the same time, which some people may have trouble with, thought signals are traveling at speeds ranging between 20 and 30 meters per second.


Hormonal Responses to Food

The endocrine system controls the release of hormones responsible for starting, stopping, slowing, and quickening digestive processes.

Learning Objectives

Describe hormonal responses to food

Key Takeaways

Key Points

  • The presence and absence of hormones that are released into the bloodstream generate specific digestive responses they either stimulate or discontinue digestive processes.
  • In hormone control, a negative feedback mechanism takes place when the stomach is empty and its acidic environment does not need to be maintained as a result, a hormone is released to stop the release of hydrochloric acid, which was previously activated to aid digestion.
  • In some cases, hormones work in tandem and sequentially to achieve important digestive functions, such as in the breakdown of acidic chyme, where hormones act in releasing the appropriate secretions in the appropriate stages of digestion.
  • When digesting certain types of foods, such as ones high in fat, hormones can control the speed of food digestion and, therefore, absorption.

Key Terms

  • endocrine system: a control system of ductless glands that secrete hormones which circulate via the bloodstream to affect cells within specific organs
  • chyme: the thick semifluid mass of partly digested food that is passed from the stomach to the duodenum
  • secretin: a peptide hormone, secreted by the duodenum, that serves to regulate its acidity
  • cholecystokinin: any of several peptide hormones that stimulate the digestion of fat and protein
  • somatostatin: a polypeptide hormone, secreted by the pancreas, that inhibits the production of certain other hormones
  • gastrin: a hormone that stimulates the production of gastric acid in the stomach

Hormonal Responses to Food

The endocrine system controls the response of the various glands in the body and the release of hormones at the appropriate times. The endocrine system’s effects are slow to initiate, but prolonged in their response, lasting from a few hours up to weeks. The system is made of a series of glands that produce chemicals called hormones. These hormones are chemical mediators released from endocrine tissue into the bloodstream where they travel to target tissue and generate a response.

One of the important factors under hormonal control is the stomach acid environment. During the gastric phase, the hormone gastrin is secreted by G cells in the stomach in response to the presence of proteins. Gastrin stimulates the release of stomach acid, or hydrochloric acid (HCl), which aids in the digestion of the majority of proteins. However, when the stomach is emptied, the acidic environment need not be maintained and a hormone called somatostatin stops the release of hydrochloric acid. This is controlled by a negative feedback mechanism.

In the duodenum, digestive secretions from the liver, pancreas, and gallbladder play an important role in digesting chyme during the intestinal phase. In order to neutralize the acidic chyme, a hormone called secretin stimulates the pancreas to produce alkaline bicarbonate solution and deliver it to the duodenum. Secretin acts in tandem with another hormone called cholecystokinin (CCK). Not only does CCK stimulate the pancreas to produce the requisite pancreatic juices, it also stimulates the gallbladder to release bile into the duodenum.

Digestive endocrine system: Hormones, such as secretin and cholecystokinin, play important roles in digestive processes. These hormones are released from endocrine tissue to generate specific controls in the digestion of chyme. As seen in the image, hormones are vital players in several bodily functions and processes.

Another level of hormonal control occurs in response to the composition of food. Foods high in lipids (fatty foods) take a long time to digest. A hormone called gastric inhibitory peptide is secreted by the small intestine to slow down the peristaltic movements of the intestine to allow fatty foods more time to be digested and absorbed.

Understanding the hormonal control of the digestive system is an important area of ongoing research. Scientists are exploring the role of each hormone in the digestive process and developing ways to target these hormones. Advances could lead to knowledge that may help to battle the obesity epidemic.


The role of neural architecture and the speed of signal propagation in the process of synchronization of bursting neurons

Synchronized neuronal activity has been observed at all levels of human and any other nervous systems and was suggested as particularly relevant in information processing and coding. In the present paper we investigate the synchronization of bursting neuronal activity. Motivated by the fact that in neural systems the interplay between the network structure and the dynamics taking place on it is closely interrelated, we develop a spatial network representation of neural architecture in which we can tune the network organization between a scale-free network with dominating long-range connections and a homogeneous network with mostly adjacent neurons connected. Our results reveal that the most synchronized response is obtained for the intermediate regime where long- as well as short-range connections constitute the neural architecture. Moreover, the optimal response is additionally enhanced when the speed of signal propagation is optimized.

