This Event is licensed under the Creative Commons BY-SA license. This license allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. If you remix, adapt, or build upon the material, you must license the modified material under identical terms.
Key Event Title
Altered neurotransmission in development
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Inhibition of voltage gate during development is leading to cognitive disorders||KeyEvent||Arthur Author (send email)||Under development: Not open for comment. Do not cite||Under Development|
Key Event Description
During axonal action potential initiation, the active depolarization propagates both towards the soma (antidromic) and down the axon (orthodromic). Because of very different passive and active membrane properties of the soma compared with the axon, the conduction velocity in the two directions is likely different in most cells. The conduction velocity of the antidromic action potential may have a significant impact on dendritic backpropagation. This in turn will affect spike-timing dependent plasticity, synaptic plasticity sensitive to the timing of dendritic action potentials relative to incoming synaptic information. The orthodromic velocity will affect the degree of synchrony of arrival of information at different postsynaptic targets of the same axon (Kress G.)
The arrival of the nerve impulse at the presynaptic terminal stimulates the release of neurotransmitter into the synaptic gap. The neuron is a secretory cell and the secretory product, the neurotransmitter, is released at the level of chemical synapses.
Neurotransmitters synthesized by the neuron are stored in the presynaptic element, inside the synaptic vesicles. In the absence of presynaptic activity, the probability of a neurotransmitter being released in the synaptic cleft is very low. This probability increases strongly when the presynaptic element is depolarized by an action potential. When an action potential reaches the axon terminal it depolarizes the membrane and opens VGSC. Sodium ions enter the cell, further depolarizing the presynaptic membrane. This depolarization causes voltage-gated Ca2+ channels to open. Calcium ions entering the cell initiate a signalling cascade that causes small membrane-bound vesicles, called synaptic vesicles, containing neurotransmitter molecules to fuse with the presynaptic membrane. Fusion of a vesicle with the presynaptic membrane causes neurotransmitter to be released into the synaptic cleft, the extracellular space between the presynaptic and postsynaptic membranes. The neurotransmitter diffuses across the synaptic cleft and binds to receptor proteins on the postsynaptic membrane. Once neurotransmission has occurred, the neurotransmitter is removed from the synaptic cleft and the postsynaptic membrane can “reset” and be ready to receive another signal. This can be accomplished in three ways: the neurotransmitter can diffuse away from the synaptic cleft, it can be degraded by enzymes in the synaptic cleft, or it can be recycled (sometimes called reuptake) by glia cells and the presynaptic neuron.
In principle neurons can excrete excitatory and inhibitory neurotransmitters, which induces in the postsynaptic membrane either a depolarisation or hyperpolarisation, respectively. In consequence this can trigger or impede the generation of a new postsynaptic action potential. Neurons integrate the various excitatory and inhibitory signals they receive from the synapses with their presynaptic network, which leads to a net signalling result for their postsynaptic neurons. The development and function of these neurotransmissions can be disturbed by various mechanisms. A useful indicative summary of potential effects of stressors on neurotransmission can be found, e.g. in the open textbook Foundations of Neuroscience by Casey Henley (https://openbooks.lib.msu.edu/neuroscience/chapter/drug-and-toxin-effects/ )
“Drugs can alter neurotransmitter synthesis pathways, either increasing or decreasing the amount of neurotransmitter made in the terminal, affecting how much transmitter is released. An example of this is administration of L-DOPA, a dopamine precursor molecule that results in increased dopamine production; it is used as a treatment for Parkinson’s Disease. Neurotransmitter packaging is another site of possible drug action. Reserpine, which has been used to treat high blood pressure, blocks the transport of the monoamine transmitters into vesicles by inhibiting the vesicular monoamine transporter (VMAT). This decreases the amount of neurotransmitter stores and the amount of neurotransmitter released in response to an action potential. The neurotransmitter receptors are another critical location for drug and toxin action. Agonists mimic neurotransmitter effects, whereas antagonists block neurotransmitter effects. Muscimol, a component of some mushrooms, is an agonist for the ionotropic GABA receptor. Bicuculine, a component of some plants, is an antagonist to this receptor and blocks the action of GABA. Additionally, many chemicals are able to modulate receptors in either a positive or negative fashion. Alcohol binds to the GABA receptor and increases the time the receptor is open when GABA binds. Finally, neurotransmitter degradation and reuptake can also be altered by drugs and toxins. Depending on the neurotransmitter, enzymes located in either the synapse or in the terminal are responsible for degradation of the transmitter, and these enzyme can be blocked by drugs. Organophosphates are found in many pesticides and prevent the action of acetylcholinesterase, the enzyme that breaks down acetylcholine in the synapse. This inhibition increases acetylcholine action on the postsynaptic neuron. Monoamine oxidase inhibitors (MAOIs) prevent monoamine oxidase from degrading the biogenic amine neurotransmitters. MAOIs have been used as antidepressants since they increase the amount of transmitter available. Additionally, drugs can prevent the reuptake of neurotransmitters into the presynaptic terminal. Cocaine blocks the dopamine transporter, which results in increased action of dopamine in the synapse. Drugs and toxins can also affect neuron function by acting outside of the synapse. For example, some chemicals change voltage-gated ion channel dynamics. Veratridine, a compound found in plants from the lily family, prevents voltage-gated sodium channels from inactivating. Initially, this causes an increase in neurotransmitter release, but it can quickly lead to excitotoxicity.”