Highlights

► We study synchronization of bursting neuronal activity on a spatial network. ► The roles of the network structure and the signal propagation speed are analysed. ► The greatest synchronization is attained at intermediate heterogeneous topologies. ► Proper signal propagation speed can additionally enhance the optimal response.


Pulse propagation and signal transduction in the hydraulic brain

Section of myelinated nerve segments. Credit: education.vetmed.vt.edu

(Medical Xpress)—When Descartes turned his critical eye to the nervous system, he reasoned that the nerves must transduce hydraulic power to control the musculature. In the circulatory system, blood is pushed comparatively slowly through the aorta, perhaps around 0.3 meters per second. Superimposed on that flow, however, is an arterial pulse wave which propagates much faster, both through the blood and the walls of the vessel. For compliant and healthy vessels that speed might be around 10 meters per second, while for more hardened arteries, it could be 15 or higher. Modern day electrophysiologists have since replaced the hydraulic model with the idea that nerves really only transmit information—electrical information no less. Yet when looking at the power supply to the leg, for example, it is still hard to ignore the fact that the main femoral artery, at a diameter scarcely a half of an inch, looks rather meager next to the "information-supplying" sciatic nerve, which may actually be more like three-quarters of an inch. A conflux of ideas from a variety of disciplines has recently led to a critical re-emergence of the more mechanical side of the nervous system. To that point, two German scientists have just published a paper in the journal, Medical Hypotheses, where they suggest that the pulse wave is the main event in nervous conduction, while the electrical show is mere epiphenomenon.

We recently discussed the increasingly popular idea that action potentials may actually be soliton waves which propagate in the membranes of axons as phase transitions with minimal loss in energy. Convincing biologists that these subtle creatures could exist in the chaotic and varied conditions inside neurons has been a challenge. However, it is harder to argue against the fact that any kind of electrochemical spike based on the rapid influx of ions will be accompanied by a significant pressure pulse. The idea that the German researchers have supported, is that these as the pressure pulses naturally decay in the viscoelastic medium of the nerve, they are refreshed by ionic input at the nodes between myelinated axon segments, or continuously in unmyelinated axons.

If you have ever been absent-minded enough to grab a live wire, or even brush up strongly against one, the sensation is unforgettable. It is not such a stretch to acknowledge that when you slam your funny bone, or more precisely the Ulnar nerve (largest unprotected nerve in your body), the resultant vibe and decay feels almost identical to a real electrical assault. Similarly, the so-called "stingers" that run down the limbs after a sharp blow to the head are familiar to most footballers, and can give one quite a shock. Unfortunately these (albeit very simplistic) macroscopic intuitions don't hold up so well when extended to the microscopic domain. Granted, when the electrochemical mechanisms that are assumed to underlie nervous conduction are looked at in detail, it becomes more difficult to disentangle the mechanical from the electric. However, as the authors observe, at some point, an attentive electrophysiologist must ask his or herself, "why are so many ion channels mechanosensitive" ?

Original view of nerves from Descartes. Credit: cns.nyu.edu

One unexpected finding of the patch clamp recording technique was that the dilation of the membrane caused by local tension leads to considerable increase in transmembrane ion flow. Impulse waves causing short extensions in the membrane can directly induce opening and closing of both voltage and ligand gated channels. The idea that the pore in these channels is a rigid tube isolated from larger membrane events is difficult to support in this context. According to the authors, it is quite likely that common mechanoreceptor devices, like the pressure- or vibration-sensitive Vater-Pacinian corpuscles of the skin, conduct signals to initiate high-speed polysynaptic muscle reflex circuits without any classical intermediary electrical conversion.

The exact conduction velocity of mechanical impulses in nerve fibers remains unknown. It is estimated that under physiologic conditions, an unamplified axoplasmic pressure pulse would decay over roughly 1 mm due to viscosity, depending on the distensibility of the axon wall. When compared to the theoretical case of an absolutely rigid wall, a typical myelin sheath may be rigid enough to support pulse speeds up to one-fifth of the estimated maximum. That speed is not to shabby when compared with some rough estimates from previous authors, which put the maximum pulse velocity under an indistensible membrane somewhere upwards of 1500 meters per second. Suddenly, the quicker than life eyeblink response, or speed of the tenderfoot stepping on a sharp shard, become a little more comprehensible.

The theory as it stands is incomplete and needs to be adapted for specific cases with real biology in mind. In different animals, and in different regions of their brains, conduction in neurons goes by different names. For example, in the cerebellum, the unmyelinated parallel fibers pack to extreme densities in a regular crystalline lattice whose reason to be defies physiologic explanation to this day. Just as we currently have no good explanation for how signals could be properly isolated in nerve bundles where seemingly random nodes of Ranvier overlap in extent and influence, it is hard to imagine parallel fibers could maintain their electrochemical, or even mechanical, autonomy within this geometry.

The pressure wave theory wields considerable predictive power when it comes to explaining some of the unique synaptic specializations found throughout the brain. When considered only from an electrochemical point of view, the huge structural synaptic investments, like those found at the neuromuscular junction (NMJ), can hardly be imagined to be driven by local, and weak, current or field effects. One might need look no further than simple-to-recreate Chaldni patterns set up in two dimensions on the surface of a taunt drum, to make the imaginative leap to a three dimensional system, with multiple vibrating players, where more extreme patterns might easily be set up to provide authorship to repeatable complex structure. For the NMJ in particular, the case has been made that at the end-plate, the comparatively enormous efflux of acetylcholine to the deeply-guttered cleft, and propagation of excitation through the transverse tubule system, are all components of a continuous mechanical amplifier.

The apparent ease with which evolving organisms manage to cobble together all manner of sensitive hearing devices becomes infinitely more explicable once we see that nature has apparently been doing this kind of thing all alone inside of neurons. The amplification and transduction through liquid channels, of barely noticeable vibrations against a background of thermal noise much greater in magnitude, is in this light, no evolutionary stumble-upon, but rather the bread and butter of neural systems, and perhaps many aspects of life in general.


Random or unpredictable fluctuations and disturbances that are not part of a signal.

An action potential interpreted as a unitary pulse signal (that is, it either is or is not present), the timing of which determines its information content. Other properties of the action potential, such as its shape or depolarization levels, are ignored.

The differences between responses that are observed when the same experiment is repeated in the same specimen (for example, in the same neuron or in the same subject).

A random process that generates binary (yes or no) events for which the probability of occurrence in any small time interval is low. The rate at which events occur completely determines the statistics of the events. Poisson processes have a Fano factor of 1.

The ratio of the variance of a variable quantity to its mean.

Stochastic process (random process)

A process that generates a series of random events.

Feedback that responds to a perturbation in the same direction as the perturbation, thereby amplifying its effect.

Regularly spaced gaps in the myelin sheath that surrounds a myelinated axon. They expose the axonal membrane to the extracellular fluid and contain large numbers of voltage-gated ion channels and thus enable conduction of the action potential.

An electrophysiological method that allows the study of the flow of current through a very small patch of cell membrane, which can contain just a single ion channel.

The ratio of how much power is contained in the signal over the power of the noise, often measured as the variance of the signal divided by the variance of the noise.

The anatomical part of a mammalian neuron that connects the cell body to the axon. Axon hillocks are the postulated primary site of action-potential initiation.

Johnson noise (thermal noise, Johnson–Nyquist noise or Nyquist noise)

The electronic noise that is generated by the thermal agitation of the charge carriers (electrons and ions) inside an electrical conductor at equilibrium, which happens regardless of any applied voltage. Johnson noise is distinguished from shot noise, which consists of additional current fluctuations that occur when a voltage is applied to a resistance and a macroscopic current starts to flow.

A type of noise that occurs when the finite number of signal particles, such as electrons or ions in an electrical circuit or photons arriving at a photoreceptor, is small enough to give rise to detectable statistical fluctuations in a measurement.

The coupling of very close or touching neurons, mediated by the electrical fields the neurons generate during electrical activity.

(CV). The ratio of the standard deviation of a variable quantity to its mean.

The probability of a vesicle being released during a synaptic-transmission event.

The presence of superfluous or duplicate information in a message.

White Gaussian noise process

A random process that generates a series of events, each Gaussian distributed. The mean and varience of the Gaussian completely determines the statistics of the series, and there is no temporal correlation between events.