Neurotoxins acting on sodium channels have similar effects, at the steady state, on increase of Na influx, depolarization, Ca2 increase, and exocytosis. Depending on the toxin binding site on sodium channels, the release of neurotransmitter can be modified qualitatively and/or quantitatively (Messensini et al. 2003).
Figure 7. Mechanism involved in synaptic neurotransmitter release (Source: Encyclopedia Britannica)
How It Is Measured or Detected
Neurotransmitter release can be measured by electrical effects of released neurotransmitter on postsynaptic membrane receptors and directly by biochemical assay. One way to estimate neurotransmitter release is to measure the postsynaptic response that it evokes. It is an indirect measure since it includes events following release, such as neurotransmitter diffusion from the pre- to the postsynaptic element, binding of neurotransmitter molecules to postsynaptic receptors and induction of the postsynaptic current. Within scientific experiments often the influence of various biological modifiers (proteins, genes, chemicals) on the neurotransmission of inhibitory neurons and excitatory neurons is tested to understand the development, function and disturbance of neuronal networks. This can be done, e.g. by methods that measure evoked inhibitory postsynaptic currents (eIPSCs) or evoked excitatory postsynaptic currents (eEPSCs) or micro postsynaptic currents (mPSCs).
All these methods require to control spontaneous action potentials and pre-synaptic currents. This allows to use the patch clamp technique applying (evoking) pre-synaptic currents and measuring resulting post-synaptic currents (ePSCs) or measure just the mPSCs (thought to represent a response that is elicited by a single vesicle of transmitter under the condition of a pre-synaptically blocked action potential). The electrophysiological technique used to measure such variables is the patch clamp (Neher & Marty, 1982) in the whole-cell recording mode, where the plasma membrane patch in the pipette is ruptured. The transverse hippocampal slice is widely used as an electrophysiological preparation to study synaptic plasticity. The optimizations of the experimental settings for these measurements represent specific measurements for the quantitative relationship between evoking pre-synaptic current and resulting post-synaptic current. This is the immediate empirical evidence for this KE and associated KERs (KER3 in this AOP) in terms of essentially and, as such, can be used in the assessment of dose-concordance to empirically support the KER (KER3).
Neurotransmitter release can also be examined using the genetically encoded synaptic transmission reporter synapto-pHluorin, which uses pH-sensitive mutants of GFP. The interiors of synaptic vesicles are acidified to a pH of approximately 5.7, an environment that keeps the pHluorins in an off state. When the vesicle fuses with the plasma membrane during exocytosis, the pH rises to extracellular levels, switching the pHluorin on and causing it to fluoresce (Figure 7.6). One of the advantages of using synapto-pHluorins is that the signal regenerates through multiple rounds of vesicle release and recycling, which permits vesicle recycling to be imaged in addition to synaptic transmission (Carter and Shieh, 2015).
Neurotransmitter release can also be evaluated in vivo by western blotting quantification or by micro dialysis and analytical quantification.
The Patch-clamping is a versatile electrophysiological tool for understanding ion channel behaviour. Every cell expresses ion channels, but the most common cells to study with patch-clamp techniques include neurons, muscle fibres, cardiomyocytes, and oocytes overexpressing single ion channels.
The patch-clamp technique involves a glass micropipette forming a tight gigaohm (GΩ) seal with the cell membrane. The micropipette contains a wire bathed in an electrolytic solution to conduct ions. The whole-cell technique involves rupturing a patch of membrane with mild suction to provide low-resistance electrical access, allowing control of transmembrane voltage. Alternatively, investigators can pull a patch of membrane away from the cell and evaluate currents through single channels via the inside-out or outside-out patch-clamp technique.
The analysis of single cell membranes can give information about how the cell membrane responds when changes to ionic strength, voltage potential, or cell type are changed. Patch clamp analysis is often studied in Petri dishes or well-plates to minimize manipulation and increase the chances of cell viability. However, planar electrical stimulation geometries can also be used for ease of use. Additionally, robotic targeting algorithms can be used to improve the rate of specified cell targeting.
Voltage clamping is performed by applying a sustained and consistent voltage to the sample to stimulate the membranes of excitable cells, initiating the ionic flow changes. These cells have a resting membrane potential and membrane resistance that must be measured and overcome before stimulation. Voltage clamping requires a voltage electrode for recording the transmembrane voltage and a current electrode for passing current through the membrane. A constant feedback loop of recording the membrane potential is generated, ensuring the cell remains at a constant potential. Once the desired potential has been stabilized, electrical potential measurements, as well as visual recordings are taken to determine the organelle functionality in the presence of various stimuli.
The current clamping technique applies a known current through the specimen and measures how the membrane potential changes. Unlike voltage clamping which seeks to maintain the membrane potential at a given value, current clamping uses a single micropipette to apply the desired current and record how the membrane potential changes in response. By administering repetitive current pulses, the membrane resistance can be calculated using Ohm’s law.