Myelin dynamics: protecting and shaping neuronal functions

Cytosolic channels in the myelin sheath are critical for axonal integrity.

Activity-dependent vesicular release and transmitter signaling regulate myelination.

Glutamate signaling adjusts oligodendroglial metabolic support to axons.

Adult myelin plasticity impacts on motor skill learning and CNS repair.

Myelinating glial cells are well-known to insulate axons and to speed up action potential propagation. Through adjustments in the axonal coverage with myelin, myelin sheath thickness and possibly nodal/internode length oligodendrocytes are involved in fine-tuning the brain's computational power throughout life. Be it motor skill learning or social behaviors in higher vertebrates, proper myelination is critical in shaping brain functions. Neurons rely on their myelinating partners not only for setting conduction speed, but also for regulating the ionic environment and fueling their energy demands with metabolites. Also, long-term axonal integrity and neuronal survival are maintained by oligodendrocytes and loss of this well-coordinated axon–glial interplay contributes to neuropsychiatric diseases. Better insight into how myelination and oligodendrocyte functions are constantly fine-tuned in the adult CNS, which includes sensing of neuronal activity and adjusting glial metabolic support, will be critical for understanding higher brain functions and cognitive decline associated with myelin abnormalities in the aging brain.


INTRODUCTION

The CaV1 family of L-type voltage-gated Ca 2+ channels (LTCCs) are widely expressed and regulate important physiological processes including insulin secretion, muscle contraction, rhythmic firing, neurotransmitter release, and transcription (Catterall, 2011). In neurons, LTCCs both provide depolarizing inward Ca 2+ current and initiate Ca 2+ -dependent intracellular signaling cascades. LTCCs are advantaged with respect to other voltage-gated Ca 2+ channels in activating neuronal gene expression programs in response to electrical excitation (Ma et al., 2013). LTCC-mediated excitation–transcription coupling alters gene expression by downstream signaling to transcription factors, including the cAMP response element–binding protein (CREB) and the nuclear factor of activated T-cells (NFAT Murphy et al., 1991 Bading et al., 1993 Graef et al., 1999 Dolmetsch et al., 2001). Persistent changes in transcription and protein translation in neurons are required for long-lasting forms of synaptic long-term potentiation (L-LTP), processes that alter neuronal circuit connectivity with a duration that extends from hours to months and is regarded as a key cellular correlate for neuronal memory (Malenka and Bear, 2004). The NMDA receptor-independent form of L-LTP requires LTCCs, as demonstrated by blockade of channel function using dihydropyridine antagonists and in CaV1.2 knockout animals (Grover and Teyler, 1990 Grover, 1998 Morgan and Teyler, 1999). For example, mice with conditional knockout of the predominant LTCC isoform in the forebrain, CaV1.2, have reduced NMDA receptor-independent L-LTP, spatial learning, and CREB activation (Moosmang et al., 2005). The importance of CaV1.2 in synaptic plasticity and brain function, not only in mice but also in humans, is evident from the link between alterations in channel function and polymorphisms of the human CACNA1C gene found in multiple neuropsychiatric disorders including Parkinson’s disease, Alzheimer’s disease, schizophrenia, and autism-spectrum disorders (Ortner and Striessnig, 2016). Therefore, a better understanding of the mechanisms that control LTCC function in neurons, especially with regard to excitation–transcription signaling, is critical to our understanding of cognition and brain function.

A-kinase anchoring protein (AKAP) 79/150 (human 79/rodent 150 also known as AKAP5) is a multidomain scaffolding protein that imparts spatial specificity and temporal precision to intracellular signaling by anchoring kinases, phosphatases, and components of the cAMP signaling pathway, including adenylyl cyclases and phosphodiesterases, to plasma membrane structures (Dell’Acqua et al., 1998 Bauman et al., 2006 Efendiev et al., 2010 Woolfrey and Dell’Acqua, 2015). In neurons, AKAP79/150 is an indispensable component of the postsynaptic proteome that regulates the phosphorylation state of postsynaptic glutamate receptors and ion channels (reviewed in Wild and Dell’Acqua, 2018) through recruitment of cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and the protein phosphatase calcineurin (CaN also known as PP2B and PPP3 Carr et al., 1992 Coghlan et al., 1995 Klauck et al., 1996 Tavalin, 2008). In particular, AKAP79/150 interacts directly with LTCCs and bidirectionally regulates channel phosphorylation and function through local anchoring of PKA and CaN (Gao et al., 1997 Oliveria et al., 2007, 2012 Dittmer et al., 2014 Murphy et al., 2014). The activity of enzymes anchored within this multiprotein complex determines the shape and size of the Ca 2+ signal that is generated by the channel. Basal-channel phosphorylation and activity are maintained by AKAP79/150–targeted PKA (Dittmer et al., 2014 Murphy et al., 2014), while AKAP79/150 targeting of CaN limits basal phosphorylation and activity and also imparts negative feedback regulation of the amount and duration of Ca 2+ entry in a process known as Ca 2+ -dependent inactivation (CDI Peterson et al., 1999 Erickson et al., 2001 Oliveria et al., 2012).

In addition to the important role of AKAP79/150 in regulating LTCC channel function, dynamic AKAP anchoring of CaN is required for NFAT signaling (Li et al., 2012). When bound to the C-terminus of LTCCs, the Ca 2+ sensor calmodulin (CaM) responds to channel Ca 2+ entry to activate AKAP79/150–anchored CaN (Oliveria et al., 2012). Upon Ca 2+ /CaM-dependent CaN activation and dissociation from the nonconsensus PxIxIT-motif of AKAP79/150, CaN dephosphorylates NFAT, exposing a nuclear localization signal that results in nuclear import (Beals et al., 1997 Li et al., 2012). The AKAP79/150 complex associates with the LTCC at least in part through a predicted coiled-coil interaction of C terminal–modified leucine zipper motifs (LZ) located between residues 408-427 of human AKAP79 (695-714 of rat orthologue AKAP150) and 2072-2102 of human CaV1.2. We previously demonstrated that point mutations introduced simultaneously into the LZ domains of both AKAP79 and CaV1.2 disrupted the AKAP–CaV1.2 interaction, resulting in a loss of PKA/CaN regulation of CaV1.2 through AKAP uncoupling from the channel (Oliveria et al., 2007).

In this study, we present new information regarding the importance of the AKAP79/150 LZ motif in regulation of NFAT-based excitation–transcription signaling. In particular, we show that the AKAP79/150 LZ motif likely functions to recruit NFAT to the AKAP–LTCC complex at the plasma membrane to promote its activation by Ca 2+ –CaN signaling.


Neural Mechanisms (Cortex)

The cerebral cortex of the brain controls voluntary respiration.

Learning Objectives

Describe the mechanism of the neural cortex in respiration control

Key Takeaways

Key Points

  • The motor cortex within the cerebral cortex of the brain controls voluntary respiration (the ascending respiratory pathway).
  • Voluntary respiration may be overridden by aspects of involuntary respiration, such as chemoreceptor stimulus, and hypothalamus stress response.
  • The phrenic nerves, vagus nerves, and posterior thoracic nerves are the major nerves involved in respiration.
  • Voluntary respiration is needed to perform higher functions, such as voice control.

Key Terms

  • The Phrenic Nerves: A set of two nerves that brings nerve impulses from the spinal cord to the diaphragm.
  • primary motor cortex: The region in the brain that initiates all voluntary muscular movement, including those for respiration.

Voluntary respiration is any type of respiration that is under conscious control. Voluntary respiration is important for the higher functions that involve air supply, such as voice control or blowing out candles. Similarly to how involuntary respiration’s lower functions are controlled by the lower brain, voluntary respiration’s higher functions are controlled by the upper brain, namely parts of the cerebral cortex.

The Motor Cortex

The primary motor cortex is the neural center for voluntary respiratory control. More broadly, the motor cortex is responsible for initiating any voluntary muscular movement.

The processes that drive its functions aren’t fully understood, but it works by sending signals to the spinal cord, which sends signals to the muscles it controls, such as the diaphragm and the accessory muscles for respiration. This neural pathway is called the ascending respiratory pathway.

Different parts of the cerebral cortex control different forms of voluntary respiration. Initiation of the voluntary contraction and relaxation of the internal and external intercostal muscles takes place in the superior portion of the primary motor cortex.

The center for diaphragm control is posterior to the location of thoracic control (within the superior portion of the primary motor cortex). The inferior portion of the primary motor cortex may be involved in controlled exhalation.