Extracellular recording and microelectrodes array.
Intracellular recordings are great for measurements of the ionic conditions in a single cell. A different set of measurements can look at changes in ion concentrations in the extracellular fluid or a group of neurons. Extracellular recordings show changes in the current or potential of several cells surrounding a microelectrode. Alterations to position and size of this electrode will change the nature of the measurement, depending on what properties are being investigated. By using two or more electrodes and a process called spike sorting, it is possible to work out the number of cells being recorded from and the activity occurring in each cell. Spike sorting uses computer algorithms to analyse the waveforms of the electrical activity from multiple electrodes and distinguish the activity of the individual neurons. Extracellular field potentials measure the electrical potential of a group of cells whose source is difficult to determine. The signals from these cells will overlap and the recording will be a sum of all of the electrical activity. These recordings are known as local field potentials.
Microelectrode arrays are chips that contain multiple electrodes through which neural signals can be recorded. They commonly have stimulating electrodes to deliver signals to a sample as well. The number of electrodes ranges from tens to thousands depending on the spatial resolution and amount of data required by the experiment. Different types of arrays can be used for a wide variety of in vitro and in vivo applications.
Microelectrode array (MEA) recordings are also used to measure electrical activity in cultured neurons (Keefer et al., 2001; Gramowski et al., 2000; Gopal, 2003; Johnstone et al., 2010). MEAs can be applied in high-throughput platforms to facilitate screening of numerous chemical compounds (McConnell et al., 2012). Using selective agonists and antagonists of different classes of receptors their response can be evaluated in a quantitative manner (Novellino et al., 2011; Hogberg et al., 2011). The MEA allow examination of general network activity, bursting activity and network connectivity and using at least 16 measures it has been demonstrated to identify negative and positive control compounds, to identify concentrations at which network failure begins and to identify selective network perturbations versus non-specific cytotoxic effects when coupled with terminal cell death assays when using different test systems from different species (human, rats, mouse; Brown et al., 2016, 2017; Frank et al., 2017; Masjosthusmann et al., 2020; Vassallo et al., 2017).
Domain of Applicability
The connections between neurons, and between neurons and downstream effector cells, occur at specialized cell junctions called synapses. Synapses can occur by direct electrical coupling between two cells, but chemical synapses, in which communication is via the release of a neurotransmitter, are more common and are involved in more complex information processing. The proteins involved in chemical synaptic transmission are much more numerous and diverse than those involved in electrical conduction In vertebrates, synaptic transmission usually travels in one direction, but ctenophore and cnidarian synapses are often bidirectional.
We divide the synapse into (a) a presynaptic module, in which calcium signals are transduced into chemical secretions (known as excitation–secretion coupling); (b) a postsynaptic module (postsynaptic density), which comprises the proteins that support the specialized postsynaptic membrane and the signalling that goes on there; and (c) a module that determines the specific wiring diagram of neurons during development (axonogenesis). For the module “c”, during development, and after injury, axons must grow and find their correct synaptic targets. The proteins responsible for this targeting include secreted and membrane-bound signals and receptors that have not been studied in an evolutionary framework as well as for the other modules. Despite its apparent specialization for neuronal signalling, the excitation–secretion system in neurons comprises many ancient gene families. However, like the transduction module, these gene families are often used differently in the various animal lineages. The proteins involved in docking and in recycling are, for the most part, conserved across eukaryotes (Liebenskind et al. 2017).
Several neurotransmitters have been found not only in animals, but also in plants and microorganisms. Thus, the presence of neurotransmitter compounds has been shown in organisms lacking a nervous system and even in unicellular organisms. Today, we have evidence that neurotransmitters, which participate in synaptic neurotransmission, are multifunctional substances participating in developmental processes of microorganisms, plants, and animals (Roschchina 2010).
The neurotransmission wiring code, which includes Excitation–Secretion Coupling, Postsynaptic Density and Axonogenesis is present across multiple taxa and is representing a fundamental brain developmental process.
Carter, M., & Shieh, J. C. (2015). Guide to research techniques in neuroscience. Academic Press.
Encyclopædia Britannica, Inc. https://www.britannica.com/science/synapse, accessed on December 2020
Liebeskind, B. J., Hofmann, H. A., Hillis, D. M., & Zakon, H. H. (2017). Evolution of animal neural systems, Annual review of ecology, evolution, and systematics, 48, 377-398, Annual Reviews, https://doi.org/10.1146/annurev-ecolsys-110316-023048.
Massensini, A. R., Romano-Silva, M. A., & Gomez, M. V. (2003). Sodium channel toxins and neurotransmitter release. Neurochemical research, 28(10), 1607-1611.
Roshchina, V. V. (2010). Evolutionary considerations of neurotransmitters in microbial, plant, and animal cells. In Microbial endocrinology (pp. 17-52). Springer, New York, NY.
Kress G.J. and Mennerick S. Action potential initiation and propagation: upstream influences on neurotransmission. Neuroscience. 2009 January 12; 158(1): 211–222. doi:10.1016/j.neuroscience.2008.03.021)