Activity has also been seen within the supplementary motor area and the premotor cortex during voluntary respiration. This is most likely due to the focus and mental preparation of the voluntary muscular movement that occurs when one decides to initiate that muscle movement.

Note that voluntary respiratory nerve signals in the ascending respiratory pathway can be overridden by chemoreceptor signals from involuntary respiration. Additionally, other structures may override voluntary respiratory signals, such as the activity of limbic center structures like the hypothalamus.

During periods of perceived danger or emotional stress, signals from the hypothalamus take over the respiratory signals and increase the respiratory rate to facilitate the fight or flight response.

Topography of the primary motor cortex: Topography of the primary motor cortex, on an outline drawing of the human brain. Each part of the primary motor cortex controls a different part of the body.

Nerves Used in Respiration

There are several nerves responsible for the muscular functions involved in respiration. There are three types of important respiratory nerves:

  • The phrenic nerves: The nerves that stimulate the activity of the diaphragm. They are composed of two nerves, the right and left phrenic nerve, which pass through the right and left side of the heart respectively. They are autonomic nerves.
  • The vagus nerve: Innervates the diaphragm as well as movements in the larynx and pharynx. It also provides parasympathetic stimulation for the heart and the digestive system. It is a major autonomic nerve.
  • The posterior thoracic nerves: These nerves stimulate the intercostal muscles located around the pleura. They are considered to be part of a larger group of intercostal nerves that stimulate regions across the thorax and abdomen. They are somatic nerves.

These three types of nerves continue the signal of the ascending respiratory pathway from the spinal cord to stimulate the muscles that perform the movements needed for respiration.

Damage to any of these three respiratory nerves can cause severe problems, such as diaphragm paralysis if the phrenic nerves are damaged. Less severe damage can cause irritation to the phrenic or vagus nerves, which can result in hiccups.


CRISPR Screening IDs Factors for Turning Stem Cells into Neurons

Genetic reprogramming can help stem cells mature into desired cell types, but it is often kludgy, which is to say, clumsy and inefficient—or worse, inexact. It may produce cells that don’t mature quite as much as they should, or that fail to represent the exact right subtype. These shortcomings may be avoided if more elegant genetic programming methods become available, methods of the sort being developed by scientists in the Duke University laboratory led by Charles Gersbach, PhD, an associate professor of biomedical engineering.

According to a new study from the Gersbach laboratory, genetic reprogramming may be improved with a CRISPR-based method called CRISPR activation (CRISPRa). The Gersbach team used CRISPRa to identify factors that could improve the efficiency with which stem cells are turned into neurons. CRISPRa, the scientists pointed out, could have a more general application. That is, CRISPRa could be extended to other cell reprogramming applications and facilitate the production of cell types other than neurons. Ultimately, CRISPRa could help researchers generate cell sources that would be useful for disease modeling, drug screening, and regenerative medicine.

The CRISPRa work was published December 1 in Cell Reports, in an article titled, “Master Regulators and Cofactors of Human Neuronal Cell Fate Specification Identified by CRISPR Gene Activation Screens.” The article describes how CRISPRa screens were used to identify transcription factors that regulate human neuronal fate specification. These factors, the article’s authors emphasized, “influence conversion rate, subtype profile, and maturation.”

“Currently, the selection of fate-determining factors for cell reprogramming applications is typically a laborious and low-throughput process,” the article’s authors wrote. “[But we used] CRISPRa screens, which offer a high-throughput approach to profile thousands of gain-of-function perturbations in a pooled format.”

CRISPR technology is most often used for genome editing. In this application, the Cas9 protein is bound to a guide RNA that directs Cas9 to cut the DNA at a specific location, leading to changes in the DNA sequence. “DNA editing has been widely used to alter gene sequences,” Gersbach noted, “but that doesn’t help in situations where the gene is turned off.”

A deactivated Cas9 (dCas9) protein, though, will attach to the DNA without cutting it. In fact, it typically won’t do anything without another molecule attached or recruited to it. As Gersbach and colleagues have reported in previous studies, the dCas9 protein may be fused to molecular domains that allow it to activate a gene and remodel chromatin structure.

Back in 2016, Gersbach and graduate student Joshua Black reported an approach to use the CRISPR-based gene activators to turn on gene networks that would convert fibroblasts, an easily accessible cell type that makes up connective tissue, to neuronal cells. This study targeted gene networks that were known to be associated with neuronal specification, but they did not generate cells with all of the properties needed to make effective disease models. The right gene networks to generate those desired cells remained unknown. They were hidden among the thousands of possibilities encoded in the human genome. So, Gersbach and Black devised a strategy to test all of the networks in a single experiment.

“[We] developed a CRISPRa screening approach to profile the contribution of all putative human transcription factors (TFs) to neuronal cell fate specification of pluripotent stem cells (PSCs),” the authors of the current study reported. “We first performed a single-factor screen to identify master regulators of neuronal fate and identified many known and previously uncharacterized TFs. We subsequently performed paired gRNA screens and identified synergistic and antagonistic TF interactions that enhance or diminish neuronal differentiation, respectively.

“Importantly, through this method, we have uncovered TFs that increase conversion efficiency and modulate neuronal gene expression programs influencing subtype specificity and maturation of in vitro–derived neurons.”

The team engineered stem cells that fluoresced red once they became neuronal. The brighter the fluorescence, the stronger the push toward a neuronal fate. Then the team made a pooled library of thousands of guide RNAs targeted to all of the genes that encode transcription factors in the human genome. Transcription factors are the master regulators of gene networks, so to make the desired neurons, they have to get all of the right transcription factors turned on.

The scientists introduced the CRISPR gene activator and guide RNA library into the stem cells so that each cell received only a single guide RNA, and therefore turned on its particular corresponding transcription factor gene target. Then the scientists sorted the cells based on how red they became and sequenced the guide RNAs in the most and least red cells, which told them which genes, when turned on, made the cells more or less neuronal.

When the scientists profiled the gene expression from the stem cells engineered with the guide RNAs, the results suggested that the corresponding cells generated more specific and more mature types of neurons. The scientists also found genes that worked together when targeted simultaneously. Moreover, the experiment revealed factors that antagonized the neuronal commitment of the stem cells, and when they used CRISPR-based repressors of those genes, they could also enhance the neuronal specification.

To confirm that the engineered cells truly recapitulated the function of more mature neurons, the scientists tested their ability to transmit electrical signals. This task was completed by Shataakshi Dube, a graduate student in the Duke University laboratory of Scott Soderling, PhD, a professor of molecular biology and chair of the department of cell biology.

Using patch clamp electrophysiology, Dube determined that the neurons engineered to activate a particular pair of transcription factor genes emitted more action potentials more frequently. That is, these neurons were more functionally mature.

“I was curious but skeptical on how neuronal these stem cells could become,” Dube said, “but it was remarkable to see how much these programmed cells looked just like normal neurons.”

A CRISPR-based system was used to activate gene networks that had been shown to help human stem cells mature into neurons. CRISPR activation led to the generation of cells with neuronal shapes and markers (left) and enhanced function and electrophysiological properties, including more frequent action potentials (right). [Gersbach Lab, Duke University] Often, programmed stem cells do not mature correctly when cultured in the lab, so researchers seeking adult neuron cells for an experiment might end up with embryonic neurons, which won’t be able to model late-onset psychiatric and neurodegenerative conditions.

“The cells might seem right at first glance,” Black explained, “but they are often missing some of the key properties you want in those cells.”

This shortcoming can be addressed, the scientists argued, with CRISPRa screening. In the current study, CRISPRa screening was used to identify a set of TFs that improved neuronal differentiation efficiency, maturation, and subtype specification. “Interestingly, the majority of these TFs did not possess neurogenic activity on their own, as assessed in our single-factor CAS-TF screen,” the scientists added. “This observation underscores the importance of synergistic TF interactions that govern cell differentiation and supports the use of unbiased methods to identify these TFs.”

The process from stem cell to mature neuronal cell took seven days, dramatically shortening the timeframe compared to other methods that take weeks or months. This faster timeline has the potential to significantly accelerate the development and testing of new therapies for neurological disorders.

“The key to this work is developing methods to use the power and scalability of CRISPR-based DNA targeting to program any function into any cell type,” Gersbach said. “By leveraging the gene networks already encoded in our genome, our control over cell biology is dramatically improved.”


Watch the video: 7 Major Factors That Control What Calories Do In Your Body (February 2023).